IMMUNOLOGY - Roit A. - Mir 2000

Chapter 9. Antigen Recognition

ANTIBODY SPECIFICITY AND AFFINITY

Antigen-antibody reactions are characterized by high Specificity. For example, measles Antibodies bind to measles Viruses and establish Immunity against this disease, but they are unable to bind to other virus species, such as polioviruses, and do not protect the body against them. The specificity of an antiserum reflects the combined specificities of the antibodies it contains, a population that may include numerous paratopes capable of binding to different epitopes or even to different PARTS OF THE same epitope (Fig. 9.8). However, if antigen A shares common epitopes with antigen B, a fraction of the antibodies specific for A will also react with B. This phenomenon is known as cross-reactivity.

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Fig. 9.8. The specificity of an antiserum reflects the specificity of its individual antibody molecules (e.g., anti-X, anti-Y, and anti-Z antibodies directed against epitopes X, Y, and Z found on different antigen molecules). Antigen A (AgA) and antigen B (AgB) share a common epitope Y. Antiserum against AgA (anti-XYZ) not only specifically binds to AgA but also cross-reacts with AgB (due to recognition of epitopes Y and X'), yet shows no reaction with AgC since it shares no common epitopes with AgA.

Antibodies recognize the outer conformation of Antigens

Naturally, antibodies do not recognize isolated chemical groups, but rather the spatial conformation of epitopes (Fig. 9.9). This occurs with striking specificity, capturing not only differences in charge distribution, optical and stereoisomerism, but also minimal variations in the primary Amino Acid Sequence (in the case of protein antigens) (Fig. 9.10). Because of this exquisite specificity, the majority of antibodies are capable of binding only to native (undenatured) antigens or to antigen fragments that retain the tertiary Structure required for the multiple interactions involved in paratope-epitope binding.

Fig. 9.9. Antiserum obtained by immunizing animals with meta-aminobenzenesulfonate as a hapten was tested for binding with the ortho and para isomers of the same hapten, as well as with three isomers (ortho, meta, and para) of two different but chemically related antigens: aminobenzenearsonate and aminobenzenecarboxylate. The antiserum reacts specifically with the hapten whose sulfonate group is in the meta position, but also shows a cross-reaction (albeit weak) with the ortho isomer of the same hapten. Even weaker cross-reactions are observed with haptens bearing an arsonate or carboxylate group in the meta position, but not in the ortho or para positions. Unlike the sulfonate group, the arsonate group is larger and possesses an additional hydrogen atom, whereas the carboxylate group is smaller than both. These findings indicate that both the overall configuration of the antigen and the specific chemical groups contained within its molecule are crucial for antibody binding.

Fig. 9.10. The Lysozyme molecule contains intrachain Disulfide Bonds (shown as red bars) that cause the polypeptide to form loops. Antisera directed against the whole lysozyme molecule (antilysozyme) and against one of its peptide loops (“antiloop”) distinguish both of these antigens, but fail to react with the same peptide in its open (reduced) form. This confirms The Significance of the antigen's tertiary structure as a factor determining antibody specificity.

Consequently, difficulties may arise in immunological research when generating specific antibodies. To simplify the process, researchers often use a specially synthesized short polypeptide of known primary sequence as an antigen, since this is easier than obtaining a sufficient quantity of native antigen through purification. However, antibodies elicited by synthetic Peptides often lack the required specificity and affinity for the antigen in its native form.

Gene modification enables The production of custom-designed antibodies

Advances in molecular biology have made it possible to produce Monoclonal Antibodies (mAbs) of predetermined specificity, affinity, and isotype, and furthermore, to engineer antibody genes containing mouse V genes and human C genes. By immunizing mice, one can induce The formation of antibodies with the desired specificity and subsequently impart human antibody species antigenicity to them. Due to the very low immunogenicity of such mAbs in humans, they can be used clinically. Even more sophisticated engineering techniques allow the framework of animal V domains to be supplemented with complementarity-determining regions (CDRs, see Ch. 8) derived from antibodies of another animal species. It is even possible to substitute specific amino acid residues within the antigen-binding site to enhance antibody affinity (Fig. 9.11).

Fig. 9.11. Mouse V genes can be joined with human C genes and the recombinant product introduced into a Cell genome, which will then synthesize chimeric “mouse-human” antibodies (1). Another antibody engineering approach involves grafting hypervariable regions (CDRs) of the desired structure onto a light chain V-region framework (2). In addition, the affinity of the antigen-binding site can be altered by introducing a point mutation into the DNA segment encoding the hypervariable loops of the V_H domain (in this case, the replacement of phenylalanine with Lysine is shown) (3).

Polyfunctional antigen-binding sites can be simultaneously complementary to different epitopes

It has recently been discovered that the same antibody can be complementary to several different antigenic determinants. These determinants compete for binding to the antibody and apparently occupy different Regions of the cavity formed by the antigen-binding site (Fig. 9.12).

Fig. 9.12. A single antigen-binding site is capable of binding to several different antigenic determinants. For example, the antigen-binding site of antibody 460 features two pockets spaced 1.2–1.4 nm apart, one of which binds a menadione (vitamin K₃) hapten group and the other a dinitrophenyl (DNP) hapten group. This binding occurs competitively, meaning that the binding of one hapten precludes the subsequent binding of the other. Antigen-binding sites capable of interacting with multiple antigenic determinants are termed polyfunctional.

The specificity of a polyclonal antibody population for a given antigen (A) does not imply that every antibody species within it interacts exclusively with A. Rather, a large proportion of these antibodies possess a polyfunctional Active Site capable of binding A, and the total reactivity toward A significantly exceeds the reactivity toward other antigens. In other words, (polyclonal) antibody specificity can be a population phenomenon—an averaged characteristic of all antibodies contained within the antiserum (Fig. 9.13).

Fig. 9.13. Antiserum specificity as a population phenomenon. A single antigen A (AgA) can bind to receptors of various B Cells specific not only for A but also for other antigens (C, D, E, F, etc.), i.e., possessing polyfunctional active sites. Each B cell activated by AgA produces antibodies specific not only for AgA but also for other antigens. However, because all activated B cells are specific for AgA, whereas not all of them are specific for other antigens, the concentration of antibodies to AgA will be high, and to other antigens—low.

High-affinity antibodies are often more effective than low-affinity ones

The affinity of antibody-antigen binding is of much more than theoretical interest. Along with avidity, it dictates the physiological and pathological effects of antibodies. High-affinity antibodies are significantly more active than low-affinity ones in a variety of biological reactions (Fig. 9.14). In animal experiments, immune complexes containing low-affinity antibodies persist in the Circulation for prolonged periods and deposit on the basement membrane of renal capillaries, impairing their function. Conversely, immune complexes formed by high-affinity antibodies are cleared from the bloodstream much more rapidly and localize within the mesangium of renal glomeruli with minimal impact on their function.

Fig. 9.14. Biological reactions in which high-affinity antibodies outperform low-affinity ones.

Antibody affinity for most T-dependent antigens increases over the course of the Immune Response. This enhancement can also be achieved through specialized immunization protocols. For instance, the serum subpopulation of high-affinity antibodies expands when the antigen is administered alongside IFNγ (Fig. 9.15). While A number of immunoadjuvants stimulate antibody production, only a few are capable of increasing antibody affinity. Given that antibody efficacy relies heavily on this property, IFNγ holds considerable potential as an adjuvant in vaccine formulations.

Fig. 9.15. Mice in the experimental group were immunized with an antigen combined with 30,000 IU of IFNγ (Ag + IFNγ), whereas control mice received the antigen alone (Ag). The affinity of the resulting antibodies was measured at early and late time points post-immunization. Regardless of when the Blood samples were drawn, the serum of the experimental mice contained a higher proportion of high-affinity antibodies (dark blue bars) compared to the control group. (Adapted from Holland, Holland and Steward, 1990. Clin. Exp. Immunol. 82: 221-226.)

Determination of Antibody Affinity

A range of Methods exists for measuring antibody affinity and antiserum avidity. The underlying principle of these Procedures is to establish conditions where the antibody-antigen interaction reaches equilibrium: Ab + Ag ⇔ AbAg, and subsequently—without disturbing this equilibrium—to measure the concentrations of free and bound antigen. This can be accomplished in various ways, such as separating free antigen from bound antigen using techniques like dialysis, Gel filtration, centrifugation, or selective precipitation, or by detecting Changes in the fluorescence of the antigen or antibodies following immune complex formation.

Using the obtained data in conjunction with the law of mass action, one can calculate the equilibrium (affinity) constant, K:

where [AbAg] is the concentration of immune complexes, (Ag) is the concentration of free antigen, and [Ab] is the concentration of unoccupied antigen-binding sites at equilibrium. When half of the antigen-binding sites are occupied by the antigen, meaning [Ab] equals [AbAg], the value of K equals 1/[Ag]. In other words, to bind 50% of the active sites, high-affinity antibodies (i.e., those with a high K) require a low antigen concentration, whereas low-affinity antibodies require a high antigen concentration.

The Affinity Distribution of Antibodies in Antiserum Deviates from Normality

In enzyme-substrate reactions, the Equilibrium Constant (K) remains constant across varying substrate concentrations. However, the binding of antibodies to a homologous antigen behaves differently: a population of polyclonal antibodies is heterogeneous regarding this constant, meaning it varies in affinity. It was previously assumed that the quantitative distribution of antibodies by affinity in any antiserum followed a normal (Gaussian) distribution. As is now established, this assumption was incorrect. Graphical analysis of affinity data for the reaction between antiserum and antigen demonstrates that the affinity distribution of antibodies does not conform to a normal distribution (Fig. 9.16). Nevertheless, the average affinity of the antibody population in a given antiserum (K₀) can be approximated quite accurately as the reciprocal of the free antigen concentration required to saturate half of all antibody antigen-binding sites: K₀ = 1/[Ag_free].

Fig. 9.16. Histogram illustrating a typical distribution of antibody affinity in an antiserum directed against a specific antigenic determinant, compared with a homogeneous affinity population of monoclonal antibodies against the same determinant. Unlike polyclonal antibodies, all monoclonal antibodies exhibit uniform affinity. The affinity distribution of polyclonal antibodies present in antiserum deviates from a normal distribution.

Evaluating the Affinity Heterogeneity of an Antibody Population is Experimentally Challenging. The affinity heterogeneity of an antibody population cannot be precisely described by a mathematical formula because the exact nature of their distribution by this parameter cannot be reliably assumed for every individual antiserum—whether it is normal, skewed, bimodal, or polymodal. To assess such heterogeneity, researchers employ fairly complex computer analyses of binding curves based on the assumption of a normal distribution. However, a simpler experimental approach that avoids this assumption has also been described. In this method, microtiter plate wells pre-coated with immobilized antigen are treated with a test antibody solution at a known concentration along with serial dilutions (solutions of progressively decreasing concentrations) of free antigen, which competitively inhibits Antibody Binding to the surface-bound antigen. The percentage of binding inhibition is then calculated for each free antigen dilution to determine the molar concentration that yields 50% inhibition (I_0.5). Expressing the results in percentiles allows for the Construction of a histogram to analyze the spread of affinity values. The Shannon index can serve as a mathematical measure to express this spread, where high values indicate a broad range of affinities within the antibody population, and low values indicate a narrow range accordingly.