Unit 3: Antigen - antibody interactions

The interaction between an antigen (Ag) and an antibody (Ab) is the non-covalent, reversible binding of an epitope to the paratope of an immunoglobulin. It underlies both natural immunity and every serological assay. This unit treats first the physical nature of that binding, then the specificity issues arising from it, then the laboratory methods that exploit it.

Defining properties of the interaction:

• Non-covalent forces: binding uses hydrogen bonds, electrostatic (ionic) bonds, van der Waals forces and hydrophobic interactions — no covalent bond forms.
• Reversibility: governed by the law of mass action; the complex forms and dissociates continuously (Ag + Ab ⇌ Ag–Ab).
• Specificity and complementarity: binding depends on shape fit ("lock and key") between epitope and the hypervariable CDR loops of the paratope.
• Epitope-based: an antibody recognises a small determinant (5–8 residues or sugars), not the whole antigen.

II. Strength of antigen–antibody interaction

Affinity, avidity and the thermodynamics of binding.

A. Affinity — single-site binding strength

The affinity constant quantifies binding at one paratope–epitope pair.

• Definition: the equilibrium association constant for one antigen-binding site.

K = [Ag–Ab] / ([Ag][Ab])

• Symbols: K = association (affinity) constant (M⁻¹); [Ag–Ab] = complex concentration; [Ag], [Ab] = free reactant concentrations.
• Magnitude: high-affinity antibodies reach K ≈ 10⁸–10¹¹ M⁻¹; low affinity ~10⁴ M⁻¹.
• Basis: the closer the epitope–paratope fit, the greater the summed non-covalent forces and the higher K.

B. Avidity — total binding strength

Avidity is the cumulative strength when multiple sites bind simultaneously.

• Definition: the overall stability of a multivalent Ag–Ab complex.
• Multivalency effect: IgM (10 binding sites) has modest per-site affinity but very high avidity; IgG has 2 sites.
• Consequence: avidity greatly exceeds the sum of individual affinities because dissociation requires all bonds to break at once.

III. Cross reactivity

When one antibody binds more than one antigen.

A. Basis and consequences

Cross reactivity occurs when an antibody raised against one antigen reacts with a structurally similar epitope on another.

• Cause: shared or near-identical epitopes on different antigens.
• Example: anti–blood group A antibodies cross-react with similar bacterial polysaccharides; heterophile antigens (Forssman) shared across species.
• Clinical use: the Weil–Felix test uses cross reactivity between Rickettsia and Proteus OX antigens.
• Drawback: it lowers assay specificity, producing false positives.

IV. Precipitation

Reaction of soluble antigen with soluble antibody to form a visible lattice.

A. Principle and lattice theory

Precipitation is the cross-linking of multivalent soluble antigen and bivalent antibody into an insoluble complex.

• Requirement: both reactants soluble; antigen multivalent, antibody at least bivalent.
• Marrack lattice hypothesis: cross-linking builds a three-dimensional network that falls out of solution.

B. The precipitin curve — zones of reaction

Precipitate amount depends on the Ag:Ab ratio.

1. Zone of antibody excess (prozone): too much antibody, little lattice, scant precipitate.
2. Zone of antigen excess (postzone): too much antigen, small soluble complexes, scant precipitate.
   • Equivalence zone: optimal Ag:Ab ratio gives maximal lattice and maximal precipitate.

C. Techniques

• Immunodiffusion (Ouchterlony): reactants diffuse through agar; precipitin lines form at equivalence, revealing identity, non-identity or partial identity.
• Radial immunodiffusion (Mancini): antigen diffuses into antibody-containing gel; ring diameter² ∝ antigen concentration.
• Immunoelectrophoresis: electrophoretic separation followed by diffusion against antiserum.

V. Agglutination

Clumping of particulate antigen by antibody.

A. Principle

Agglutination is the visible aggregation of particle-bound antigens cross-linked by antibody.

• Antigen form: particulate (cells, latex beads), unlike soluble antigen in precipitation.
• Best agglutinin: IgM, owing to its 10 valencies and large size.
• Prozone effect: excess antibody can inhibit clumping, as in precipitation.

B. Types

• Direct (active) agglutination: antigen is naturally on the particle surface (e.g. ABO blood typing of red cells).
• Passive (indirect) agglutination: soluble antigen is adsorbed onto carrier particles (latex, RBCs) to make it visible.
• Hemagglutination inhibition: free antigen blocks antibody, preventing RBC clumping — used to titre viruses such as influenza.
• Coombs (antiglobulin) test: anti-human globulin agglutinates cells coated with non-agglutinating (incomplete) IgG.

VI. Radioimmunoassay (RIA)

Quantitation by competition with a radiolabelled antigen.

A. Principle and procedure

RIA measures antigen concentration by competitive binding against a radioactive tracer.

• Label: a radioisotope, typically ¹²⁵I, on a known amount of antigen.
• Competition: unlabelled (sample) antigen and labelled antigen compete for limited antibody.

Ab + Ag* (labelled) + Ag (unlabelled)
→ more sample Ag ⇒ less bound Ag* ⇒ lower bound radioactivity

• Readout: bound-label signal is inversely proportional to sample antigen concentration.
• Sensitivity: detects picogram levels — hormones (insulin), drugs.

B. Applications and limitations

• Uses: endocrine assays, therapeutic drug monitoring.
• Limitations: radioactive hazard, short isotope half-life, waste disposal — largely displaced by ELISA.

VII. Enzyme linked immunosorbent assay (ELISA)

Enzyme-generated colour replaces radioactivity for quantitation.

A. Principle

ELISA uses an enzyme-conjugated reagent whose substrate yields a measurable colour proportional to bound analyte.

• Solid phase: antigen or antibody adsorbed to microtitre wells.
• Enzyme labels: horseradish peroxidase (HRP) or alkaline phosphatase.
• Substrate: e.g. TMB or p-nitrophenyl phosphate; colour read by spectrophotometer.

B. Formats

1. Direct / indirect: antigen coated; detected by enzyme-antibody (direct) or by primary then enzyme-labelled secondary antibody (indirect).
2. Sandwich: capture antibody binds antigen, then a labelled detection antibody binds a second epitope — signal ∝ antigen.
   • Competitive ELISA: sample antigen competes with labelled antigen; signal inversely ∝ concentration.
   • Application: HIV screening, pregnancy (hCG), cytokine measurement.

VIII. Immunofluorescence

Localisation of antigen using fluorochrome-tagged antibody.

A. Principle

Immunofluorescence detects antigen by tagging antibody with a dye that emits visible light under excitation.

• Fluorochromes: fluorescein isothiocyanate (FITC, green emission) and rhodamine (red).
• Detection: viewed under a fluorescence microscope.

B. Types

1. Direct: primary antibody itself is fluorochrome-labelled — one step, less sensitive.
2. Indirect: unlabelled primary antibody detected by a labelled anti-immunoglobulin secondary — amplified signal, more sensitive.
   • Applications: autoantibody detection (ANA test), identifying microbes and surface markers.

IX. Immunoblot (Western blot)

Detection of a specific protein after electrophoretic separation and transfer.

A. Principle and steps

The immunoblot identifies an antigen by size after separation, transfer to a membrane and antibody probing.

• Separation: SDS-PAGE resolves proteins by molecular weight.
• Transfer: proteins electro-blotted onto nitrocellulose or PVDF membrane.
• Blocking: non-fat milk or BSA saturates free sites to prevent non-specific binding.
• Probing: primary antibody binds target; enzyme- or fluorophore-labelled secondary antibody visualises a band at the correct molecular weight.
• Application: confirmatory HIV testing, detection of specific antibodies in Lyme disease.

X. Immunohistochemistry (IHC)

Antigen localisation within tissue sections.

A. Principle

IHC visualises antigens in their anatomical context on fixed tissue using labelled antibodies.

• Sample: formalin-fixed, paraffin-embedded or frozen tissue sections on slides.
• Antigen retrieval: heat or enzyme treatment unmasks epitopes hidden by fixation.
• Label: an enzyme (HRP with DAB substrate giving a brown deposit) or a fluorophore (then termed immunofluorescence on tissue).
• Detection: direct (labelled primary) or indirect (labelled secondary), often amplified with avidin–biotin complexes.

B. Applications and distinction

• Uses: tumour classification, identifying oestrogen/HER2 receptors in breast cancer, distinguishing cell lineages.
• Contrast with IF: IHC typically gives a permanent coloured, light-microscope-visible product; immunofluorescence gives transient fluorescent signal requiring a fluorescence microscope.
• Value: preserves tissue architecture, so antigen expression is mapped to specific cells and structures.