5.2 The Immune System and Immunoglobulins

Immune responses arise from cooperation between specialised cells and proteins

Innate immunity uses skin and mucosal barriers, phagocytes, natural killer cells and complement for a rapid, low-specificity response
Adaptive immunity selectively activates lymphocytes with diverse antigen receptors, giving high specificity and memory
The two arms are linked by antigen presentation and cytokine signalling rather than separate systems
Antigen = a molecule recognised by antibody or receptor; immunogen = a substance that actually elicits a response, so not every antigen is a strong immunogen
Epitope = the local site recognised by an antibody, either a continuous sequence or a conformational patch
Antibodies recognise the free antigen surface, whereas αβ T-cell receptors recognise peptide bound to MHC
B cells express a BCR after gene rearrangement, then clonally expand and differentiate into plasma and memory cells
Somatic hypermutation, affinity maturation and class switching change effector function but do not themselves create new epitope recognition
Antibody actions: neutralisation, opsonisation, classical complement activation and Fc-receptor-mediated regulation

Table 5.2-1Components of innate and adaptive immunity

Figure 5.2-21Flow linking antigen detection to antibody production by B cells; the real response involves more cell types and signals.

Figure 5.2-22Simplified model of the timing difference between primary and secondary responses; magnitudes and time axis are not patient data.

Figure 5.2-28Concept diagram of B-cell clonal selection and differentiation; the real process depends on several cells and signals.

Figure 5.2-29Concept diagram comparing the three main effector functions of antibodies.

An antibody has two identical antigen-binding sites

An IgG monomer has two identical heavy chains (about 50 kDa) and two identical light chains (about 25 kDa), about 150 kDa in total
Disulfide bonds between heavy chains and between heavy and light chains, plus non-covalent contacts, hold the fold together
Paired V_H and V_L domains at the arm tips form one antigen-binding site each, so a monomer has two
Fab carries antigen recognition; Fc engages Fc receptors and some complement components to mediate effector function
The hinge allows the arms to move; limited papain digestion yields two Fab fragments and one Fc fragment
Five isotypes (IgG, IgM, IgA, IgE, IgD) are defined by heavy-chain constant regions
Secreted IgM is mostly pentameric and secreted IgA mostly dimeric, so the claim that every antibody is bivalent is inaccurate

Table 5.2-2The five antibody isotypes: typical form and role

Figure 5.2-23Educational diagram of bivalent antigen binding and the Fab and Fc functions; atomic coordinates and scale are not reproduced.

Figure 5.2-24Functional division into variable and constant regions and disulfide bonds; six CDR loops form the centre of the binding surface.

Figure 5.2-25Conceptual outcome of papain cleavage of IgG; cleavage positions and fragment shapes are simplified.

Figure 5.2-27Representative quaternary forms of the five antibody classes; steric hindrance may prevent all sites being occupied at once.

Antibodies bind a specific antigen strongly and reversibly

Selective recognition rests on partial complementarity of shape, charge distribution, polar groups and hydrophobic surface
Binding combines hydrogen bonds, electrostatic interactions, van der Waals forces and the hydrophobic effect, with no new covalent bond
Rearrangement of the hydration layer and conformational change also contribute to the binding free energy
Specificity = ability to distinguish targets; affinity = strength at one site with one epitope; avidity = combined multivalent stability
Kd = [Ab][Ag]/[Ab·Ag] = koff/kon, and θ = [Ag]/(Kd + [Ag]), so θ = 0.5 when [Ag] = Kd
Example: Kd 2 nM with 6 nM free antigen gives θ = 6/(2 + 6) = 0.75
kon has units M⁻¹s⁻¹ and koff units s⁻¹; two antibodies with the same Kd can still associate and dissociate at different rates
Example: kon 2.0×10⁵ and koff 2.0×10⁻⁴ give Kd 1.0×10⁻⁹ M, and t₁/₂ = ln2/koff
Cross-reactivity is partial binding to similar epitopes; high concentrations of similar proteins, matrix interference and non-specific adsorption cause unexpected signal
Specificity validation is essential when choosing a detection antibody

Figure 5.2-26Complementarity between the epitope on the antigen surface and the antibody paratope.

Figure 5.2-30Four classes of interaction observed at the antigen–antibody interface.

Figure 5.2-31Calculated occupancy for single sites with different Kd (assumes equilibrium, independent sites and excess free antigen).

Figure 5.2-32Educational simulated curves showing relative changes in the association and dissociation steps.

Figure 5.2-33Conceptual comparison of monovalent affinity with avidity from multivalent binding; real avidity depends on spacing, density and flexibility.

Figure 5.2-34Simulated matrix of relative antibody signal against different antigens; off-diagonal signal indicates cross-reactivity.

Antigen–antibody interactions underpin important assays

ELISA reads antigen–antibody complex formation through an enzymatic colour reaction, in direct, indirect, sandwich or competitive format
A sandwich assay uses capture and detection antibodies that recognise different parts of the target
In a competitive ELISA the signal falls as target concentration rises
Blocking and washing at each step remove non-specific binding and excess reagent; stay within the validated range of the standard curve
Example: A450 0.690 with A = 0.060 + 0.090C gives 7.0 ng/mL, so a 5-fold dilution corresponds to 35 ng/mL in the original sample
Lateral flow devices form test (T) and control (C) lines as labelled particles move through a porous membrane
The control line mainly confirms sample migration and device function, and reading rules differ between products
Western blot separates proteins by electrophoresis, transfers them to a membrane and detects with primary and labelled secondary antibodies; position gives approximate mass and intensity a relative amount
Immunoprecipitation recovers a target with binding partners, immunofluorescence shows location and flow cytometry compares markers across many cells
Diagnostic sensitivity = TP/(TP+FN), specificity = TN/(TN+FP) and positive predictive value = TP/(TP+FP)
Example: prevalence 1%, sensitivity 90%, specificity 95% gives 90 true and 495 false positives per 10,000, so PPV ≈ 15.4%
Reliability requires positive and negative controls, blanks, standards, replicates and a defined measuring range
Distinguish analytical specificity from clinical specificity, and limit of detection (LOD) from limit of quantification (LOQ)
A monoclonal antibody comes from one clone with a single specificity: consistent, but vulnerable to epitope change or masking
A polyclonal antibody is a mixture from several clones: robust signal from multiple epitopes, but batch variation and cross-reactivity need care

Table 5.2-3Antibody-based methods compared

Figure 5.2-35Four representative ELISA formats; the immobilised partner and label position are conceptual examples.

Figure 5.2-36Binding principle of the sandwich ELISA; capture and detection antibodies must recognise different accessible epitopes.

Figure 5.2-37Classroom standard curve; in the linear range A = 0.060 + 0.090C (C in ng/mL). Not a real kit characteristic.

Figure 5.2-38Test and control lines of a typical antigen-detection lateral flow device; reading follows the product instructions.

Figure 5.2-39Stepwise concept flow of the western blot; bands are teaching models.

Figure 5.2-40Immunoprecipitation favours protein recovery; immunofluorescence favours visualising location in cells.

Figure 5.2-41Role of controls and replicate measurements that must be checked before interpreting results.

Figure 5.2-42Positive predictive value model for a hypothetical test with sensitivity 90% and specificity 95%; not representative of clinical performance.

Figure 5.2-43Classification of true and false positives and negatives, with the denominator of each index.

Figure 5.2-44Examples of diagnostics, protein research, cell analysis and antibody medicines that rely on antigen–antibody recognition.

References

**Table 5.2-1**Components of innate and adaptive immunity
Component	Representative cells and molecules	Core role
Innate immunity	Neutrophils, macrophages, dendritic cells, NK cells, complement	Early defence, antigen presentation, pathogen clearance
Humoral adaptive immunity	B cells, plasma cells, antibodies	Neutralisation, tagging and clearance of extracellular targets
Cell-mediated adaptive immunity	CD4⁺ helper T cells, CD8⁺ cytotoxic T cells	Regulation of the response; killing of infected cells
Immunological memory	Memory B cells, memory T cells	Rapid and efficient response on re-exposure

**Figure 5.2-21**Flow linking antigen detection to antibody production by B cells; the real response involves more cell types and signals.

**Figure 5.2-22**Simplified model of the timing difference between primary and secondary responses; magnitudes and time axis are not patient data.

**Figure 5.2-28**Concept diagram of B-cell clonal selection and differentiation; the real process depends on several cells and signals.

**Figure 5.2-29**Concept diagram comparing the three main effector functions of antibodies.

**Table 5.2-2**The five antibody isotypes: typical form and role
Isotype	Typical form	Main biological role
IgG	Mostly monomer (two binding sites)	Neutralisation and opsonisation in blood and tissue; some complement activation; placental transfer
IgM	Secreted form mostly pentameric	Early response; high avidity; complement activation
IgA	Secreted form mostly dimeric	Mucosal and secretion defence in gut and airway
IgE	Monomer	Mast cell and basophil Fc receptors; allergy and parasite defence
IgD	Mostly monomer	Mainly the antigen receptor of mature naive B cells

**Figure 5.2-23**Educational diagram of bivalent antigen binding and the Fab and Fc functions; atomic coordinates and scale are not reproduced.

**Figure 5.2-24**Functional division into variable and constant regions and disulfide bonds; six CDR loops form the centre of the binding surface.

**Figure 5.2-25**Conceptual outcome of papain cleavage of IgG; cleavage positions and fragment shapes are simplified.

**Figure 5.2-27**Representative quaternary forms of the five antibody classes; steric hindrance may prevent all sites being occupied at once.

**Figure 5.2-26**Complementarity between the epitope on the antigen surface and the antibody paratope.

**Figure 5.2-30**Four classes of interaction observed at the antigen–antibody interface.

**Figure 5.2-31**Calculated occupancy for single sites with different Kd (assumes equilibrium, independent sites and excess free antigen).

**Figure 5.2-32**Educational simulated curves showing relative changes in the association and dissociation steps.

**Figure 5.2-33**Conceptual comparison of monovalent affinity with avidity from multivalent binding; real avidity depends on spacing, density and flexibility.

**Figure 5.2-34**Simulated matrix of relative antibody signal against different antigens; off-diagonal signal indicates cross-reactivity.

**Table 5.2-3**Antibody-based methods compared
Method	Main target observed	Strength	Typical limitation
Sandwich ELISA	Concentration of the target antigen	Quantitative; many samples	Two antibodies must bind different epitopes at once
Indirect ELISA	Specific antibody in the sample	Antibody-response testing	Cross-reactivity; timing of sampling
Rapid antigen test	Presence of target (partly semi-quantitative)	Fast point-of-care reading	Limit of detection; sampling; product-specific reading
Western blot	Specific protein band	Detection with mass information	Off-target bands; non-linear quantification
Immunofluorescence	Spatial distribution of the target	Visualises cellular location	Depends on antibody, fixation and permeabilisation

**Figure 5.2-35**Four representative ELISA formats; the immobilised partner and label position are conceptual examples.

**Figure 5.2-36**Binding principle of the sandwich ELISA; capture and detection antibodies must recognise different accessible epitopes.

**Figure 5.2-37**Classroom standard curve; in the linear range A = 0.060 + 0.090C (C in ng/mL). Not a real kit characteristic.

**Figure 5.2-38**Test and control lines of a typical antigen-detection lateral flow device; reading follows the product instructions.

**Figure 5.2-39**Stepwise concept flow of the western blot; bands are teaching models.

**Figure 5.2-40**Immunoprecipitation favours protein recovery; immunofluorescence favours visualising location in cells.

**Figure 5.2-41**Role of controls and replicate measurements that must be checked before interpreting results.

**Figure 5.2-42**Positive predictive value model for a hypothetical test with sensitivity 90% and specificity 95%; not representative of clinical performance.

**Figure 5.2-43**Classification of true and false positives and negatives, with the denominator of each index.

**Figure 5.2-44**Examples of diagnostics, protein research, cell analysis and antibody medicines that rely on antigen–antibody recognition.