outline the principles of using monoclonal antibodies in the diagnosis of disease and in the treatment of disease

Immunity – Antibodies and Vaccination (Cambridge IGCSE/A‑Level)

1. Overview of the Immune System

• Innate (non‑specific) immunity

  • Physical & chemical barriers – skin, mucous membranes, gastric acid.
  • Phagocytes – neutrophils and macrophages engulf microbes, generate an oxidative burst (ROS) and kill the pathogen.
  • Complement cascade – opsonisation, membrane‑attack complex.
  • Inflammatory mediators – histamine, prostaglandins, cytokines.

• Adaptive (specific) immunity

  • Lymphocytes – B‑cells (antibody producers) and T‑cells (cell‑mediated).
  • Antibodies – bind antigens with high specificity.
  • Memory cells – provide faster, stronger secondary responses.

• Interaction between innate and adaptive systems

  • Antigen presentation by dendritic cells/macrophages to naïve T‑cells (via MHC).
  • Cytokine signalling coordinates the response.

2. Antigens – Self vs. Non‑self

An antigen is any molecule that can be specifically recognised by an antibody or a T‑cell receptor. The immune system distinguishes:

• Self‑antigens – normal body components; usually ignored (tolerance).
• Non‑self‑antigens – foreign proteins, polysaccharides, lipids, or nucleic acids that trigger an immune response.

3. Antibody Structure, Classes & Fc Functions

All antibodies are immunoglobulins (Ig) composed of two identical heavy chains and two identical light chains forming a Y‑shaped molecule.

• Variable (V) region – binds a specific epitope.
• Constant (C) region – determines class and mediates effector functions via the Fc fragment.

4. Primary vs. Secondary Immune Responses

1. Primary response (first exposure)

   • Antigen is captured by phagocytes → processed and presented on MHC II.
   • Naïve CD4⁺ T‑cells are activated → help B‑cells.
   • Naïve B‑cells differentiate into plasma cells (mainly IgM) and memory B‑cells.
   • Lag phase ≈ 5–7 days before detectable antibodies appear.
   • Affinity is low; IgM dominates.

2. Secondary (memory) response (re‑exposure)

   • Memory B‑cells rapidly differentiate into plasma cells.
   • IgG (or IgA/IgE, depending on antigen) is produced in large quantities.
   • Affinity maturation (somatic hypermutation) yields high‑affinity antibodies.
   • Response begins within 1–3 days, is stronger and longer‑lasting.

5. Memory Cells & Why They Improve Protection

• Memory B‑cells retain the rearranged V(D)J genes and undergo rapid proliferation on re‑encounter.
• Memory T‑cells (both CD4⁺ and CD8⁺) persist in peripheral tissues, ready to secrete cytokines or kill infected cells.
• Result: higher antibody titres, faster class‑switch to IgG, and quicker clearance of the pathogen.

6. Active vs. Passive Immunity

7. Vaccination – Principles, Types & Real‑World Examples

Vaccines mimic infection to trigger the primary‑secondary response pattern without causing disease.

Herd Immunity

When a sufficient proportion of a population is immune, transmission of the pathogen is interrupted, providing indirect protection to those who are susceptible. The required coverage depends on the basic reproduction number (R₀) of the disease.

8. Monoclonal Antibodies (mAbs) – Production Overview

1. Immunise a mouse (or other suitable animal) with the target antigen.
2. Harvest spleen cells and isolate a single B‑cell that produces the desired antibody.
3. Fuse the B‑cell with an immortal myeloma cell → hybridoma.
4. Select hybridomas that secrete the specific antibody (screening by ELISA or flow cytometry).
5. Clone the selected hybridoma to obtain a monoclonal population.
6. Harvest and purify the identical antibody molecules for research, diagnostic or therapeutic use.

9. Principles of Using Monoclonal Antibodies in Diagnosis

• Specificity – each mAb recognises a single epitope, minimising cross‑reactivity.
• Sensitivity – high affinity (low K_d) enables detection of minute antigen quantities.
• Reproducibility – unlimited supply of identical antibodies guarantees consistent test performance.
• Label versatility – mAbs can be conjugated to enzymes, fluorophores, or radioisotopes for a range of assay formats.

Common Diagnostic Formats

1. ELISA (Enzyme‑Linked Immunosorbent Assay) – capture mAb immobilised on a plate; patient sample added; bound antigen detected with a second enzyme‑conjugated mAb. Quantitative.
2. Rapid Lateral‑Flow (Immunochromatographic) Test – coloured mAb migrates with the sample; a visible line forms if antigen is present (e.g., pregnancy test, COVID‑19 rapid antigen test).
3. Immunohistochemistry (IHC) – tissue sections stained with a labelled mAb to visualise localisation of disease markers (e.g., HER2 in breast‑cancer biopsies).
4. Flow Cytometry – fluorescent mAbs bind cell‑surface antigens; fluorescence intensity quantifies specific cell populations (e.g., CD4⁺ T‑cell counts in HIV monitoring).
5. Western Blot – after SDS‑PAGE separation, a specific mAb probes the target protein, confirming its presence.

10. Principles of Using Monoclonal Antibodies in Treatment

• Neutralisation – mAb binds a pathogen or toxin, blocking its interaction with host cells (e.g., anti‑rabies mAbs).
• Receptor blockade – prevents ligand binding to a cell‑surface receptor (e.g., anti‑TNF‑α in rheumatoid arthritis).
• Induction of cytotoxicity

  • Complement‑dependent cytotoxicity (CDC) – Fc activates the complement cascade.
  • Antibody‑dependent cellular cytotoxicity (ADCC) – Fc engages NK cells or macrophages via Fcγ receptors.

• Targeted delivery (antibody‑drug conjugates, ADCs) – a cytotoxic drug, toxin or radioisotope is chemically linked to the mAb, delivering the payload directly to diseased cells (e.g., Brentuximab vedotin).

Key Considerations for Therapeutic Use

1. Humanisation – grafting murine complementarity‑determining regions (CDRs) onto a human IgG framework reduces immunogenicity.
2. Pharmacokinetics – Fc engineering (e.g., FcRn binding enhancement) or PEGylation can prolong half‑life and improve tissue distribution.
3. Safety monitoring – watch for infusion reactions, cytokine‑release syndrome, and off‑target effects.
4. Resistance mechanisms – tumour cells may down‑regulate the target antigen; combination therapies are often employed to overcome escape.

11. Examples of Diagnostic and Therapeutic Monoclonal Antibodies

12. Practical Activity – Designing a Sandwich ELISA (AO2 & AO3)

Task: Design a sandwich ELISA to detect the malaria antigen HRP2 in patient blood.

1. Select antibodies – two non‑overlapping monoclonal anti‑HRP2 antibodies; label the detection antibody with horseradish peroxidase (HRP).
2. Coating – add 100 µL of capture antibody (2 µg mL⁻¹) to each well; incubate overnight at 4 °C.
3. Blocking – block with 5 % skimmed milk or BSA to prevent non‑specific binding.
4. Sample addition – add diluted patient plasma (or standard HRP2 dilutions) and incubate 1 h at 37 °C.
5. Detection – add HRP‑conjugated detection antibody; incubate 1 h.
6. Substrate – add TMB (tetramethylbenzidine); stop reaction with 1 M H₂SO₄.
7. Readout – measure absorbance at 450 nm using a plate reader.

Data analysis: Plot a standard curve (absorbance vs. HRP2 concentration) and determine the concentration in unknown samples by interpolation. Calculate the limit of detection (LOD) as 3 × standard deviation of the blank.

Evaluation points (AO2):

• Sources of error – incomplete blocking, pipetting inaccuracies, cross‑reactivity.
• Specificity – monoclonal antibodies minimise false‑positive results.
• Sensitivity – optimisation of antibody concentrations and incubation times improves LOD.
• Reproducibility – repeat the assay on different days to assess intra‑assay variation.

13. Cross‑Topic Links (Connecting to Other Syllabus Areas)

• DNA → Antibody production – V(D)J recombination generates antibody diversity; somatic hypermutation refines affinity.
• Cell membranes – antigen‑antibody binding involves non‑covalent interactions at the cell surface.
• Enzymes – HRP used in ELISA catalyses a colour‑producing reaction.
• Energy metabolism – rapid proliferation of B‑cells during a secondary response requires increased ATP production (glycolysis & oxidative phosphorylation).

14. Summary

The immune system integrates innate barriers, phagocytic killing, and a sophisticated adaptive response that produces highly specific antibodies. Understanding antibody structure, class‑specific functions, and the sequence of primary and secondary responses provides the foundation for appreciating how vaccines generate long‑lasting protection. Monoclonal antibodies embody this knowledge in the laboratory and clinic: they enable ultra‑specific diagnostic assays and serve as powerful, targeted therapeutics when engineered for optimal affinity, reduced immunogenicity, and appropriate effector functions. Mastery of these principles equips students to meet Cambridge IGCSE/A‑Level objectives for immunity, experimental design, and critical evaluation of biotechnological applications.