Immunology

Immunology

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Board Coverage AQA Paper 2 | Edexcel A Paper 2 | OCR (A) Paper 2 | CIE Paper 4

1. Overview of the Immune System

1.1 Types of Immunity

The immune system can be divided into two broad categories based on specificity and memory:

Feature	Innate (Non-Specific) Immunity	Adaptive (Specific) Immunity
Specificity	Non-specific; responds to a range of pathogens	Highly specific; targets particular antigens
Memory	No immunological memory	Memory cells enable faster secondary response
Speed of response	Immediate (minutes to hours)	Slower initial response (days); faster on second exposure
Cells involved	Phagocytes, neutrophils, mast cells, NK cells	T lymphocytes (T cells), B lymphocytes (B cells)
Molecules involved	Complement proteins, interferons, antimicrobial peptides	Antibodies, cytokines, perforin, granzymes
Physical barriers	Skin, mucous membranes, stomach acid, lysozyme	Not applicable

The immune response proceeds in a coordinated sequence. Innate immunity provides the first line of defence, acting immediately to limit infection. Adaptive immunity is activated more slowly but provides targeted, long-lasting protection.

1.2 Antigens and Pathogens

A pathogen is any organism that causes disease: bacteria, viruses, fungi, protoctista, and helminths (parasitic worms).

An antigen is a molecule (usually a protein or glycoprotein) on the surface of a pathogen or on a transplanted cell that is recognised as foreign by the immune system, provoking an immune response. Each antigen has a specific epitope (the precise molecular region recognised by an antibody or T-cell receptor).

Self-antigens are molecules on the surface of an individual's own cells that are normally tolerated by the immune system. The ability to distinguish self from non-self is fundamental to immune function. Failure of this distinction leads to autoimmune diseases (see Section 6).

Non-self antigens include:

• Antigens on the surface of invading pathogens.
• Toxins produced by pathogens.
• Antigens on the surface of transplanted tissue (triggering transplant rejection).
• Allergens (antigens that trigger allergic responses).

2. Innate Immunity

2.1 Physical and Chemical Barriers

The first line of defence prevents pathogen entry entirely:

Barrier	Mechanism
Skin	Physical barrier; keratinised epidermis; sebum (antimicrobial lipids); low pH ($\approx 5.5$)
Mucous membranes	Trap pathogens; mucus contains lysozyme (breaks bacterial cell walls); cilia move mucus outwards
Stomach acid	$\mathrm{HCl}$ (pH $\approx 2$) denatures proteins and kills most ingested pathogens
Tears and saliva	Contain lysozyme; flush pathogens from eyes and mouth
Commensal bacteria	Compete with pathogens for nutrients and space; produce antimicrobial substances
Blood clotting	Seals wounds, preventing pathogen entry into tissues

2.2 Phagocytosis

Phagocytosis is the engulfment and digestion of pathogens by specialised white blood cells called phagocytes (neutrophils and macrophages). It is a non-specific mechanism -- phagocytes recognise and destroy any foreign material.

Stages of phagocytosis:

1. Chemotaxis: phagocytes are attracted to the site of infection by chemical signals released by damaged cells and pathogens (e.g., bacterial products, complement fragments $\mathrm{C3a}$ and $\mathrm{C5a}$).

2. Recognition and attachment: the phagocyte recognises non-self antigens on the pathogen surface. Phagocytes have pattern recognition receptors (PRRs) that bind to pathogen-associated molecular patterns (PAMPs) -- conserved molecular structures found on many pathogens (e.g., lipopolysaccharide on Gram-negative bacteria).

3. Engulfment: the phagocyte extends pseudopodia around the pathogen, enclosing it in a membrane-bound vesicle called a phagosome.

4. Digestion: the phagosome fuses with a lysosome (a vesicle containing hydrolytic enzymes) to form a phagolysosome. Lysosomal enzymes (lysozyme, proteases, lipases, nucleases) break down the pathogen.

5. Exocytosis: indigestible material is expelled from the cell by exocytosis.

6. Antigen presentation: after digestion, phagocytes (particularly macrophages and dendritic cells) process some of the pathogen's antigens and display them on their own cell surface using major histocompatibility complex (MHC) molecules. This is a critical bridge between innate and adaptive immunity, as displayed antigens activate T lymphocytes.

Macrophages vs neutrophils:

Feature	Neutrophils	Macrophages
Lifespan	Short (hours to days)	Long (months)
Location	Circulate in blood; migrate to infection	Reside in tissues (liver, spleen, lymph nodes, lungs)
Antigen presentation	Limited	Yes -- display antigens on MHC molecules
Number in blood	Most abundant white blood cell ($60$--$70\%$)	Less abundant ($2$--$8\%$)
Response	First to arrive at infection site	Arrive later; sustain long-term response

2.3 Inflammation

Inflammation is a localised, non-specific immune response to tissue damage or infection. It is characterised by the classic signs: rubor (redness), calor (heat), tumor (swelling), dolor (pain), and functio laesa (loss of function).

The inflammatory response cascade:

1. Tissue damage releases chemical mediators: histamine (from mast cells and basophils), prostaglandins, and cytokines (e.g., interleukins, tumour necrosis factor).

2. Histamine causes vasodilation of arterioles supplying the damaged tissue. This increases blood flow to the area, causing redness and heat. The increased blood flow delivers more phagocytes, oxygen, and nutrients.

3. Histamine also increases the permeability of capillary walls, allowing plasma proteins (including complement and clotting factors) and phagocytes to leak into the tissue fluid. This causes swelling (oedema) and pain (due to pressure on nerve endings).

4. Phagocyte migration: neutrophils adhere to the inner wall of blood vessels near the site of damage (margination), then squeeze through the capillary wall by diapedesis and migrate towards the site of infection by chemotaxis.

5. Fibrin formation: a blood clot may form to wall off the infected area, preventing the spread of pathogens.

6. Resolution: once the infection is cleared, dead cells and debris are removed by phagocytes. Tissue repair begins.

warning

Common Pitfall Students often confuse the roles of histamine and antibodies in the inflammatory response. Histamine is released by mast cells immediately upon tissue damage and causes the vasodilation and increased permeability characteristic of inflammation. Antibodies are produced later by B cells as part of the adaptive immune response and are not involved in the initial inflammatory cascade.

2.4 The Complement System

The complement system is a collection of approximately 20 plasma proteins that enhance (complement) the immune response. They are synthesised in the liver and circulate in an inactive form.

When activated (by antigen-antibody complexes or by pathogen surfaces), complement proteins act in a cascade:

1. Opsonisation: complement protein $\mathrm{C3b}$ binds to the pathogen surface, making it more easily recognised and engulfed by phagocytes (acts as an opsonin).

2. Chemotaxis: $\mathrm{C3a}$ and $\mathrm{C5a}$ attract phagocytes to the site of infection.

3. Membrane attack complex (MAC): complement proteins $\mathrm{C5}$ through $\mathrm{C9}$ assemble into a pore-forming complex that inserts into the pathogen's cell membrane, creating transmembrane channels. Ions and water flow freely through these channels, causing the pathogen to swell and burst (lysis). The MAC is effective against Gram-negative bacteria and enveloped viruses.

3. Adaptive Immunity: The Cell-Mediated Response

3.1 T Lymphocytes

T lymphocytes (T cells) are produced in the bone marrow but migrate to and mature in the thymus gland. During maturation, T cells undergo a selection process: cells that recognise self-antigens too strongly are eliminated (negative selection), preventing autoimmune reactions. Only T cells that can recognise foreign antigens presented on self-MHC molecules are released into circulation.

T cells have specific T-cell receptors (TCRs) on their surface, each binding to a specific antigen presented by an MHC molecule. Unlike antibodies, TCRs cannot bind free antigen -- they can only recognise antigens displayed on the surface of another cell.

There are two major classes of T cells:

• T helper cells ($\mathrm{CD4^+}$): coordinate the immune response by releasing chemical messengers (cytokines) that stimulate other immune cells. They recognise antigens presented by MHC class II molecules on antigen-presenting cells (macrophages, dendritic cells, B cells).
• T killer cells (cytotoxic T cells, $\mathrm{CD8^+}$): directly kill infected body cells and cancer cells by inducing apoptosis (programmed cell death). They recognise antigens presented by MHC class I molecules on the surface of any nucleated cell.

3.2 Antigen Presentation

Antigen-presenting cells (APCs) -- primarily macrophages and dendritic cells -- are the bridge between innate and adaptive immunity:

1. An APC phagocytoses a pathogen and partially digests it.
2. The pathogen's antigens are processed and combined with MHC molecules inside the cell.
3. The antigen-MHC complex is displayed on the APC's cell surface.
4. A T helper cell with a complementary TCR binds to the antigen-MHC complex.
5. The APC also provides co-stimulatory signals (e.g., cytokines) that fully activate the T helper cell.

3.3 Activation of T Helper Cells

When a T helper cell binds to its specific antigen on an APC:

1. The T helper cell is activated and undergoes clonal expansion: it divides repeatedly by mitosis to produce a large clone of identical T helper cells, all specific to the same antigen.
2. The activated T helper cells release cytokines that:
   • Stimulate B cells to divide and differentiate (linking to the humoral response).
   • Stimulate phagocytes to increase phagocytic activity.
   • Activate T killer cells.

3.4 T Killer Cells and Apoptosis

T killer cells destroy host cells that have been infected by viruses or have become cancerous (displaying abnormal antigens on their MHC class I molecules).

The mechanism of killing:

1. The T killer cell's TCR binds to the specific antigen displayed on the target cell's MHC class I.
2. The T killer cell releases perforin, a protein that inserts into the target cell membrane and forms pores (transmembrane channels).
3. Through these pores, granzymes (protease enzymes) enter the target cell.
4. Granzymes activate caspases (enzymes that drive apoptosis) inside the target cell.
5. The target cell undergoes apoptosis: its DNA is fragmented, the cell shrinks and breaks into membrane-bound fragments (apoptotic bodies) that are phagocytosed by macrophages. This is important because it prevents the release of new virus particles and limits damage to surrounding tissue.

After the infection is cleared, most effector T cells die by apoptosis. A small population differentiates into memory T cells that persist for years, providing rapid secondary response on re-exposure to the same antigen.

warning

Common Pitfall Students often write that T killer cells "engulf" or "digest" pathogens. They do not. T killer cells induce apoptosis in infected host cells. Phagocytosis is carried out by phagocytes (neutrophils and macrophages), not by T killer cells.

4. Adaptive Immunity: The Humoral Response

4.1 B Lymphocytes and Antibodies

B lymphocytes (B cells) are produced and mature in the bone marrow. Each B cell displays antibodies on its surface that act as receptors specific to a particular antigen. There are approximately $10^7$ to $10^9$ different B cell clones, each recognising a different antigen.

B cells are responsible for the humoral response (antibody-mediated immunity), which targets extracellular pathogens (bacteria, viruses in body fluids, and toxins).

4.2 Activation and Clonal Selection of B Cells

1. A B cell with surface antibodies complementary to a specific antigen binds to that antigen.
2. The antigen is internalised, processed, and displayed on the B cell's MHC class II molecules.
3. An activated T helper cell (specific to the same antigen) binds to the antigen on the B cell and releases cytokines.
4. These cytokines stimulate the B cell to undergo clonal selection and clonal expansion: the B cell divides repeatedly by mitosis, producing a large clone of identical B cells.
5. Most of these clone cells differentiate into plasma cells; some differentiate into memory B cells.

4.3 Plasma Cells and Antibody Production

Plasma cells are short-lived (days to weeks) but produce and secrete enormous quantities of antibodies -- up to 2000 antibody molecules per second. These antibodies circulate in the blood and lymph, binding to the specific antigen that triggered the response.

Memory B cells persist for months to years. On re-exposure to the same antigen, they divide rapidly and differentiate into plasma cells, producing antibodies much faster and in greater quantities than during the primary response.

4.4 Antibody Structure

Antibodies (immunoglobulins, Ig) are Y-shaped quaternary proteins consisting of four polypeptide chains: two identical heavy chains and two identical light chains, held together by disulfide bonds and non-covalent interactions.

Each antibody has:

• Two identical antigen-binding sites (at the tips of the Y), formed by the variable regions ($\mathrm{V}$ regions) of the heavy and light chains. The amino acid sequence in the variable region differs between antibodies, determining antigen specificity.
• A constant region ($\mathrm{C}$ region) that is the same within each antibody class (IgG, IgA, IgM, IgE, IgD). The constant region determines the antibody's effector functions (e.g., which cells and complement proteins it can bind).
• A hinge region allowing flexibility, enabling the two antigen-binding sites to attach to antigens at varying distances.

4.5 Classes of Antibodies

Class	Structure	Location	Function
IgG	Monomer	Blood, lymph, tissue fluid	Most abundant; crosses placenta; opsonisation; neutralisation
IgA	Monomer or dimer	Secretions (saliva, tears, breast milk, mucus)	Protects mucosal surfaces; dimer form in secretions
IgM	Pentamer	Blood	First antibody produced in primary response; effective agglutination
IgE	Monomer	Bound to mast cells	Triggers allergic responses (histamine release); defence against parasites
IgD	Monomer	B cell surface	Acts as B cell receptor; role not fully understood

4.6 Mechanisms of Antibody Action

Antibodies do not kill pathogens directly. They act by several mechanisms:

1. Agglutination: antibodies bind to antigens on the surface of pathogens, cross-linking them into clumps. This prevents the pathogens from spreading, makes it easier for phagocytes to engulf multiple pathogens at once, and blocks pathogen attachment to host cells.

2. Opsonisation: the constant region (Fc region) of the antibody is recognised by phagocytes, which have Fc receptors. Antibodies coating a pathogen act as a molecular "flag" that enhances phagocytosis.

3. Neutralisation: antibodies bind to toxins or viral surface proteins, preventing them from damaging host cells or entering host cells. Antibodies against viruses can block the viral attachment proteins that bind to host cell receptors.

4. Activation of complement: the Fc region of IgG and IgM antibodies can initiate the classical complement pathway, leading to opsonisation (C3b), chemotaxis (C3a, C5a), and membrane attack complex formation (C5--C9).

5. Antibody-dependent cell-mediated cytotoxicity (ADCC): the Fc region of antibodies bound to a target cell is recognised by natural killer (NK) cells, which then kill the target cell.

5. The Primary and Secondary Immune Responses

5.1 Primary Response

The primary response occurs on first exposure to a specific antigen:

• Latent period: there is a delay of approximately 5--10 days before a significant antibody concentration is detectable. This is because:
  • The naive B cell specific to the antigen must be located and activated.
  • Clonal expansion takes time.
  • Plasma cell differentiation and antibody production must occur.
• Antibody concentration: rises slowly, reaches a relatively low peak, then declines as plasma cells die.
• IgM is the predominant antibody class initially.
• Some B cells differentiate into memory B cells and some T cells into memory T cells, providing long-term immunity.

5.2 Secondary Response

The secondary (anamnestic) response occurs on subsequent exposure to the same antigen:

• Latent period: much shorter (1--3 days). Memory B cells are already present and rapidly divide and differentiate into plasma cells.
• Antibody concentration: rises more rapidly, reaches a much higher peak, and persists for longer.
• IgG is the predominant antibody class.
• The secondary response is the basis of immunological memory and vaccination.

Feature	Primary Response	Secondary Response
Latent period	5--10 days	1--3 days
Peak antibody level	Relatively low	Much higher (10--100 times)
Antibody class	IgM predominates initially	IgG predominates
Antibody persistence	Declines within weeks	Persists for months to years
Memory cells produced	Yes	Yes (additional)
Cell type involved	Naive B cells	Memory B cells

warning

Common Pitfall Students often state that "antibodies kill pathogens." Antibodies do not kill pathogens directly. They mark pathogens for destruction by other mechanisms (phagocytosis, complement lysis, neutralisation of toxins). The antibody itself is a signalling molecule that tags the pathogen and triggers other components of the immune system.

6. Autoimmune Diseases

6.1 Mechanism of Autoimmunity

Autoimmune diseases occur when the immune system fails to distinguish self from non-self and attacks the body's own tissues. This involves the activation of autoreactive T or B cells that have escaped negative selection in the thymus or bone marrow.

Potential triggers for autoimmune disease:

• Molecular mimicry: a pathogen antigen resembles a self-antigen, so antibodies and T cells raised against the pathogen cross-react with self-tissues.
• Genetic predisposition: certain HLA (MHC) alleles increase susceptibility to autoimmune diseases.
• Hormonal factors: autoimmune diseases are more common in females, suggesting oestrogen may play a role.
• Environmental factors: infections, stress, and toxins may trigger autoimmunity in genetically susceptible individuals.

6.2 Examples of Autoimmune Diseases

Disease	Target Tissue	Mechanism and Effects
Type 1 diabetes mellitus	Pancreatic beta cells (islets of Langerhans)	T killer cells destroy insulin-producing beta cells; insulin deficiency causes elevated blood glucose
Rheumatoid arthritis	Synovial membranes of joints	Antibodies attack joint linings; inflammation, pain, and joint destruction
Multiple sclerosis (MS)	Myelin sheath of nerve fibres	T cells attack myelin; loss of myelin disrupts nerve impulse transmission
Systemic lupus erythematosus (SLE)	Multiple tissues (skin, kidneys, joints)	Antibodies against nuclear antigens; widespread tissue damage
Myasthenia gravis	Acetylcholine receptors at neuromuscular junctions	Antibodies block ACh receptors; muscle weakness and fatigue

For the management of Type 1 diabetes, see Homeostasis.

7. Vaccination

7.1 Principles of Vaccination

Vaccination is the deliberate introduction of antigenic material into the body to stimulate an adaptive immune response without causing disease. The immune system produces memory cells, so that subsequent exposure to the actual pathogen triggers a rapid secondary response that prevents or reduces the severity of illness.

Types of vaccine:

Type	Description	Examples
Live attenuated	Weakened (attenuated) form of the pathogen; replicates slowly but does not cause disease	MMR (measles, mumps, rubella); oral polio
Inactivated (killed)	Pathogen killed by heat or chemicals; cannot replicate	Influenza (some formulations); cholera
Subunit (recombinant)	Purified antigens from the pathogen; often produced by recombinant DNA technology	Hepatitis B; HPV (human papillomavirus)
Toxoid	Inactivated toxin that retains antigenicity	Tetanus; diphtheria
mRNA	mRNA encoding a pathogen antigen; host cells translate the mRNA and display the antigen	COVID-19 (Pfizer-BioNTech, Moderna)
Viral vector	Harmless virus engineered to carry genes encoding pathogen antigens	COVID-19 (Oxford-AstraZeneca); Ebola

7.2 Herd Immunity

Herd immunity occurs when a sufficiently high proportion of the population is immune to an infectious disease (through vaccination or previous infection), reducing the probability of transmission to susceptible individuals. If each infected individual transmits the pathogen to fewer than one susceptible person on average ($R_0 < 1$), the disease will decline and eventually be eliminated.

The proportion of the population that must be immune to achieve herd immunity depends on the basic reproduction number ($R_0$):

$\text{Herd immunity threshold} = 1 - \frac{1}{R_0}$

Worked Example. Measles has $R_0 \approx 15$ (highly contagious).

$\text{Threshold} = 1 - \frac{1}{15} = 1 - 0.067 = 0.933 = 93.3\%$

Approximately 93% of the population must be immune to measles to prevent outbreaks. This is why measles vaccination coverage targets must be very high.

For comparison:

• Polio ($R_0 \approx 6$): threshold $\approx 83\%$
• COVID-19 (Omicron, $R_0 \approx 10$): threshold $\approx 90\%$
• Seasonal influenza ($R_0 \approx 2$): threshold $\approx 50\%$

7.3 Limitations and Challenges of Vaccination

• Antigenic variation: pathogens such as influenza virus and HIV mutate rapidly, changing their surface antigens. Vaccines developed against one strain may not be effective against new strains, requiring annual vaccine updates.
• Individuals who cannot be vaccinated: infants, the immunocompromised, and those with severe allergies to vaccine components rely on herd immunity for protection.
• Vaccine hesitancy: misinformation and lack of public trust can reduce vaccination coverage below the herd immunity threshold, leading to outbreaks of preventable diseases.
• Ethical considerations: mandatory vaccination policies must balance individual autonomy with public health benefits.

warning

Common Pitfall Students sometimes state that vaccination provides "artificial passive immunity." Vaccination provides artificial active immunity because it stimulates the body's own immune system to produce antibodies and memory cells. Passive immunity (e.g., maternal antibodies crossing the placenta, or injection of pre-made antibodies) provides temporary protection but does not create memory cells.

8. ELISA (Enzyme-Linked Immunosorbent Assay)

8.1 Principle

ELISA is a quantitative biochemical assay that uses antibodies linked to an enzyme to detect and measure the concentration of a specific antigen (or antibody) in a sample. The enzyme catalyses a colour change reaction, and the intensity of the colour is proportional to the amount of antigen or antibody present.

8.2 Direct ELISA (Detecting Antigen)

1. Antigen from the sample is bound to the bottom of a well in a microtitre plate.
2. A primary antibody specific to the antigen is added and binds to the antigen.
3. A secondary antibody (linked to an enzyme such as horseradish peroxidase) is added and binds to the primary antibody.
4. A substrate is added. The enzyme converts the substrate into a coloured product.
5. The absorbance (colour intensity) is measured with a colorimeter/plate reader.
6. A standard curve (absorbance vs. known antigen concentration) is used to determine the concentration of antigen in the unknown sample.

8.3 Indirect ELISA (Detecting Antibodies)

Used, for example, in HIV testing:

1. HIV antigen is bound to the bottom of the well.
2. The patient's blood serum is added. If HIV antibodies are present, they bind to the antigen.
3. An enzyme-linked secondary antibody (anti-human immunoglobulin) is added and binds to any patient antibodies present.
4. Substrate is added. Colour develops only if patient antibodies are present.
5. Absorbance is measured and compared to controls.

8.4 Worked Example: Quantitative ELISA

A standard curve is prepared using known concentrations of antigen, and the absorbance of each is measured:

Antigen concentration ($\mathrm{ng\ cm^{-3}}$)	0	5	10	20	40	80
Absorbance (at 450 nm)	0.05	0.12	0.23	0.42	0.78	1.45

An unknown patient sample gives an absorbance of 0.55. Determine the antigen concentration.

From the standard curve, absorbance of 0.55 lies between the 20 ($0.42$) and 40 ($0.78$) data points. Using linear interpolation between these two points:

$\text{Concentration} = 20 + (40 - 20) \times \frac{0.55 - 0.42}{0.78 - 0.42} = 20 + 20 \times \frac{0.13}{0.36} = 20 + 7.2 = 27.2\ \mathrm{ng\ cm^{-3}}$

The patient sample contains approximately $27.2\ \mathrm{ng\ cm^{-3}}$ of antigen.

warning

Common Pitfall In ELISA, the colour change is catalysed by an enzyme attached to the antibody, not by the antigen itself. Students sometimes write that "the antigen changes colour." The enzyme on the secondary antibody converts the substrate to a coloured product; the intensity of colour indicates how much antigen-antibody complex is present.

9. Monoclonal Antibodies

9.1 Production of Monoclonal Antibodies (Hybridoma Method)

Monoclonal antibodies are identical antibodies produced by a single clone of B cells, all specific to the same epitope on an antigen. They are produced using the hybridoma technique (Kohler and Milstein, 1975):

1. A mouse is injected with the target antigen, stimulating its B cells to produce antibodies.
2. B cells are extracted from the mouse's spleen.
3. These B cells are fused with myeloma cells (cancerous B cells that divide indefinitely) using polyethylene glycol (PEG) or electrofusion.
4. The resulting hybridomas combine the antibody-producing ability of B cells with the immortality of myeloma cells.
5. Hybridomas are cultured in HAT medium (containing hypoxanthine, aminopterin, and thymidine). Unfused myeloma cells die (they lack HGPRT, an enzyme needed to use hypoxanthine); unfused B cells die naturally (they have a limited lifespan).
6. Individual hybridoma cells are separated (by limiting dilution) and cultured in separate wells.
7. Each clone is tested for antibody production against the target antigen.
8. The clone producing the desired antibody is expanded in culture or in mice (ascites fluid).

9.2 Applications of Monoclonal Antibodies

Application	Mechanism
Pregnancy testing	Monoclonal antibodies bind to hCG (human chorionic gonadotropin) in urine; a colour change indicates pregnancy
Cancer treatment	Monoclonal antibodies (e.g., trastuzumab/Herceptin) bind to receptors on cancer cells, blocking growth signals and marking them for immune destruction
Diagnosis of disease	ELISA and lateral flow tests use monoclonal antibodies to detect specific disease antigens or antibodies
Drug delivery	Monoclonal antibodies can be conjugated to drugs or radioactive isotopes and targeted to specific cells (e.g., cancer cells), minimising side effects
Transplant rejection	Monoclonal antibodies (e.g., OKT3) suppress the immune response against transplanted organs by binding to T cells
Purification of substances	Monoclonal antibodies attached to a column can purify specific proteins from a mixture (affinity chromatography)

9.3 Ethical Issues of Monoclonal Antibody Production

• Use of animals: the hybridoma method requires mice for immunisation, spleen cell extraction, and (in some cases) antibody production in ascites fluid. This raises animal welfare concerns.
• Humanised antibodies: early monoclonal antibodies were entirely mouse-derived, causing human anti-mouse antibody (HAMA) responses when used clinically. Modern techniques produce humanised or fully human monoclonal antibodies by genetic engineering, reducing this problem.
• Cost: monoclonal antibody therapies are extremely expensive to develop and manufacture, raising questions about access and equity in healthcare.
• Side effects: monoclonal antibodies can cause immune reactions, infusion reactions, and off-target effects.

10. HIV and AIDS

10.1 How HIV Affects the Immune System

Human immunodeficiency virus (HIV) specifically targets T helper cells ($\mathrm{CD4^+}$):

1. The viral envelope glycoprotein (gp120) binds to the $\mathrm{CD4}$ receptor on T helper cells, along with a co-receptor (CCR5 or CXCR4).
2. The virus enters the T helper cell by fusion with the cell membrane.
3. Viral RNA is reverse-transcribed into DNA by reverse transcriptase.
4. Viral DNA is integrated into the host cell's genome by integrase.
5. The host cell is hijacked to produce new viral particles, which bud from the cell membrane, destroying the T helper cell.

The progressive loss of T helper cells devastates the immune system because T helper cells coordinate both the cell-mediated and humoral responses. Without sufficient T helper cells:

• B cells are not properly stimulated to produce antibodies.
• T killer cells are not activated.
• Macrophage activity is reduced.

The resulting state of immunodeficiency is called AIDS (acquired immune deficiency syndrome), where the individual becomes susceptible to opportunistic infections (infections that a healthy immune system would normally control, e.g., Pneumocystis pneumonia, Mycobacterium tuberculosis, Kaposi's sarcoma).

10.2 Transmission and Prevention

HIV is transmitted through:

• Exchange of bodily fluids (blood, semen, vaginal fluid, breast milk).
• Routes: unprotected sexual contact, sharing contaminated needles, mother-to-child (during birth or breastfeeding), contaminated blood transfusions (now rare due to screening).

Prevention strategies: condom use, needle exchange programmes, antiretroviral therapy (ART) to reduce viral load and transmission risk, pre-exposure prophylaxis (PrEP).

11. Interferons and the Antiviral Response

11.1 Interferons

Interferons are cytokines produced by virus-infected cells as part of the innate immune response. They are the first line of defence against viral infection before the adaptive response is activated.

Mechanism:

1. When a cell is infected by a virus, it produces and secretes interferons.
2. Interferons bind to receptors on neighbouring cells, triggering an antiviral state in those cells.
3. The antiviral state involves: production of enzymes that degrade viral mRNA; inhibition of viral protein synthesis; activation of NK cells (which kill virus-infected cells).

Interferons also enhance the antigen-presenting function of APCs and stimulate T cell activity, bridging innate and adaptive immunity.

Practice Problems

Details

Problem 1

Describe the process of phagocytosis. Explain how phagocytes are able to distinguish between self and non-self cells. (6 marks)

Answer. Phagocytosis involves six stages. (1) Chemotaxis: phagocytes migrate towards the site of infection following chemical gradients (complement fragments, bacterial products). (2) Recognition: phagocytes use pattern recognition receptors (PRRs) to bind to pathogen-associated molecular patterns (PAMPs) on non-self cells. PAMPs include molecules such as lipopolysaccharide (on Gram-negative bacteria) and peptidoglycan (on all bacteria) that are not found on host cells. (3) Engulfment: pseudopodia extend around the pathogen, enclosing it in a phagosome. (4) Lysosome fusion: the phagosome fuses with a lysosome containing hydrolytic enzymes, forming a phagolysosome. (5) Digestion: lysosomal enzymes (lysozyme, proteases) break down the pathogen. (6) Exocytosis: waste material is expelled. Phagocytes distinguish self from non-self through PRRs that bind to molecular patterns unique to pathogens (PAMPs). Healthy self-cells display "self" markers (MHC molecules and specific glycoproteins) that inhibit phagocytosis.

If you get this wrong, revise: Phagocytosis

Details

Problem 2

Explain the difference between the primary and secondary immune responses. Include reference to the cells involved, the speed of response, and the antibody classes produced. (5 marks)

Answer. The primary response occurs on first exposure to an antigen. Naive B cells must be activated (by binding the antigen and receiving cytokine signals from T helper cells), undergo clonal expansion, and differentiate into plasma cells. This takes 5--10 days (the latent period), and antibody levels peak at a relatively low concentration. IgM is the predominant antibody class initially. After the infection is cleared, memory B and T cells persist. The secondary response occurs on re-exposure to the same antigen. Memory B cells already exist, so the latent period is shorter (1--3 days), clonal expansion is faster, and antibody levels reach a much higher peak (10--100 times the primary peak). IgG predominates. The response is faster, stronger, and longer-lasting because the immune system has already been "primed" by the primary exposure.

If you get this wrong, revise: The Primary and Secondary Immune Responses

Details

Problem 3

A patient is tested for HIV antibodies using an indirect ELISA. Explain why this test detects antibodies rather than the virus itself, and describe the steps of the test. (6 marks)

Answer. The test detects antibodies because antibodies are produced in large quantities during the immune response and persist in the blood, whereas the virus itself may be present at very low concentrations that are difficult to detect directly. Additionally, the window period (when antibodies are first detectable) is well-characterised. Steps: (1) HIV antigen is bound to the bottom of a microtitre plate well. (2) The patient's blood serum is added. If HIV antibodies are present, they bind to the immobilised antigen. (3) The well is washed to remove unbound material. (4) An enzyme-linked secondary antibody (anti-human immunoglobulin) is added and binds to any patient antibodies present. (5) The well is washed again. (6) A substrate is added. If the enzyme is present (because patient antibodies were bound), the substrate is converted to a coloured product. (7) Absorbance is measured; a value above a defined threshold indicates a positive result.

If you get this wrong, revise: ELISA

Details

Problem 4

Explain how monoclonal antibodies are produced using the hybridoma technique. Why is this method necessary instead of simply culturing B cells? (5 marks)

Answer. B cells cannot be cultured indefinitely because they have a limited lifespan and undergo apoptosis after a few divisions. The hybridoma technique overcomes this by fusing B cells with myeloma cells (cancerous B cells that divide indefinitely). Steps: (1) A mouse is injected with the target antigen to stimulate antibody production. (2) B cells are extracted from the spleen. (3) B cells are fused with myeloma cells using polyethylene glycol. (4) The mixture is cultured in HAT medium, which allows only hybridomas (B cells + myeloma cells) to survive: unfused myeloma cells die because they lack HGPRT (needed to use hypoxanthine), and unfused B cells die because they have a limited lifespan. (5) Individual hybridoma clones are separated by limiting dilution and tested for antibody production. (6) The clone producing the desired antibody is expanded. This produces an unlimited supply of identical (monoclonal) antibodies specific to a single epitope.

If you get this wrong, revise: Production of Monoclonal Antibodies

Details

Problem 5

Measles has a basic reproduction number ($R_0$) of approximately 15. (a) Calculate the herd immunity threshold for measles. (b) If a school has 1200 students and the measles vaccination rate is 92%, is the school population above or below the herd immunity threshold? How many additional students need to be vaccinated to reach the threshold? (c) Explain why falling vaccination rates can lead to outbreaks of measles even in populations with high overall vaccination coverage.

Answer. (a) Herd immunity threshold $= 1 - \frac{1}{R_0} = 1 - \frac{1}{15} = 1 - 0.0667 = 0.933 = 93.3\%$.

(b) 92% of 1200 = 1104 vaccinated students. The threshold requires 93.3% = 1119.6, so at least 1120 students. An additional $1120 - 1104 = 16$ students need to be vaccinated.

(c) Herd immunity requires uniform distribution of immune individuals. If vaccination rates cluster geographically or socially (e.g., communities with low vaccination rates due to religious or philosophical objections), pockets of susceptible individuals can exist within an otherwise well-vaccinated population. These pockets allow the virus to spread locally, leading to outbreaks. This is why high vaccination coverage must be maintained across all communities, not just on average.

If you get this wrong, revise: Herd Immunity

12. Active and Passive Immunity

12.1 Types of Acquired Immunity

Feature	Active Immunity	Passive Immunity
Source	Own immune system produces antibodies and memory cells	Pre-made antibodies received from another individual
Speed of response	Slow (requires activation and clonal expansion)	Immediate (antibodies already present)
Duration	Long-term (memory cells persist for years)	Short-term (antibodies degraded within weeks/months)
Memory	Yes	No
Examples	Natural infection, vaccination	Maternal antibodies crossing placenta; antivenom/antisera injections

Natural active immunity: acquired through infection with a pathogen. The immune system mounts a primary response, followed by immunological memory.

Artificial active immunity: acquired through vaccination. The immune system responds to a vaccine antigen as if it were a real pathogen, producing memory cells without causing disease.

Natural passive immunity: antibodies transferred from mother to baby across the placenta (IgG) and in breast milk (IgA). Provides temporary protection during the first few months of life, when the infant's own immune system is immature. This is why breast-feeding is recommended for the first 6 months.

Artificial passive immunity: injection of pre-formed antibodies (immunoglobulins) from an immune individual or animal. Examples: antivenom for snake bites, antitoxin for diphtheria, hepatitis B immune globulin, tetanus antitoxin. Used for rapid, short-term protection when there is no time for active immunisation (e.g., after a snake bite or exposure to rabies).

12.2 Comparison of Natural and Artificial Immunity

Type	Natural	Artificial
Active immunity	Infection	Vaccination
Passive immunity	Maternal antibodies (placenta, milk)	Antiserum/antivenom injection

13. Types of Vaccine and Their Mechanisms

13.1 Live Attenuated Vaccines

Live attenuated vaccines contain a weakened (attenuated) form of the pathogen that can replicate within the host but does not cause disease. Because the pathogen replicates, it presents a wide range of antigens to the immune system over an extended period, stimulating both humoral and cell-mediated immunity. This typically produces strong, long-lasting immunity with one or two doses.

Examples: MMR (measles, mumps, rubella), oral polio vaccine (Sabin), varicella (chickenpox), yellow fever.

Advantages: strong, long-lasting immune response; mimics natural infection; often requires fewer doses; can induce mucosal immunity (oral vaccines).

Disadvantages: requires cold chain storage (refrigeration); may revert to virulent form (rare); cannot be given to immunocompromised individuals; shorter shelf life.

13.2 Inactivated Vaccines

Inactivated vaccines contain pathogens that have been killed by heat or chemical treatment (e.g., formaldehyde). They cannot replicate, so they present a limited range of antigens and typically require adjuvants (substances that enhance the immune response) and booster doses to achieve durable immunity.

Examples: influenza (some formulations), cholera, hepatitis A, rabies, polio (Salk, injected).

Advantages: safer than live vaccines; no risk of reversion; can be given to immunocompromised individuals.

Disadvantages: weaker immune response; typically requires multiple doses and boosters; does not induce cell-mediated immunity as effectively as live vaccines.

13.3 Subunit and Recombinant Vaccines

Subunit vaccines contain purified antigens from the pathogen rather than the whole pathogen. Recombinant subunit vaccines are produced by genetically engineering organisms (usually yeast or mammalian cells) to express specific pathogen proteins.

Examples: hepatitis B (produced in yeast), HPV (human papillomavirus, produced in yeast), acellular pertussis (whooping cough), meningococcal group C.

Advantages: very safe (cannot cause disease); no genetic material from the pathogen; suitable for immunocompromised individuals.

Disadvantages: weaker immune response than whole-pathogen vaccines; requires adjuvants; may not stimulate cell-mediated immunity; production can be expensive.

13.4 Toxoid Vaccines

Toxoid vaccines contain inactivated bacterial toxins (toxoids) that retain their antigenicity but not their toxicity. The immune system produces antibodies that neutralise the toxin.

Examples: tetanus, diphtheria.

13.5 mRNA Vaccines

mRNA vaccines contain messenger RNA encoding a pathogen antigen (e.g., the spike protein of SARS-CoV-2). The mRNA is encapsulated in lipid nanoparticles (LNPs) that fuse with host cell membranes, delivering the mRNA into the cytoplasm. The host cell's ribosomes translate the mRNA into the antigen protein, which is displayed on the cell surface and recognised by the immune system.

Examples: Pfizer-BioNTech (Comirnaty), Moderna (Spikevax) -- both for COVID-19.

Advantages: rapid design and production (weeks rather than years); no live pathogen involved; mRNA does not enter the nucleus or interact with host DNA; strong immune response; no risk of infection.

Disadvantages: requires ultra-cold storage (initially $-70\ ^\circ\mathrm{C}$ for Pfizer); mRNA is rapidly degraded; requires lipid nanoparticle delivery; long-term safety data still being collected.

13.6 Viral Vector Vaccines

Viral vector vaccines use a harmless, modified virus (typically an adenovirus) that carries the gene for a pathogen antigen. The vector enters host cells and delivers the gene, which is transcribed and translated, producing the antigen protein.

Examples: Oxford-AstraZeneca (ChAdOx1 nCoV-19), Johnson & Johnson (Ad26.COV2.S) -- both for COVID-19; Ebola vaccine (rVSV-ZEBOV).

Advantages: induces both humoral and cell-mediated immunity; no live pathogen; relatively stable storage.

Disadvantages: pre-existing immunity to the vector may reduce efficacy; potential for rare adverse events (e.g., thrombosis with thrombocytopenia syndrome with adenoviral vectors).

14. Lymphoid Organs and Tissue Distribution

14.1 Primary and Secondary Lymphoid Organs

Primary lymphoid organs are where lymphocytes mature and become functional:

Organ	Lymphocyte Type	Function
Bone marrow	B cells, T cells (produced)	B cell maturation; production of all blood cells (haematopoiesis)
Thymus	T cells	T cell maturation; positive and negative selection

In the thymus, developing T cells undergo:

• Positive selection: T cells whose TCRs can recognise self-MHC molecules survive. T cells that cannot bind to self-MHC die by apoptosis.
• Negative selection: T cells whose TCRs bind too strongly to self-antigens presented by self-MHC are eliminated (prevents autoimmunity). Approximately 95% of developing T cells die in the thymus.

Secondary lymphoid organs are where lymphocytes encounter antigens and are activated:

Organ	Function
Lymph nodes	Filter lymph; site of antigen presentation and B/T cell activation
Spleen	Filters blood; removes old/damaged red blood cells; immune response to blood-borne antigens
Mucosa-associated lymphoid tissue (MALT)	Immune defence at mucosal surfaces (gut, respiratory tract, urogenital tract); includes Peyer's patches in the ileum
Tonsils	Immune defence at the entrance to the respiratory and digestive tracts

14.2 Lymphocyte Circulation

Lymphocytes continuously circulate between the blood, lymph, and secondary lymphoid organs. This surveillance ensures that pathogens are detected rapidly wherever they enter the body. The process:

1. Lymphocytes enter a lymph node via the bloodstream (through specialised blood vessels called high endothelial venules).
2. They pass through the lymph node, scanning for antigens presented by APCs.
3. If no antigen is encountered, they leave via the efferent lymphatic vessel and return to the blood via the thoracic duct.
4. This cycle repeats continuously, ensuring surveillance of the entire body.

When a lymphocyte encounters its specific antigen, it is retained in the lymph node and undergoes clonal expansion.

15. Antibody Structure in Detail

15.1 Domains and Regions

The antibody molecule has several structural domains:

• Variable domain ($V_H$, $V_L$): at the N-terminus of each chain; contains the antigen-binding site (complementarity-determining regions, CDRs). The amino acid sequence varies between antibodies, determining specificity.
• Constant domain ($C_H$1--3, $C_L$): at the C-terminus; the amino acid sequence is the same within each antibody class. Determines the effector functions.
• Hinge region: between $C_H$1 and $C_H$2; provides flexibility, allowing both arms to bind to antigens at varying distances.

15.2 Antibody Diversity

The human immune system can produce approximately $10^{11}$ different antibody specificities from a limited number of genes through several mechanisms:

1. V(D)J recombination: during B cell development in the bone marrow, random combinations of Variable (V), Diversity (D), and Joining (J) gene segments are assembled to form the variable region of the heavy and light chains.
2. Somatic hypermutation: after antigen activation, B cells in the germinal centres of lymph nodes accumulate mutations in the variable region at a rate approximately $10^6$ times higher than the background mutation rate. Mutations that increase antibody affinity are selected for (affinity maturation).
3. Class switching: activated B cells can change the constant region of the antibody they produce (e.g., from IgM to IgG) without changing the variable region (antigen specificity). This is directed by cytokines from T helper cells.

15.3 The Five Antibody Classes: Functional Comparison

Class	Heavy Chain	Structure	Key Properties
IgG	$\gamma$	Monomer	Most abundant in serum; crosses placenta; opsonises; activates complement
IgA	$\alpha$	Mono/dimer	In secretions (saliva, milk, mucus); protects mucosal surfaces
IgM	$\mu$	Pentamer	First antibody in primary response; efficient agglutination
IgE	$\epsilon$	Monomer	Binds to mast cells; triggers histamine release; anti-parasitic
IgD	$\delta$	Monomer	B cell surface receptor; function not fully understood

15.4 Antibody-Antigen Binding

The antibody-antigen interaction is non-covalent and involves several types of bond:

• Hydrogen bonds: between polar groups on the antibody CDRs and the antigen epitope.
• Electrostatic interactions (ionic bonds): between charged amino acid side chains.
• Van der Waals forces: weak, transient attractions between atoms in close proximity.
• Hydrophobic interactions: between non-polar side chains.

The binding is reversible and the strength depends on the affinity (how tightly a single antibody binding site binds to a single epitope) and avidity (the overall strength of binding when multiple binding sites interact with multiple epitopes, as in IgM).

16. Immune System Disorders

16.1 Immunodeficiency

Primary immunodeficiency: genetic disorders affecting immune system components. Examples:

• Severe combined immunodeficiency (SCID): caused by mutations affecting cytokine receptors or enzymes required for V(D)J recombination. Both T and B cell function are severely impaired. Affected individuals are highly susceptible to infections and require a sterile environment ("bubble boy" syndrome). Treated by bone marrow transplant or gene therapy.
• X-linked agammaglobulinaemia (Bruton's disease): mutation in the BTK gene prevents B cell maturation. Patients have very low antibody levels and recurrent bacterial infections. Treated with regular immunoglobulin replacement therapy.

Secondary (acquired) immunodeficiency: caused by external factors:

• HIV/AIDS: the virus destroys T helper cells, progressively devastating cell-mediated and humoral immunity.
• Malnutrition: deficiencies in protein, zinc, vitamin A, and iron impair immune function.
• Immunosuppressive drugs: corticosteroids, chemotherapy drugs, drugs given after organ transplantation.
• Ageing: the immune system gradually declines (immunosenescence), leading to increased susceptibility to infections and reduced vaccine efficacy in the elderly.

16.2 Hypersensitivity (Allergy)

Hypersensitivity is an exaggerated immune response to a normally harmless antigen (allergen).

Type I hypersensitivity (immediate, IgE-mediated):

1. First exposure: the allergen stimulates B cells to produce IgE antibodies.
2. IgE binds to the surface of mast cells and basophils (via Fc receptors).
3. Second exposure: the allergen cross-links adjacent IgE molecules on the mast cell surface.
4. This triggers degranulation: the mast cell releases histamine, heparin, and other inflammatory mediators.
5. Histamine causes vasodilation, increased capillary permeability, mucus production, and smooth muscle contraction.
6. Symptoms: hay fever (rhinitis), asthma, eczema, anaphylaxis.

Anaphylaxis is a severe, systemic Type I reaction that can be fatal. It causes rapid airway constriction, swelling of the throat and tongue, hypotension, and shock. Treatment: intramuscular adrenaline (epinephrine) to counteract vasodilation and bronchoconstriction.

Type IV hypersensitivity (delayed, T cell-mediated):

• Caused by T killer cells (Type IVc) or T helper cells (Type IVd) reacting against antigens.
• Onset is delayed (24--72 hours after exposure).
• Examples: contact dermatitis (poison ivy, nickel), the tuberculin skin test (Mantoux test), graft rejection.
• No antibodies are involved -- this is a cell-mediated immune response.

17. Transplant Immunology

17.1 Types of Transplant

Type	Donor-Recipient Relationship	Rejection Risk	Example
Autograft	Self	None	Skin graft from thigh to face
Isograft	Identical twin	Very low	Skin graft between identical twins
Allograft	Same species, unrelated	Moderate	Kidney transplant, heart transplant
Xenograft	Different species	Very high	Pig heart valve in humans

17.2 Rejection Mechanisms

Transplant rejection occurs when the recipient's immune system recognises the donor tissue as foreign:

• Hyperacute rejection (minutes to hours): caused by pre-existing antibodies in the recipient that react against donor antigens (e.g., ABO blood group antigens on endothelial cells). Prevented by ABO matching.
• Acute rejection (days to weeks): T cell-mediated rejection. Recipient T cells recognise donor MHC molecules (HLA antigens) as foreign and mount a cell-mediated immune response against the graft. Prevented by HLA matching and immunosuppressive drugs (ciclosporin, tacrolimus, azathioprine).
• Chronic rejection (months to years): gradual loss of graft function due to chronic inflammation and fibrosis. Antibodies and T cells against donor HLA contribute. No effective treatment; may require re-transplantation.

17.3 Prevention of Rejection

• Tissue typing: matching donor and recipient HLA (human leukocyte antigen) types to minimise antigenic differences.
• Blood group matching: ensuring donor and recipient have compatible ABO blood groups.
• Immunosuppressive drugs: drugs that suppress the recipient's immune system to prevent rejection (e.g., ciclosporin, which inhibits T cell activation by blocking calcineurin; corticosteroids, which reduce inflammation; azathioprine, which inhibits lymphocyte proliferation). These drugs have significant side effects: increased susceptibility to infections, increased cancer risk, kidney toxicity.

18. Evolutionary Arms Race: Pathogens and the Immune System

18.1 Pathogen Evasion Strategies

Pathogens have evolved numerous strategies to evade the immune system:

Strategy	Mechanism	Example
Antigenic variation	Pathogen alters its surface antigens, evading pre-existing antibodies	Influenza virus (antigenic drift and shift)
Antigenic mimicry	Pathogen surface antigens resemble host molecules, reducing immune detection	Streptococcus pyogenes (M protein resembles host proteins)
Capsule formation	Polysaccharide capsule prevents phagocytosis	Streptococcus pneumoniae, Neisseria meningitidis
Intracellular survival	Pathogen lives inside host cells, hidden from antibodies	Mycobacterium tuberculosis, viruses
Immunosuppression	Pathogen produces proteins that suppress the immune response	HIV (destroys T helper cells); EBV (inactivates CTLs)
Biofilm formation	Bacteria in biofilms are resistant to phagocytosis and antibiotics	Pseudomonas aeruginosa in cystic fibrosis patients

18.2 Antigenic Drift and Shift in Influenza

Antigenic drift: gradual, minor changes in the viral surface proteins (haemagglutinin and neuraminidase) due to the accumulation of point mutations. This is why seasonal flu vaccines are updated annually. The immune system partially recognises the new strain, so disease is usually milder than for a completely novel strain.

Antigenic shift: a major, sudden change in the viral surface proteins caused by the reassortment of genome segments when two different influenza virus strains infect the same cell (e.g., human and avian influenza strains exchanging RNA segments). This produces a novel virus to which the human population has no pre-existing immunity. Antigenic shift can cause pandemics (e.g., H1N1 "swine flu" in 2009).

19. Immunological Techniques

19.1 ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA is used to detect and quantify antigens or antibodies in a sample.

Direct ELISA (detecting antigen):

1. The antigen (e.g., a viral protein) is immobilised on the surface of a microtiter plate well.
2. A primary antibody specific to the antigen is added. It binds to the antigen.
3. An enzyme-linked secondary antibody is added. It binds to the primary antibody.
4. A substrate is added. The enzyme catalyses a colour change. The intensity of the colour is proportional to the amount of antigen present.

Indirect ELISA (detecting antibody, e.g., HIV testing):

1. The antigen (e.g., HIV envelope protein) is immobilised on the plate.
2. The patient's serum (which may contain anti-HIV antibodies) is added. If antibodies are present, they bind to the antigen.
3. An enzyme-linked secondary antibody (anti-human IgG) is added. It binds to any patient antibodies present.
4. Substrate is added. A colour change indicates the presence of anti-HIV antibodies in the patient's serum.

19.2 Monoclonal Antibodies

Monoclonal antibodies (mAbs) are identical antibodies produced by a single clone of B cells (specifically, a single hybridoma cell). They are produced by:

1. Immunising a mouse with the target antigen.
2. Collecting B cells from the mouse's spleen.
3. Fusing the B cells with myeloma cells (cancerous B cells that divide indefinitely) using polyethylene glycol (PEG) or electrofusion. The resulting hybridoma cells combine the antibody-producing ability of the B cell with the immortality of the myeloma cell.
4. Screening hybridomas to identify those producing the desired antibody.
5. Cloning the selected hybridoma by limiting dilution to ensure a monoclonal population.
6. Culturing the hybridoma in bulk (in vivo in mice or in vitro in bioreactors) to harvest the monoclonal antibody.

19.3 Applications of Monoclonal Antibodies

Application	How mAbs Are Used
Pregnancy testing	mAbs against hCG (human chorionic gonadotropin) bound to a colour indicator on a test strip
Cancer treatment	mAbs that bind to tumour-specific antigens (e.g., trastuzumab/Herceptin binds to HER2 receptor on breast cancer cells, blocking growth signals)
Diagnosis	mAbs tagged with radioactive isotopes or fluorescent dyes to locate tumours (imaging)
Autoimmune disease	mAbs that block inflammatory cytokines (e.g., infliximab blocks TNF-$\alpha$ in rheumatoid arthritis)
Transplant rejection	mAbs that suppress the immune response (e.g., basiliximab blocks IL-2 receptor, preventing T cell activation)
Drug delivery	mAbs conjugated to drugs or toxins, delivering them specifically to cancer cells (reducing side effects)

19.4 Advantages and Disadvantages of Monoclonal Antibodies

Advantages	Disadvantages
Highly specific (bind to one target antigen)	Expensive to produce
Can be produced in unlimited quantities	Mouse-derived mAbs may trigger immune response in humans (HAMA response)
Consistent quality between batches	Ethical concerns about animal use (mice for immunisation)
Versatile (many applications)	Development is time-consuming (months to years)

20. Vaccination: Principles and Challenges

20.1 Types of Vaccines

Vaccine Type	Description	Examples	Advantages	Disadvantages
Live attenuated	Weakened pathogen that can replicate but cannot cause disease	MMR, oral polio (Sabin)	Strong immune response (closely mimics natural infection); often provides lifelong immunity with one or two doses	Risk of reversion to virulence (rare); cannot be given to immunocompromised patients; requires cold chain
Inactivated (killed)	Whole pathogen killed by heat or chemicals	Influenza (injected), cholera	Safe (cannot replicate); no risk of reversion	Weaker immune response; requires adjuvants and booster doses
Subunit	Purified antigenic components of the pathogen	Hepatitis B (surface antigen), HPV	Very safe; well-defined composition	Weak immune response without adjuvant; expensive to produce purified antigens
Toxoid	Inactivated toxin	Tetanus, diphtheria	Safe; effective against toxin-mediated diseases	Does not prevent infection, only toxin effects; requires boosters
Conjugate	Polysaccharide antigen linked to a protein carrier	Hib, meningococcal, pneumococcal	Effective in young children (who respond poorly to polysaccharide alone)	More complex to manufacture
mRNA	mRNA encoding a viral surface protein, wrapped in a lipid nanoparticle	COVID-19 (Pfizer-BioNTech, Moderna)	Rapid development; no live virus; strong immune response; no risk of integration into genome (mRNA is quickly degraded)	Requires cold chain ($-20$ to $-80$ degrees C); new technology with limited long-term data
Viral vector	Harmless virus engineered to carry gene for a pathogen antigen	COVID-19 (Oxford-AstraZeneca, Janssen)	Strong immune response; does not require cold chain as strict as mRNA	Pre-existing immunity to the vector may reduce effectiveness
Recombinant	Genetically engineered protein	HPV (Gardasil), hepatitis B	Safe; well-defined; scalable production	May require adjuvants

20.2 Herd Immunity

Herd immunity (community immunity) occurs when a sufficiently high proportion of the population is immune to an infectious disease (through vaccination or previous infection), providing indirect protection to those who are not immune.

The herd immunity threshold depends on the basic reproduction number ($R_0$):

$H = 1 - \frac{1}{R_0}$

Where $H$ = proportion of the population that must be immune for herd immunity.

Disease	$R_0$	Herd Immunity Threshold
Measles	12--18	92--94%
Polio	5--7	80--86%
COVID-19 (Omicron)	8--10	87--90%
Seasonal influenza	1.5--3	33--67%
Smallpox	3--5	67--80%

Herd immunity is important because it protects individuals who cannot be vaccinated (newborns, immunocompromised patients, elderly with weakened immune systems). However, vaccine hesitancy and misinformation can reduce vaccination rates below the herd immunity threshold, allowing outbreaks of previously controlled diseases.

20.3 Why We Need Booster Vaccinations

The primary immune response produces memory cells (memory B and T cells), but antibody levels decline over time (months to years, depending on the vaccine). Booster vaccinations:

• Stimulate memory cells to rapidly produce a large secondary response.
• Increase antibody affinity (affinity maturation continues with each exposure).
• Broaden the immune response (memory B cells produce antibodies against different epitopes over time).
• Protect against new variants (updated vaccines for influenza, COVID-19).

21. Autoimmune Diseases

21.1 Mechanism

Autoimmune diseases occur when the immune system fails to distinguish self-antigens from foreign antigens and attacks the body's own tissues. The exact cause is not fully understood but involves a combination of:

• Genetic predisposition: certain HLA (MHC) alleles increase susceptibility (e.g., HLA-B27 is associated with ankylosing spondylitis).
• Environmental triggers: infections (molecular mimicry), hormones, stress.
• Failure of self-tolerance: during T cell development in the thymus, T cells that strongly react with self-antigens are normally deleted (negative selection). If this process fails, self-reactive T cells escape into the circulation.

21.2 Examples

Disease	Autoantigen	Affected Tissue	Symptoms
Type 1 diabetes	$\beta$ cell antigens (e.g., GAD65, insulin)	Pancreatic $\beta$ cells	Hyperglycaemia, ketoacidosis
Rheumatoid arthritis	Collagen type II, citrullinated proteins	Joint synovium	Joint pain, swelling, destruction
Multiple sclerosis	Myelin basic protein	Myelin sheath of neurons	Muscle weakness, paralysis, visual disturbances
Myasthenia gravis	Acetylcholine receptors	Neuromuscular junction	Muscle weakness, ptosis, difficulty swallowing
Systemic lupus erythematosus (SLE)	Nuclear antigens (DNA, histones)	Multiple organs (skin, kidneys, joints, brain)	Butterfly rash, joint pain, kidney failure
Coeliac disease	Gliadin (wheat protein), tissue transglutaminase	Small intestine lining	Diarrhoea, malabsorption, weight loss

21.3 Treatment Strategies

Strategy	How It Works
Immunosuppression	Drugs that suppress the immune system (e.g., corticosteroids, methotrexate, cyclophosphamide) reduce inflammation and tissue damage
Monoclonal antibodies	Anti-TNF-$\alpha$ (infliximab), anti-IL-6 (tocilizumab) block specific inflammatory cytokines
Plasmapheresis	Removal of autoantibodies from the blood (used in myasthenia gravis)
Antigen-specific tolerance	Experimental approaches to re-educate the immune system to tolerate self-antigens
Dietary modification	Gluten-free diet for coeliac disease eliminates the triggering antigen

22. Allergies and Hypersensitivity

22.1 Type I Hypersensitivity (Allergic Reactions)

Type I hypersensitivity involves IgE antibodies and mast cells:

1. Sensitisation: on first exposure to the allergen (e.g., pollen, peanut protein, house dust mite faeces), B cells produce IgE specific to the allergen. IgE binds to Fc receptors on the surface of mast cells (in connective tissue) and basophils (in blood).
2. Re-exposure: the allergen cross-links IgE molecules on the mast cell surface, triggering degranulation.
3. Degranulation: mast cells release:
   • Histamine: causes vasodilation (redness, swelling), increased capillary permeability (oedema), bronchoconstriction, mucus secretion.
   • Heparin: anticoagulant.
   • Proteases: tissue damage.
4. Symptoms: range from mild (hay fever: sneezing, itchy eyes, runny nose) to severe (anaphylaxis: life-threatening swelling of the airway, drop in blood pressure, cardiovascular collapse).

22.2 Anaphylaxis Treatment

Anaphylaxis is treated with intramuscular adrenaline (epinephrine):

• Adrenaline causes vasoconstriction (increasing blood pressure).
• It causes bronchodilation (relieving airway constriction).
• It reduces capillary permeability (reducing swelling).
• It suppresses further mast cell degranulation (via $\beta_2$-adrenergic receptors).

Auto-injector devices (e.g., EpiPen) deliver a pre-measured dose of adrenaline for emergency use.

22.3 Desensitisation (Immunotherapy)

Allergen immunotherapy involves gradually exposing the patient to increasing doses of the allergen over months to years. This aims to:

• Shift the immune response from a Th2 (IgE-producing) profile to a Th1 (IgG4-producing) profile.
• Increase the production of regulatory T cells that suppress the allergic response.
• Induce IgG "blocking antibodies" that intercept the allergen before it can reach mast cell-bound IgE.

Immunotherapy is effective for insect venom allergies, allergic rhinitis (hay fever), and some food allergies, but it carries a risk of triggering anaphylaxis and must be carried out under medical supervision.

warning

Common Pitfall Students often confuse antibodies (proteins produced by B cells/plasma cells) with antigens (molecules that trigger an immune response). Remember: Antibody is produced by the immune system in response to an Antigen. Antibodies are Y-shaped proteins; antigens can be proteins, polysaccharides, or other molecules on the surface of pathogens.

26. The Inflammatory Response: Detailed Mechanism

26.1 Steps of Inflammation

When tissue is damaged (by infection, trauma, heat, or chemicals):

1. Vasodilation: histamine (released by mast cells and basophils) and other inflammatory mediators (prostaglandins, bradykinin) cause arterioles in the affected area to dilate. This increases blood flow, causing redness (rubor) and heat (calor).

2. Increased capillary permeability: histamine causes the endothelial cells of capillaries to contract slightly, creating gaps between them. Fluid (plasma, containing proteins such as complement and antibodies) leaks into the tissue, causing swelling (oedema, tumor). This fluid also contains fibrinogen, which may clot to isolate the area.

3. Chemotaxis: complement fragments (C3a, C5a) and cytokines released by damaged cells create a chemical gradient that attracts phagocytes (neutrophils first, then monocytes/macrophages) to the site of infection.

4. Phagocytosis: neutrophils and macrophages engulf and digest pathogens and dead cells. Neutrophils are short-lived (die after 1--2 days), forming pus (dead neutrophils, dead bacteria, tissue debris).

5. Tissue repair: once the infection is cleared, macrophages release growth factors that stimulate tissue repair and regeneration.

6. Resolution: anti-inflammatory cytokines (IL-10, TGF-$\beta$) suppress the inflammatory response, and the tissue returns to normal.

26.2 Inflammatory Mediators

Mediator	Source	Effect
Histamine	Mast cells, basophils	Vasodilation; increased capillary permeability; stimulates pain receptors
Prostaglandins	Mast cells, damaged cells	Vasodilation; pain sensitisation (makes nerves more sensitive to pain); fever
Bradykinin	Plasma protein cascade	Vasodilation; increased permeability; pain
Serotonin	Platelets	Vasoconstriction (in some tissues); pain; contributes to platelet aggregation
Cytokines (IL-1, IL-6, TNF-$\alpha$)	Macrophages, T cells	Promote inflammation; activate immune cells; cause fever; stimulate acute-phase protein production by the liver

26.3 Chronic Inflammation

If the inflammatory response persists (due to persistent infection, autoimmune reactions, or exposure to irritants such as cigarette smoke), it becomes chronic inflammation. Chronic inflammation can cause tissue damage and is implicated in many diseases:

Disease	Cause of Chronic Inflammation	Tissue Damage
Rheumatoid arthritis	Autoimmune attack on joint synovium	Joint destruction
Atherosclerosis	Oxidised LDL in artery walls	Plaque formation; artery narrowing
Asthma	Allergic inflammation of airways	Airway remodelling; bronchoconstriction
Crohn's disease	Autoimmune attack on gut epithelium	Bowel wall thickening; ulceration
Chronic bronchitis	Cigarette smoke irritation	Mucus hypersecretion; airway obstruction

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tip

Diagnostic Test

23. The Complement System

23.1 Overview

The complement system is a cascade of approximately 30 plasma proteins that enhances (complements) the ability of antibodies and phagocytes to clear pathogens. It is part of the innate immune system but can be activated by antibodies (bridging innate and adaptive immunity).

23.2 Pathways of Activation

Pathway	Trigger	Key Components
Classical	Antigen-antibody complexes (IgM or IgG)	C1q, C1r, C1s, C4, C2
Alternative	Spontaneous hydrolysis of C3 on microbial surfaces (no antibody required)	C3, Factor B, Factor D, properdin
Lectin	Mannose-binding lectin (MBL) binding to mannose on microbial surfaces	MBL, MASP-1, MASP-2, C4, C2

All three pathways converge on the formation of C3 convertase, which cleaves C3 into C3a and C3b.

23.3 Effects of Complement Activation

1. Opsonisation: C3b binds to the surface of pathogens, marking them for phagocytosis. Phagocytes have C3b receptors that recognise and bind to C3b-coated pathogens.
2. Inflammation: C3a and C5a are anaphylatoxins -- they trigger mast cell degranulation (releasing histamine), increase vascular permeability, and attract phagocytes (chemotaxis).
3. Membrane attack complex (MAC): C5b initiates the formation of the MAC (C5b, C6, C7, C8, and multiple C9 molecules), which inserts into the pathogen's cell membrane, forming a pore. Ions and water flow through the pore, causing the pathogen to swell and lyse.

24. The Lymphatic System in Detail

24.1 Structure

The lymphatic system consists of:

• Lymph (tissue fluid that has entered lymphatic vessels).
• Lymphatic vessels: a network of thin-walled vessels (similar to veins but with thinner walls and more valves) that drain excess tissue fluid from body tissues and return it to the blood via the thoracic duct and right lymphatic duct.
• Lymph nodes: small, bean-shaped structures along the lymphatic vessels that filter lymph and contain lymphocytes and macrophages. They are concentrated in the neck, armpits, groin, and abdomen.
• Lymphoid organs: spleen, thymus, tonsils, Peyer's patches (in the small intestine).

24.2 The Spleen

The spleen is the largest lymphoid organ. It filters blood (not lymph) and:

• Removes old or damaged red blood cells (by macrophages in the red pulp).
• Contains lymphocytes (in the white pulp) that mount immune responses against blood-borne pathogens.
• Stores platelets (approximately one-third of the body's platelets).
• Acts as a blood reservoir (can release stored blood during haemorrhage).

People without a spleen (splenectomy, e.g., after trauma) are at increased risk of infections from encapsulated bacteria (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae) and are usually vaccinated against these and given prophylactic antibiotics.

24.3 The Thymus

The thymus is located in the upper chest, behind the sternum. It is where T lymphocytes mature:

1. Positive selection: thymocytes (immature T cells) that can recognise self-MHC molecules survive. Those that cannot recognise self-MHC die by apoptosis (approximately 90% of thymocytes die in the thymus).
2. Negative selection: thymocytes that strongly react with self-antigens presented by self-MHC are eliminated (preventing autoimmunity).

Only approximately 2--5% of thymocytes survive both selection processes and are released into the circulation as naive T cells. The thymus is largest in childhood and gradually shrinks (involution) after puberty, being replaced by adipose tissue.

25. Cytokines: Chemical Messengers of the Immune System

25.1 Types and Functions

Cytokine	Produced By	Function
Interleukin-1 (IL-1)	Macrophages, dendritic cells	Activates T helper cells; causes fever (acts on hypothalamus); promotes inflammation
Interleukin-2 (IL-2)	T helper cells	Stimulates T cell proliferation and differentiation; activates NK cells
Interleukin-4 (IL-4)	T helper 2 cells	Stimulates B cell proliferation and antibody class switching to IgE
Interleukin-10 (IL-10)	T regulatory cells, macrophages	Anti-inflammatory; suppresses immune responses
Interferon-$\alpha$/$\beta$	Virus-infected cells	Induces antiviral state in neighbouring cells; activates NK cells
Interferon-$\gamma$	T helper 1 cells, NK cells	Activates macrophages; enhances antigen presentation; promotes Th1 responses
Tumour necrosis factor-$\alpha$ (TNF-$\alpha$)	Macrophages, T cells	Promotes inflammation; induces apoptosis in tumour cells; causes fever and cachexia
Transforming growth factor-$\beta$ (TGF-$\beta$)	T regulatory cells, platelets	Suppresses immune responses; promotes tissue repair; inhibits T cell proliferation

25.2 Cytokine Storm

A cytokine storm (cytokine release syndrome) occurs when the immune system produces excessive amounts of pro-inflammatory cytokines, causing widespread inflammation and tissue damage. This can occur in severe viral infections (e.g., COVID-19, Spanish flu, SARS), sepsis, and as a side effect of some immunotherapies (CAR-T cell therapy).

Consequences:

• Massive release of TNF-$\alpha$, IL-1, IL-6, and interferons.
• Systemic inflammation: fever, widespread capillary leakage (causing oedema and hypotension), multi-organ failure.
• Treatment: anti-cytokine therapies (e.g., tocilizumab, an IL-6 receptor antagonist; anakinra, an IL-1 receptor antagonist).

26. Immunological Memory and Vaccination Strategies

26.1 Primary and Secondary Immune Responses Compared

Feature	Primary Response	Secondary Response
Time to peak antibody production	5--10 days	1--3 days
Peak antibody concentration	Relatively low	Much higher (10--100x primary)
Antibody class	Mainly IgM initially, then IgG	Mainly IgG (memory B cells produce high-affinity IgG)
Antibody affinity	Relatively low	Higher (affinity maturation has occurred)
Duration	Short-lived	Long-lived (months to years)
Memory cells produced	Yes (memory B and T cells)	Memory cells re-stimulated and expanded

26.2 Types of Vaccines

Vaccine Type	Description	Examples	Advantages	Disadvantages
Live attenuated	Weakened but live pathogen	MMR, chickenpox, BCG	Strong immune response; often single dose; cell-mediated and humoral immunity	Risk of reversion to virulence (rare); not suitable for immunocompromised
Inactivated (killed)	Dead pathogen	Influenza (injected), cholera, hepatitis A	Cannot cause disease; safer for immunocompromised	Weaker immune response; requires adjuvant; usually requires boosters
Subunit (protein)	Purified antigens from pathogen	Hepatitis B, HPV, acellular pertussis	Very safe; well-defined composition	May not elicit strong cellular immunity; requires adjuvant
Toxoid	Inactivated toxin	Tetanus, diphtheria	Safe; effective against toxin-mediated diseases	Does not prevent infection, only toxin effects
Conjugate	Polysaccharide antigen linked to a carrier protein	MenC, MenACWY, pneumococcal (PCV)	Effective in infants (T-dependent response)	More expensive to produce
mRNA	mRNA encoding a pathogen antigen	COVID-19 (Pfizer-BioNTech, Moderna)	Rapid development; strong immune response; no live virus	Requires cold storage; relatively new technology
Viral vector	Harmless virus delivers antigen gene	COVID-19 (AstraZeneca, Janssen), Ebola	Strong immune response; mimics natural infection	Pre-existing immunity to vector may reduce efficacy

26.3 Herd Immunity

Herd immunity (community immunity) occurs when a sufficient proportion of the population is immune to an infectious disease (through vaccination or previous infection), providing indirect protection to individuals who are not immune.

The herd immunity threshold depends on the basic reproduction number ($R_0$):

$\text{Herd immunity threshold} = 1 - \frac{1}{R_0}$

Disease	$R_0$	Herd Immunity Threshold
Measles	12--18	92--94%
Polio	5--7	80--86%
COVID-19 (Omicron)	8--10	87--90%
Seasonal influenza	1.5--3	33--67%
Diphtheria	4--6	75--83%

warning

Common Pitfall Students often think herd immunity means no one can get the disease. Herd immunity reduces the probability of transmission but does not eliminate risk entirely. Non-immune individuals (too young for vaccination, immunocompromised, vaccine contraindications) can still be infected if exposed. Additionally, immunity can wane over time, and new variants may partially escape existing immunity.

26.4 Antigenic Variation and Vaccine Challenges

Some pathogens evade immune memory through antigenic variation:

• Antigenic drift: minor mutations in surface antigens (HA and NA in influenza virus) that accumulate over time. Requires annual reformulation of influenza vaccines.
• Antigenic shift: major change in surface antigens due to reassortment of gene segments when two different influenza viruses infect the same cell. Can cause pandemics (e.g., H1N1 in 2009).
• HIV: extremely high mutation rate ($\approx 3 \times 10^{-5}$ mutations per base per replication cycle); rapid antigenic variation means the virus evades antibody responses. No effective vaccine yet.
• Trypanosoma brucei (African sleeping sickness): periodically switches its variant surface glycoprotein (VSG) coat, with over 1,000 different VSG genes available. The immune system is always "one step behind."

27. Immunological Techniques in Biotechnology

27.1 Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS)

Flow cytometry passes cells singly through a laser beam, measuring light scattering and fluorescence. FACS adds the ability to physically sort cells based on these properties:

• Cells are labelled with fluorescently-tagged antibodies specific to surface markers (e.g., CD4 for T helper cells, CD8 for cytotoxic T cells, CD19 for B cells).
• As each cell passes through the laser, forward scatter (cell size) and side scatter (cell granularity) are measured, along with fluorescence intensity for each antibody.
• Applications: counting immune cell subsets in blood (immunophenotyping); diagnosing leukaemia and lymphoma; sorting stem cells for transplantation.

27.2 Western Blotting

Western blotting detects specific proteins in a sample:

1. Proteins are separated by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) based on molecular weight.
2. Proteins are transferred (blotted) onto a membrane (nitrocellulose or PVDF).
3. The membrane is incubated with a primary antibody specific to the target protein.
4. The membrane is incubated with a secondary antibody (conjugated to an enzyme such as horseradish peroxidase, HRP).
5. A substrate is added that produces a detectable signal (chemiluminescence or colour change) where the target protein is located.

Applications: confirming protein expression; detecting HIV antibodies in blood samples (HIV Western blot).

27.3 Immunohistochemistry (IHC)

IHC uses antibodies to detect specific antigens in tissue sections:

1. A thin section of tissue is fixed and mounted on a slide.
2. Endogenous peroxidase is blocked (to prevent false-positive signals).
3. The section is incubated with a primary antibody specific to the target antigen.
4. A secondary antibody conjugated to an enzyme (HRP) is applied.
5. A chromogenic substrate (e.g., DAB, which produces a brown precipitate) is added, revealing the location of the target antigen.

Applications: cancer diagnosis (detecting tumour markers such as HER2 in breast cancer, CD markers in lymphoma); identifying pathogens in tissue sections.

28. Autoimmune Diseases: Detailed Analysis

28.1 Mechanisms of Autoimmunity

Autoimmune diseases occur when the immune system attacks the body's own tissues. Possible mechanisms:

1. Molecular mimicry: a pathogen antigen resembles a self-antigen, so antibodies or T cells that recognise the pathogen also attack self-tissue (e.g., Streptococcus pyogenes M protein resembles cardiac myosin, leading to rheumatic fever).
2. Release of hidden antigens: normally sequestered antigens (e.g., lens proteins in the eye, sperm antigens in the testes) are released into the bloodstream due to injury, triggering an immune response.
3. Failure of central tolerance: during T cell development in the thymus, autoreactive T cells are normally eliminated by negative selection. If this process fails, autoreactive T cells enter the circulation.
4. Failure of regulatory T cells (Tregs): Tregs normally suppress autoimmune responses. Reduced Treg function can lead to loss of self-tolerance.

28.2 Specific Autoimmune Diseases

Disease	Autoantigen	Target Tissue	Symptoms	Treatment
Type 1 diabetes	$\beta$ cell antigens (GAD, IA-2, insulin)	Pancreatic $\beta$ cells	Hyperglycaemia, polyuria, polydipsia, ketoacidosis	Insulin replacement
Rheumatoid arthritis	Citrullinated proteins, immunoglobulin (rheumatoid factor)	Synovial joints (especially hands, wrists)	Joint pain, swelling, stiffness; joint destruction	Anti-inflammatory drugs; DMARDs (methotrexate); biologics (anti-TNF-$\alpha$)
Multiple sclerosis (MS)	Myelin basic protein (MBP), proteolipid protein	Myelin sheath in CNS	Visual disturbances, muscle weakness, numbness, fatigue, paralysis	Corticosteroids (acute attacks); interferon-$\beta$; disease-modifying therapies
Myasthenia gravis	Nicotinic ACh receptors	Neuromuscular junction	Muscle weakness, ptosis, difficulty swallowing	AChE inhibitors; immunosuppressants; thymectomy
Coeliac disease	Tissue transglutaminase (tTG), gliadin (gluten protein)	Small intestine (villi)	Diarrhoea, bloating, weight loss, malabsorption, anaemia	Gluten-free diet
Systemic lupus erythematosus (SLE)	Nuclear antigens (DNA, histones)	Multiple organs (joints, kidneys, skin, heart)	Butterfly rash, joint pain, kidney damage, fatigue	Corticosteroids; immunosuppressants; antimalarials (hydroxychloroquine)

28.3 Allergies (Type I Hypersensitivity)

Allergies are exaggerated immune responses to harmless antigens (allergens):

1. Sensitisation (first exposure): the allergen is taken up by antigen-presenting cells and presented to T helper cells. Th2 cells stimulate B cells to produce IgE antibodies specific to the allergen. IgE binds to mast cells and basophils (via Fc receptors).
2. Re-exposure: the allergen cross-links IgE on mast cells, triggering degranulation (release of pre-formed mediators) and synthesis of new mediators.

Mediator	Source	Effects
Histamine	Mast cells, basophils	Vasodilation; increased capillary permeability (oedema); smooth muscle contraction (bronchoconstriction); mucus secretion; itching
Heparin	Mast cells	Anticoagulant (prevents blood clotting at the site of inflammation)
Leukotrienes (C4, D4, E4)	Mast cells	Slow, sustained bronchoconstriction; increased vascular permeability; attracts eosinophils
Prostaglandin D2	Mast cells	Bronchoconstriction; vasodilation

Anaphylaxis: a severe, systemic allergic reaction that is potentially fatal. Symptoms: urticaria (hives), angioedema (swelling of lips, tongue, throat), bronchoconstriction (difficulty breathing), hypotension (shock), gastrointestinal symptoms. Treatment: intramuscular adrenaline (epinephrine) -- causes vasoconstriction (increases blood pressure), bronchodilation, and reduces capillary permeability.

29. The Complement System

29.1 Overview

The complement system is a cascade of plasma proteins that enhances (complements) the ability of antibodies and phagocytes to clear pathogens. There are three pathways:

Pathway	Trigger	Key Proteins	Outcome
Classical pathway	Antibody-antigen complexes (IgM or IgG bound to pathogen)	C1q, C1r, C1s, C4, C2, C3	C3 convertase formed; leads to opsonisation, inflammation, MAC formation
Alternative pathway	Spontaneous hydrolysis of C3 on microbial surfaces (pathogen surfaces lack regulatory proteins)	C3, Factor B, Factor D, Properdin	C3 convertase formed; amplification loop
Lectin pathway	Mannose-binding lectin (MBL) binds to mannose residues on pathogen surface	MBL, MASP-1, MASP-2, C4, C2	Similar to classical pathway; C3 convertase formed

All three pathways converge on C3 convertase, which cleaves C3 into C3a and C3b.

29.2 Functions of Complement

Function	Mechanism	Importance
Opsonisation	C3b binds to pathogen surface; phagocytes have C3b receptors (CR1)	Enhances phagocytosis; the most important function of complement
Inflammation	C3a and C5a are anaphylatoxins: they stimulate mast cells to release histamine, causing vasodilation and increased capillary permeability	Recruits immune cells to the site of infection
Chemotaxis	C5a is a potent chemoattractant for neutrophils	Directs phagocytes to the site of infection
Membrane attack complex (MAC)	C5b, C6, C7, C8, and multiple C9 molecules form a pore (approximately 10 nm diameter) in the pathogen membrane	Lyses the pathogen by allowing uncontrolled movement of ions and water (osmotic lysis)

29.3 Regulation of Complement

Complement is tightly regulated to prevent damage to host cells:

Regulatory Protein	Function
C1 inhibitor (C1-INH)	Inhibits C1r and C1s (classical pathway)
Factor H	Binds to host cell surfaces, promoting decay of C3 convertase (alternative pathway)
Decay-accelerating factor (DAF, CD55)	Promotes decay of C3 and C5 convertases on host cell surfaces
Membrane cofactor protein (MCP, CD46)	Acts as a cofactor for Factor I, which cleaves C3b to inactive iC3b on host cells
CD59 (protectin)	Prevents MAC formation on host cells by inhibiting C9 polymerisation

Deficiency in complement regulatory proteins can cause disease: deficiency in CD59 or DAF causes paroxysmal nocturnal haemoglobinuria (PNH), where red blood cells are lysed by complement because they lack regulatory protection.

30. HIV/AIDS: Immunology

30.1 HIV Structure and Life Cycle

HIV (human immunodeficiency virus) is a retrovirus:

Component	Description
Envelope	Lipid bilayer derived from host cell membrane; contains viral glycoproteins gp120 (binds CD4) and gp41 (mediates fusion)
Capsid	Protein coat containing two copies of single-stranded RNA genome and reverse transcriptase
Reverse transcriptase	RNA-dependent DNA polymerase; converts viral RNA into DNA
Integrase	Inserts viral DNA into host chromosome
Protease	Cleaves viral polyproteins into functional proteins

Life cycle:

1. Attachment: gp120 binds to CD4 receptor and a co-receptor (CCR5 or CXCR4) on T helper cells.
2. Entry: gp41 mediates fusion of the viral envelope with the host cell membrane.
3. Reverse transcription: reverse transcriptase converts viral RNA into double-stranded DNA.
4. Integration: integrase inserts viral DNA into the host chromosome (provirus).
5. Latency: the provirus may remain dormant for months to years.
6. Activation: when the infected T cell is activated, the provirus is transcribed, producing new viral RNA and proteins.
7. Assembly and budding: new viral particles are assembled and bud from the host cell membrane, killing the cell.

30.2 Progression from HIV to AIDS

Stage	CD4 Count	Viral Load	Symptoms
Acute infection (2--4 weeks)	Normal to decreased	Very high	Flu-like illness (fever, rash, sore throat, swollen lymph nodes)
Clinical latency (2--10 years)	Gradually declining (350--500 cells/$\mathrm{mm^3}$)	Stable (set point)	Usually asymptomatic; gradual immune deterioration
AIDS	< 200 cells/$\mathrm{mm^3}$	High	Opportunistic infections (PCP pneumonia, TB, Kaposi's sarcoma, candidiasis); wasting; dementia

30.3 Antiretroviral Therapy (ART)

Drug Class	Target	Example
Nucleoside reverse transcriptase inhibitors (NRTIs)	Mimic nucleotides; cause chain termination during DNA synthesis	AZT (zidovudine), tenofovir
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)	Bind to and inhibit reverse transcriptase allosterically	Efavirenz, nevirapine
Protease inhibitors	Inhibit viral protease; prevent cleavage of viral polyproteins	Ritonavir, lopinavir
Integrase inhibitors	Block integration of viral DNA into host chromosome	Raltegravir, dolutegravir
Entry inhibitors	Block viral entry (CCR5 antagonists, fusion inhibitors)	Maraviroc (CCR5 antagonist), enfuvirtide (fusion inhibitor)

ART typically uses a combination of 3 or more drugs from at least 2 classes (combination therapy). This prevents the development of drug resistance (the virus would need to simultaneously develop resistance to multiple drugs, which is statistically very unlikely). ART does not cure HIV but can reduce viral load to undetectable levels, allowing normal life expectancy and preventing transmission (U = U: undetectable = untransmittable).

31. Blood Groups and Transfusion

31.1 ABO Blood Group System

Blood groups are determined by the presence or absence of antigenic glycoproteins (A and B antigens) on the surface of red blood cells:

Blood Group	Antigens on RBC	Antibodies in Plasma	Can Receive From	Can Donate To
A	A	Anti-B	A, O	A, AB
B	B	Anti-A	B, O	B, AB
AB	A and B	Neither	A, B, AB, O (universal recipient)	AB
O	Neither	Anti-A and Anti-B	O (universal donor)	A, B, AB, O

31.2 Rhesus (Rh) System

The RhD antigen is the most clinically significant of the Rh antigens:

Rh Status	Antigens on RBC	Antibodies	Notes
Rh positive (Rh+)	RhD present	None (unless sensitised)	~85% of UK population
Rh negative (Rh-)	RhD absent	None (unless sensitised)	~15% of UK population

Haemolytic disease of the newborn (HDN):

• If an Rh- mother carries an Rh+ foetus, some foetal RBCs may cross the placenta during birth.
• The mother's immune system produces anti-RhD antibodies.
• In a subsequent pregnancy with an Rh+ foetus, maternal anti-RhD antibodies can cross the placenta and destroy foetal RBCs (haemolytic disease).
• Prevention: anti-D immunoglobulin is given to the Rh- mother within 72 hours of birth of an Rh+ baby, destroying any foetal RBCs before the mother can mount an immune response.

31.3 Blood Typing Procedure

1. Place a drop of blood on a slide in two positions.
2. Add anti-A serum to one drop and anti-B serum to the other.
3. Observe agglutination (clumping):

Result with Anti-A	Result with Anti-B	Blood Group
Agglutination	No agglutination	A
No agglutination	Agglutination	B
Agglutination	Agglutination	AB
No agglutination	No agglutination	O

32. The Lymphatic System

32.1 Structure and Function

The lymphatic system is a network of vessels, nodes, and organs that returns excess interstitial fluid to the blood, transports lipids, and provides immune defence:

Component	Description	Function
Lymphatic capillaries	Blind-ended vessels in tissues; more permeable than blood capillaries	Collect interstitial fluid (tissue fluid that has not been reabsorbed into blood capillaries)
Lymphatic vessels	Larger vessels with valves (prevent backflow)	Transport lymph (the fluid in lymphatic vessels) towards the heart
Lymph nodes	Bean-shaped structures along lymphatic vessels; contain lymphocytes and macrophages	Filter lymph; lymphocytes proliferate in response to antigens (swelling during infection)
Spleen	Largest lymphoid organ; contains white pulp (lymphocytes) and red pulp (red blood cells, macrophages)	Filters blood; removes old/damaged RBCs; immune response to blood-borne pathogens
Thymus	Located in the upper chest; large in childhood, shrinks after puberty	T cell maturation and selection
Tonsils	Lymphoid tissue at the back of the throat	Trap pathogens entering via the mouth and nose
Thoracic duct	Main lymphatic vessel; drains lymph from most of the body into the left subclavian vein	Returns lymph to the blood circulation

32.2 Formation of Tissue Fluid and Lymph

At the arterial end of a capillary:

• Blood hydrostatic pressure ($\approx 35\ \mathrm{mmHg}$) forces fluid out of the capillary.
• Blood oncotic pressure ($\approx 25\ \mathrm{mmHg}$, due to plasma proteins) pulls fluid back in.
• Net outward pressure $= 35 - 25 = 10\ \mathrm{mmHg}$ (fluid leaves capillary).

At the venous end:

• Blood hydrostatic pressure has dropped ($\approx 15\ \mathrm{mmHg}$).
• Blood oncotic pressure remains ($\approx 25\ \mathrm{mmHg}$).
• Net inward pressure $= 25 - 15 = 10\ \mathrm{mmHg}$ (fluid returns to capillary).

Not all fluid that leaves the capillary is reabsorbed. The excess (approximately 3 litres per day) drains into the lymphatic capillaries and is returned to the blood via the thoracic duct.

32.3 Oedema

Oedema (swelling) occurs when excess tissue fluid accumulates:

Cause	Mechanism
Increased blood hydrostatic pressure	Heart failure (right ventricular failure increases venous pressure, reducing fluid return); venous thrombosis
Decreased blood oncotic pressure	Liver disease (reduced albumin synthesis); nephrotic syndrome (protein loss in urine); malnutrition (protein deficiency)
Increased capillary permeability	Inflammation (histamine increases permeability); allergic reactions; infection
Lymphatic obstruction	Filariasis (elephantiasis -- Wuchereria bancrofti blocks lymphatic vessels); tumour compression; surgical removal of lymph nodes

33. Vaccination: Types and Principles

33.1 Herd Immunity Calculations

The proportion of the population that must be immune to achieve herd immunity depends on $R_0$:

$\text{Herd immunity threshold} = 1 - \frac{1}{R_0}$

Worked example: Measles has $R_0 \approx 15$.

$\text{Threshold} = 1 - \frac{1}{15} = 0.933 = 93.3\%$

Approximately 93.3% of the population must be immune to prevent measles transmission. If vaccine coverage is below this threshold, outbreaks can still occur.

33.2 Vaccine Efficacy and Effectiveness

Term	Definition
Vaccine efficacy	Reduction in disease risk in a controlled clinical trial (ideal conditions)
Vaccine effectiveness	Reduction in disease risk in real-world conditions (accounting for compliance, logistics, population differences)

Example: A trial of a COVID-19 vaccine reports 95% efficacy. This means:

• 100 cases occurred in the placebo group.
• 5 cases occurred in the vaccinated group.
• Risk ratio $= 5/100 = 0.05$.
• Vaccine efficacy $= (1 - 0.05) \times 100 = 95\%$.

33.3 Why Booster Vaccinations Are Needed

1. Waning immunity: antibody levels decline over time after vaccination. Booster doses restore protective antibody levels.
2. Antigenic drift: RNA viruses (influenza, SARS-CoV-2) accumulate mutations that alter surface antigens, reducing the effectiveness of existing antibodies. Updated vaccines are needed to match circulating strains.
3. New variants: emerging variants with significant antigenic changes may partially escape immunity from previous vaccination or infection.

33.4 Passive vs Active Immunity

Feature	Active Immunity	Passive Immunity
How acquired	Exposure to antigen (infection or vaccination)	Transfer of pre-formed antibodies
Time to develop	Days to weeks (immune response must develop)	Immediate (antibodies are already present)
Duration	Long-term (months to years; immunological memory)	Short-term (weeks to months; antibodies are degraded; no memory)
Examples	Vaccination; natural infection	Maternal antibodies crossing the placenta (IgG) or in breast milk (IgA); antivenom; monoclonal antibody therapy

34. Inflammation: The Innate Response in Detail

34.1 The Five Cardinal Signs of Inflammation

Sign	Latin	Cause
Redness (rubor)	Rubor	Vasodilation increases blood flow to the area
Heat (calor)	Calor	Increased blood flow brings warm blood from the body core
Swelling (tumour)	Tumour	Increased capillary permeability allows fluid to leak into tissues (oedema)
Pain (dolor)	Dolor	Swelling compresses nerve endings; prostaglandins and bradykinin sensitise nociceptors
Loss of function (functio laesa)	Functio laesa	Swelling and pain restrict movement of the affected area

34.2 Steps in the Inflammatory Response

1. Tissue damage: physical injury, infection, or chemical damage damages cells, releasing inflammatory mediators.
2. Vasodilation: histamine (from mast cells and basophils) causes arterioles to dilate, increasing blood flow to the area.
3. Increased capillary permeability: histamine and bradykinin cause endothelial cells of capillaries to contract, widening the gaps between them. Fluid (plasma) leaks into the tissue, causing oedema. The leaked fluid contains antibodies, complement proteins, and clotting factors.
4. Phagocyte migration (chemotaxis): neutrophils are the first phagocytes to arrive (within minutes). They are attracted by chemotactic factors (complement C5a, bacterial products, cytokines). Later, monocytes arrive and differentiate into macrophages (larger, more powerful phagocytes).
5. Phagocytosis: phagocytes engulf and digest pathogens and dead cells.
6. Tissue repair: once the infection is cleared, macrophages release growth factors that stimulate tissue repair and regeneration.

34.3 Inflammatory Mediators

Mediator	Source	Effects
Histamine	Mast cells, basophils	Vasodilation; increased capillary permeability; bronchoconstriction; itching
Bradykinin	Plasma protein cascade (kinin system)	Vasodilation; increased permeability; pain (sensitises nociceptors)
Prostaglandins (PGE2, PGD2)	Mast cells, macrophages	Vasodilation; pain and fever; potentiates the effects of histamine and bradykinin
Leukotrienes (B4, C4, D4)	Mast cells, basophils	Chemotaxis (attract neutrophils); bronchoconstriction; increased permeability
TNF-$\alpha$	Macrophages, T cells	Activates endothelium (expresses adhesion molecules for leukocyte binding); activates macrophages; causes fever; induces apoptosis
Interleukin-1 (IL-1)	Macrophages	Fever (acts on hypothalamus); activates T cells; stimulates acute-phase protein production by the liver

35. The Mononuclear Phagocyte System

35.1 Overview

The mononuclear phagocyte system (MPS) is a network of phagocytic cells derived from bone marrow precursors:

Cell Type	Location	Function
Monocyte	Blood (circulating)	Precursor of tissue macrophages; can migrate into tissues
Macrophage	Tissues (liver = Kupffer cells; lungs = alveolar macrophages; brain = microglia; bone = osteoclasts; skin = Langerhans cells)	Phagocytosis; antigen presentation; cytokine production; tissue repair
Dendritic cell	Tissues (skin, mucosal surfaces, lymph nodes)	Most potent antigen-presenting cell; bridges innate and adaptive immunity; presents antigen to naive T cells in lymph nodes

35.2 Macrophage Functions

Function	Description
Phagocytosis	Engulfs and digests pathogens and dead cells
Antigen presentation (MHC class II)	Presents processed antigens to CD4+ T helper cells
Cytokine secretion	Produces IL-1, IL-6, TNF-$\alpha$; recruits and activates other immune cells
Wound healing	Produces growth factors (PDGF, TGF-$\beta$) that stimulate tissue repair
Iron recycling	Recycles iron from haemoglobin in old red blood cells (via ferroportin)
Tumour surveillance	Macrophages can recognise and destroy tumour cells (part of cancer immunosurveillance)

36. Autoimmune Diseases

36.1 What Are Autoimmune Diseases?

Autoimmune diseases occur when the immune system fails to distinguish self from non-self and attacks the body's own tissues:

Disease	Autoantigen	Affected Tissue	Symptoms
Type 1 diabetes mellitus	Pancreatic $\beta$-cell antigens (e.g., GAD65, IA-2, insulin)	Pancreatic islets of Langerhans	Destruction of $\beta$ cells; no insulin production; hyperglycaemia; ketoacidosis
Rheumatoid arthritis	Collagen type II, citrullinated proteins	Synovial joints	Chronic inflammation of synovium; joint destruction; pain, swelling, stiffness
Multiple sclerosis (MS)	Myelin basic protein (MBP), proteolipid protein	Myelin sheath of CNS neurons	Demyelination; impaired nerve conduction; muscle weakness, vision loss, coordination problems
Myasthenia gravis	Acetylcholine receptors (AChR) at neuromuscular junction	Skeletal muscle motor end plates	Muscle weakness and fatigue; ptosis (drooping eyelids); difficulty swallowing
Systemic lupus erythematosus (SLE)	Nuclear antigens (DNA, histones)	Multiple organs (skin, kidneys, joints, heart, brain)	Butterfly rash; joint pain; kidney inflammation; fatigue; anaemia
Coeliac disease	Tissue transglutaminase (tTG); gliadin (wheat protein)	Small intestine (villi)	Villous atrophy; malabsorption; diarrhoea; weight loss; anaemia

36.2 Mechanisms of Autoimmunity

Mechanism	Description
Molecular mimicry	Pathogen antigen resembles self-antigen; antibodies cross-react with self-tissue (e.g., Group A Streptococcus M protein resembles cardiac myosin $\to$ rheumatic fever)
Release of sequestered antigens	Tissues normally hidden from the immune system (e.g., lens of the eye, sperm in testes) are exposed by injury; immune system mounts response against them
Failure of regulatory T cells ($\mathrm{T_{reg}}$)	$\mathrm{T_{reg}}$ cells normally suppress self-reactive lymphocytes; deficiency leads to loss of self-tolerance
Polyclonal B cell activation	Some pathogens (e.g., EBV) activate many B cells non-specifically, including self-reactive clones

warning

Common Pitfall Type 1 diabetes is autoimmune (immune system destroys $\beta$ cells). Type 2 diabetes is metabolic (insulin resistance). Do not confuse them. Also, coeliac disease is an autoimmune condition triggered by gluten, not a food allergy.

37. Vaccination in Detail

37.1 Types of Vaccines

Vaccine Type	Description	Examples
Live attenuated	Weakened form of the pathogen; replicates within the host but does not cause disease	MMR (measles, mumps, rubella); oral polio vaccine (Sabin); BCG (tuberculosis)
Inactivated (killed)	Dead pathogen; cannot replicate; generally less effective than live vaccines (requires booster doses)	Inactivated polio vaccine (Salk); influenza (some formulations); hepatitis A
Subunit (acellular)	Purified antigens from the pathogen (proteins, polysaccharides)	HPV vaccine; acellular pertussis; hepatitis B
Toxoid	Inactivated toxin (retains antigenicity but not toxicity)	Tetanus; diphtheria
Conjugate	Polysaccharide antigen linked to a carrier protein (enhances immune response, especially in infants)	Meningitis C; pneumococcal; Hib (Haemophilus influenzae type b)
mRNA	Synthetic mRNA encoding a pathogen antigen; host cells translate the mRNA to produce the antigen	COVID-19 (Pfizer-BioNTech, Moderna)
Viral vector	Harmless virus carries genetic material encoding a pathogen antigen	COVID-19 (Oxford-AstraZeneca, Janssen); Ebola

37.2 Herd Immunity

Herd immunity occurs when a sufficient proportion of the population is immune to an infectious disease, providing indirect protection to those who are not immune.

Disease	$R_0$ (Basic Reproduction Number)	Herd Immunity Threshold ($1 - \frac{1}{R_0}$)
Measles	12--18	92--94%
Polio	5--7	80--86%
COVID-19 (Omicron)	8--10	87--90%
Seasonal influenza	1.5--3	33--67%
Smallpox	5--7	80--86%

$\text{Herd immunity threshold} = 1 - \frac{1}{R_0}$

38. HIV/AIDS and the Immune System

38.1 HIV Structure and Life Cycle

Feature	Description
Type	Retrovirus (RNA genome)
Genome	Two single-stranded RNA molecules + reverse transcriptase enzyme
Envelope	Lipid bilayer derived from host cell membrane; viral glycoproteins (gp120 and gp41)
Host cell	Helper T cells (CD4+ T cells), macrophages, dendritic cells

Step	Description
1. Attachment	gp120 binds to CD4 receptor and CCR5/CXCR4 co-receptor on host cell
2. Fusion	gp41 mediates fusion of viral envelope with host cell membrane
3. Entry	Viral RNA and reverse transcriptase enter the host cell
4. Reverse transcription	Reverse transcriptase converts viral RNA to cDNA (complementary DNA)
5. Integration	Viral cDNA is integrated into the host cell's genome by integrase enzyme (becomes a provirus)
6. Transcription	Host cell machinery transcribes proviral DNA to viral mRNA
7. Translation	Host cell ribosomes synthesise viral proteins
8. Assembly	New viral particles assemble at the host cell membrane
9. Budding	New virions bud from the host cell membrane, acquiring their envelope

38.2 HIV Disease Progression

Stage	Time	CD4+ T Cell Count	Symptoms
Acute infection	2--4 weeks after exposure	Drops rapidly then partially recovers	Flu-like illness (fever, rash, sore throat, swollen lymph nodes); high viral load
Clinical latency (asymptomatic)	2--10+ years	Gradually declining (500--1200 cells/$\mu$L normal)	No symptoms; virus continues to replicate at low levels
AIDS	When CD4+ count < 200 cells/$\mu$L	Severely depleted	Opportunistic infections (PCP pneumonia, TB, candidiasis, Kaposi's sarcoma); weight loss; death without treatment

38.3 Antiretroviral Therapy (ART)

Drug Class	Target	Examples
NRTIs (nucleoside reverse transcriptase inhibitors)	Competitive inhibition of reverse transcriptase	AZT (zidovudine), lamivudine
NNRTIs (non-nucleoside reverse transcriptase inhibitors)	Allosteric inhibition of reverse transcriptase	Efavirenz, nevirapine
Protease inhibitors	Inhibit HIV protease (prevents processing of viral proteins)	Ritonavir, lopinavir
Integrase inhibitors	Inhibit HIV integrase (prevents proviral integration)	Raltegravir, dolutegravir
Entry inhibitors	Block gp120 binding or gp41-mediated fusion	Maraviroc (CCR5 antagonist), enfuvirtide

39. The Lymphatic System

39.1 Structure and Function

Component	Description	Function
Lymph capillaries	Blind-ended tubes in tissues; permeable to large molecules and cells	Collect excess tissue fluid (interstitial fluid) that leaks out of blood capillaries
Lymph vessels	Larger vessels with valves (like veins); carry lymph towards the heart	Transport lymph back to the bloodstream; one-way flow due to valves and skeletal muscle pump
Lymph nodes	Bean-shaped structures along lymph vessels (concentrated in neck, armpit, groin, abdomen)	Filter lymph; contain lymphocytes and macrophages; site of immune response initiation
Lymph	Fluid in lymph vessels (composition similar to blood plasma but with less protein and more lymphocytes)	Returns fluid and proteins to the blood; transports fats (in lacteals of small intestine); transports immune cells
Spleen	Largest lymphoid organ; filters blood (not lymph)	Removes old/damaged RBCs; stores platelets; contains lymphocytes; immune surveillance of blood
Thymus	Gland in the chest (above the heart); large in children, shrinks in adults	Site of T lymphocyte maturation; T cells learn to distinguish self from non-self

39.2 Lymph Formation and Return

Step	What Happens
1. Ultrafiltration	Blood pressure at the arterial end of capillaries forces fluid out (water, dissolved solutes, small molecules)
2. Tissue fluid	The fluid that surrounds cells; exchanges gases, nutrients, and waste with cells via diffusion
3. Reabsorption	At the venous end of capillaries, lower blood pressure and higher oncotic pressure draw ~90% of tissue fluid back in
4. Lymph formation	The remaining ~10% of tissue fluid enters lymph capillaries and becomes lymph
5. Lymph return	Lymph flows through lymph vessels, passes through lymph nodes, and enters the subclavian veins (via the thoracic duct and right lymphatic duct)

40. Inflammation: The Inflammatory Response

40.1 The Four Cardinal Signs of Inflammation

Sign	Latin	Cause
Redness	Rubor	Vasodilation (increased blood flow to the area)
Heat	Calor	Increased blood flow; increased metabolic activity of immune cells
Swelling	Tumour	Increased capillary permeability (fluid and proteins leak into tissues)
Pain	Dolor	Swelling compresses nerve endings; prostaglandins and bradykinin sensitise pain receptors

40.2 Steps in the Inflammatory Response

Step	What Happens
1. Tissue damage	Pathogens, physical injury, or chemical irritation damage cells
2. Mast cell degranulation	Mast cells in the damaged tissue release histamine, heparin, and other mediators
3. Vasodilation	Histamine causes arterioles in the area to dilate; more blood flows to the area
4. Increased capillary permeability	Histamine makes capillary walls more permeable; plasma proteins (fibrinogen, complement) and fluid leak into the tissue
5. Phagocyte migration	Neutrophils and monocytes are attracted to the area by chemotaxis (following chemical gradients from damaged cells and bacteria); neutrophils arrive first (within hours); monocytes differentiate into macrophages (arrive later, within days)
6. Phagocytosis	Phagocytes engulf and digest pathogens and dead cells
7. Resolution	Once the infection is cleared, anti-inflammatory cytokines (IL-10, TGF-$\beta$) promote tissue repair and resolution of inflammation

41. ABO Blood Groups

41.1 Blood Group Genetics

Blood Group	Antigen on RBC	Antibody in Plasma	Genotype
A	Antigen A	Anti-B	$\mathrm{I^AI^A}$ or $\mathrm{I^Ai}$
B	Antigen B	Anti-A	$\mathrm{I^BI^B}$ or $\mathrm{I^Bi}$
AB	Antigen A and Antigen B	Neither anti-A nor anti-B	$\mathrm{I^AI^B}$
O	Neither antigen A nor antigen B	Both anti-A and anti-B	$\mathrm{ii}$

41.2 Blood Transfusion Compatibility

Donor Blood Group	Can Donate To	Can Receive From
O	A, B, AB, O (universal donor)	O only
A	A, AB	O, A
B	B, AB	O, B
AB	AB only	O, A, B, AB (universal recipient)

41.3 Why Blood Group Matters

• If a person receives blood with an antigen they do not have, their antibodies will cause agglutination (clumping) of the donor RBCs.
• Agglutination can block blood vessels, causing tissue damage, kidney failure, and death.
• The rhesus (Rh) system is also important: RhD+ individuals have the D antigen; RhD- individuals do not. An RhD- mother carrying an RhD+ baby can develop anti-D antibodies (if fetal RBCs enter maternal circulation), which can cause haemolytic disease of the newborn in subsequent pregnancies.

42. Types of Immunity

42.1 Natural vs Artificial Immunity

	Active Immunity	Passive Immunity
Natural	Infection with pathogen; immune system produces its own antibodies and memory cells (long-lasting; e.g., chickenpox)	Antibodies transferred from mother to baby across placenta (IgG) or in breast milk (IgA); provides temporary protection (weeks to months)
Artificial	Vaccination; immune system produces its own antibodies and memory cells in response to a vaccine (long-lasting; may require booster doses)	Injection of pre-formed antibodies (e.g., antivenom for snakebite; monoclonal antibodies for cancer treatment); provides immediate but temporary protection (weeks)

42.2 Key Differences

Feature	Active Immunity	Passive Immunity
Source of antibodies	Produced by the individual's own B cells/plasma cells	Received from another individual (or manufactured)
Memory cells produced	Yes (long-term protection)	No (short-term protection only)
Time to become effective	Slow (days to weeks for primary response)	Immediate (instant protection)
Duration	Long-lasting (years to lifetime)	Short-lasting (weeks to months)
Uses	Vaccination; natural infection	Antivenom; post-exposure prophylaxis (rabies immunoglobulin); neonatal protection

43. Monoclonal Antibodies

43.1 Production (Hybridoma Technology)

Step	Description
1	A mouse is injected with an antigen (the target)
2	B cells from the mouse's spleen produce antibodies against the antigen
3	These B cells are fused with tumour cells (myeloma cells) using polyethylene glycol (PEG)
4	The resulting hybrid cells are called hybridomas
5	Hybridomas combine the antibody-producing ability of B cells with the immortality of tumour cells
6	Hybridomas are screened to find the one producing the desired antibody
7	The selected hybridoma is cloned (grown in culture or in mice) to produce large quantities of identical (monoclonal) antibodies

43.2 Uses of Monoclonal Antibodies

Application	Description	Example
Pregnancy testing	Monoclonal antibodies bind to hCG (human chorionic gonadotropin) in urine	Pregnancy test kits (blue line appears if hCG is present)
Cancer treatment	Monoclonal antibodies bind to specific antigens on cancer cells; can deliver drugs, block growth signals, or flag cells for immune destruction	Trastuzumab (Herceptin) for HER2-positive breast cancer; rituximab for B-cell lymphoma
Diagnosis	Monoclonal antibodies used in ELISA tests, Western blotting, and immunohistochemistry to detect specific proteins	Detecting HIV antibodies in blood; detecting specific pathogens
Transplant rejection	Monoclonal antibodies can suppress the immune response against transplanted organs	Basiliximab (targets IL-2 receptor on T cells; prevents T cell activation)

44. Primary and Secondary Immune Responses

44.1 Comparison

Feature	Primary Immune Response	Secondary Immune Response
Timing	Slow (4--7 days for antibodies to appear; peak at ~10--14 days)	Fast (1--3 days for antibodies to appear; peak at ~3--5 days)
Antibody concentration	Lower peak	Much higher peak (10--100x higher)
Antibody class	Mainly IgM initially; then IgG	Mainly IgG (faster class switching)
Antibody affinity	Lower affinity (antibodies are less specific)	Higher affinity (somatic hypermutation has improved binding)
Memory cells	Produced at the end of the response (plasma cells die; memory B and T cells persist)	Memory cells are reactivated; produce large numbers of plasma cells rapidly
Duration of protection	Memory cells persist for years to lifetime	Depends on antigen; some vaccines require booster doses

44.2 Why Vaccination Works

Vaccination exploits the memory of the secondary immune response:

1. A vaccine introduces an antigen (weakened pathogen, protein subunit, mRNA) to the body.
2. The primary response is activated; memory B and T cells are produced.
3. When the real pathogen enters the body later, the secondary response is triggered.
4. Antibodies are produced rapidly and in large quantities; the pathogen is destroyed before it can cause disease.

45. Complement System

45.1 What Is the Complement System?

The complement system is a cascade of plasma proteins that enhances (complements) the immune response:

Pathway	Trigger	Key Steps
Classical pathway	Antibody-antigen complexes (IgG or IgM bound to pathogen surface)	C1 binds to antibodies $\to$ activates C4 and C2 $\to$ C3 convertase (C4b2b) $\to$ activates C3 $\to$ C5 convertase (C4b2b3b) $\to$ activates C5, C6, C7, C8, C9
Alternative pathway	Microbial surface molecules (no antibody required)	Spontaneous hydrolysis of C3 $\to$ C3b binds to microbial surface $\to$ Factor B and Factor D activate C3 convertase $\to$ amplification loop
Lectin pathway	Mannose-binding lectin (MBL) binds to mannose residues on microbial surfaces	MBL-associated serine proteases (MASPs) activate C4 and C2 (similar to classical pathway)

45.2 Effects of Complement Activation

Effect	Description
Opsonisation	C3b binds to the pathogen surface; phagocytes have C3b receptors; enhances phagocytosis
Inflammation	C3a and C5a are anaphylatoxins; they cause mast cell degranulation (histamine release); increase vascular permeability; attract phagocytes (chemotaxis)
Membrane attack complex (MAC)	C5b, C6, C7, C8, and multiple C9 molecules form a pore in the pathogen's cell membrane; water and ions enter; cell lysis (particularly effective against Gram-negative bacteria)

46. Tissue Types and the Immune System

46.1 Types of T Lymphocytes

Type	Surface Marker	Function
Helper T cells (CD4+)	CD4	Coordinate the immune response; activate B cells (help them produce antibodies); activate cytotoxic T cells; activate macrophages (enhance phagocytosis); secrete cytokines
Cytotoxic T cells (CD8+)	CD8	Kill virus-infected cells and cancer cells; recognise viral antigens presented on MHC class I; release perforin and granzymes (induce apoptosis of target cell)
Regulatory T cells ($\mathrm{T_{reg}}$, CD4+ CD25+ FoxP3+)	CD4, CD25, FoxP3	Suppress immune responses; maintain self-tolerance; prevent autoimmune reactions; secrete anti-inflammatory cytokines (IL-10, TGF-$\beta$)
Memory T cells	--	Long-lived; provide rapid secondary response upon re-exposure to the same antigen

46.2 MHC Class I vs MHC Class II

Feature	MHC Class I	MHC Class II
Found on	All nucleated cells (not RBCs)	Antigen-presenting cells (macrophages, dendritic cells, B cells)
Presents	Endogenous antigens (viral proteins from inside the cell)	Exogenous antigens (engulfed and processed from outside the cell)
Recognised by	CD8+ cytotoxic T cells	CD4+ helper T cells
Role	Alerts the immune system to intracellular infections (viruses) and cancer	Activates helper T cells to coordinate the immune response

47. Antibody Structure and Function

47.1 Antibody (Immunoglobulin) Structure

Region	Description
Variable region ($\mathrm{V}_\mathrm{H}$ and $\mathrm{V}_\mathrm{L}$)	The antigen-binding site; unique to each B cell clone; determines which antigen the antibody recognises
Constant region ($\mathrm{C}_\mathrm{H}$ and $\mathrm{C}_\mathrm{L}$)	Determines the antibody class (isotype) and effector function; the same across all antibodies of the same class
Heavy chain	Two identical heavy chains per antibody; contains one variable and three constant domains
Light chain	Two identical light chains per antibody; contains one variable and one constant domain
Hinge region	Flexible region between Fab and Fc portions; allows the antibody to bind to antigens at different angles
Disulphide bonds	Covalent bonds between heavy chains and between heavy and light chains; hold the antibody together

47.2 Antibody Functions

Function	Mechanism
Neutralisation	Antibodies bind to toxins, viruses, or bacteria and block their ability to enter cells or damage tissues
Opsonisation	Antibodies bind to the surface of pathogens; phagocytes have Fc receptors that recognise the antibody Fc region; enhances phagocytosis
Agglutination	Antibodies bind to multiple pathogens, causing them to clump together; clumped pathogens are easier for phagocytes to engulf
Activation of complement	Antibodies bound to the pathogen surface activate the classical complement pathway (C1 binds to the Fc region)
Antibody-dependent cellular cytotoxicity (ADCC)	Natural killer (NK) cells recognise the Fc region of antibodies bound to infected cells; NK cells release perforin and granzymes, killing the infected cell

47.3 Antibody Classes

Class	Structure	Location	Function
IgG	Monomer	Blood, lymph, tissue fluid	Most abundant antibody in blood; crosses the placenta; opsonisation; complement activation; secondary immune response
IgM	Pentamer (5 units joined by a J chain)	Blood, lymph	First antibody produced in primary response; very effective at complement activation (10 Fc regions); too large to cross the placenta
IgA	Dimer (in secretions); monomer (in blood)	Mucosal surfaces (saliva, tears, breast milk, gut lining)	Prevents pathogen attachment to epithelial surfaces; provides passive immunity to newborns via breast milk
IgE	Monomer	Bound to mast cells and basophils	Allergic reactions (cross-links IgE on mast cells $\to$ degranulation $\to$ histamine release); defence against parasites (helminths)
IgD	Monomer	B cell surface	Function of membrane-bound IgD is not fully understood; may act as a B cell receptor

48. The Human Microbiome

48.1 What Is the Microbiome?

The human microbiome is the collection of all microorganisms (bacteria, archaea, fungi, viruses) that live on and in the human body.

Site	Approximate Bacterial Density	Dominant Phyla
Skin	~10$^6$ per cm2	Staphylococcus, Corynebacterium, Propionibacterium
Mouth	~10$^9$ per mL saliva	Streptococcus, Prevotella, Fusobacterium
Gut (large intestine)	~10$^{11}$ per gram of faeces	Bacteroidetes, Firmicutes (especially Clostridium, Lactobacillus, Bifidobacterium)
Vagina	~10$^9$ per mL	Lactobacillus dominant (produces lactic acid, maintains low pH)

48.2 Functions of the Gut Microbiome

Function	Description
Digestion	Breaks down complex carbohydrates (cellulose, pectin) that human enzymes cannot digest; produces short-chain fatty acids (butyrate, propionate, acetate) that are absorbed and used as energy by colonocytes
Vitamin synthesis	Produces vitamin K (essential for blood clotting) and B vitamins (B12, folate)
Immune development	Stimulates development of gut-associated lymphoid tissue (GALT); trains the immune system; helps establish immune tolerance
Pathogen resistance	Competes with pathogens for nutrients and attachment sites; produces bacteriocins (antimicrobial peptides) that kill competing bacteria
Metabolic effects	Influences energy harvest (people with different gut microbiota extract different amounts of energy from the same food); links to obesity, insulin resistance, and fatty liver disease

48.3 Factors That Disrupt the Microbiome

Factor	Effect
Antibiotics	Kill beneficial bacteria; can cause C. difficile overgrowth; may take months for microbiome to recover
Diet	High-fibre diet promotes diversity; high-sugar/high-fat diet reduces diversity; fermented foods (yogurt, kefir, sauerkraut) introduce beneficial bacteria
Birth method	Vaginal birth exposes the newborn to the mother's microbiota; Caesarean section delays colonisation; associated with higher rates of asthma, allergies, and obesity

49. ELISA (Enzyme-Linked Immunosorbent Assay)

49.1 Principle

ELISA uses antibodies conjugated to an enzyme to detect the presence and quantity of a specific antigen (or antibody) in a sample. The enzyme catalyses a colour-change reaction whose intensity is proportional to the amount of target molecule present.

49.2 Direct ELISA Steps

Step	Description
1. Coat	The well of a microtitre plate is coated with the antigen of interest
2. Block	A blocking agent (e.g., BSA or milk protein) is added to block any remaining binding sites on the well surface (prevents non-specific binding)
3. Add primary antibody	An antibody specific to the antigen is added; it binds to the immobilised antigen
4. Wash	Unbound antibody is washed away
5. Add enzyme-linked secondary antibody	A second antibody (with an enzyme attached) binds to the primary antibody
6. Wash	Unbound secondary antibody is washed away
7. Add substrate	A colourless substrate is added; the enzyme converts it to a coloured product
8. Measure	The colour intensity is measured using a colorimeter or spectrophotometer; the absorbance is proportional to the amount of antigen present

49.3 Applications

Application	What Is Detected
Disease diagnosis	HIV antibodies in patient blood; SARS-CoV-2 antigens; hepatitis B surface antigen
Pregnancy testing	hCG (human chorionic gonadotropin) in urine
Drug testing	Detecting drugs of abuse or performance-enhancing substances
Food testing	Detecting allergens (e.g., peanut protein) or food-borne pathogens

50. Types of Immunity

50.1 Active vs Passive Immunity

Feature	Active Immunity	Passive Immunity
What is introduced	Antigen (or pathogen)	Antibodies (pre-made)
Response required	The body's own immune system produces antibodies and memory cells	No immune response required; pre-made antibodies provide immediate protection
Time to become effective	Slow (days to weeks for antibody production)	Immediate
Duration of protection	Long-term (years to lifetime; memory cells persist)	Short-term (weeks to months; antibodies are broken down and not replaced)
Example (natural)	Catching chickenpox and recovering	Antibodies crossing the placenta from mother to foetus; IgA in breast milk
Example (artificial)	Vaccination (e.g., MMR, COVID-19)	Injection of antibodies (e.g., anti-venom for snake bites; anti-tetanus serum)

50.2 Natural vs Artificial Immunity

Feature	Natural	Artificial
Definition	Immunity acquired through normal life events	Immunity acquired through medical intervention
Active natural	Exposure to pathogen; recovery gives immunity (e.g., catching measles)	--
Active artificial	--	Vaccination (e.g., BCG vaccine for tuberculosis)
Passive natural	Antibodies from mother (placenta, breast milk)	--
Passive artificial	--	Injection of antibodies (e.g., rabies immunoglobulin after a dog bite)

51. The Complement System

51.1 What Is the Complement System?

The complement system is a collection of ~30 proteins found in blood plasma that work together to enhance (complement) the immune response. They are produced by the liver and circulate in an inactive form.

51.2 Three Pathways of Activation

Pathway	Trigger	Description
Classical pathway	Antigen-antibody complexes (IgG or IgM bound to antigen)	C1 binds to the antibody; activates C4 and C2; forms C3 convertase (C4b2b)
Alternative pathway	Microbial cell surface molecules (e.g., LPS on Gram-negative bacteria)	Spontaneous hydrolysis of C3; factor B and factor D form C3 convertase (C3bBb)
Lectin pathway	Mannose-binding lectin (MBL) binds to mannose sugars on microbial surfaces	MBL-associated serine proteases (MASPs) activate C4 and C2; same as classical pathway from this point

51.3 Effects of Complement Activation

Effect	Description
Opsonisation	C3b coats the surface of the pathogen; phagocytes have C3b receptors; binding is enhanced; phagocytosis is more efficient
Inflammation	C3a and C5a act as anaphylatoxins; they cause mast cells to release histamine; increase vascular permeability; attract phagocytes to the site of infection (chemotaxis)
Membrane attack complex (MAC)	C5b initiates the formation of a pore (channel) in the pathogen's cell membrane (C5b, C6, C7, C8, multiple C9); the MAC allows water and ions to enter the pathogen; the pathogen swells and lyses (bursts)

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