Micromolecule Immunocytochemistry

TECHNICAL NOTE: Micromolecule Immunocytochemistry

Epitope types

The epitope is the set of atom groups in 3-space that form the "target" of the IgG antigen binding site (the paratope). A single conventional IgG has two identical paratopes which preferentially bind an epitope. There are, in principle, two kinds of epitopes or antigenic determinants: (1) linear and (2) non-linear determinants. The latter are composed of juxtaposed groups that are not necessarily adjacent in the native molecule but become so by folding. This is most common for polypeptide and protein determinants. The power of the monoclonal antibody technology resides in the specificity of those paratopes for a specific epitope on a target molecule that may have hundreds of possible determinant configurations. Thus two different antibodies often target exactly the same protein but at very different epitopes.

The dimensions of a typical epitope is often given as roughly 6-residues in size but can be much smaller - 2 or 3 residues - while retaining high preference for the corresponding paratope. Importantly, haptens based on single molecules coupled to carriers are nearly always linear determinants: molecular configurations that are characterized by adjacency of interaction sites and restricted mobilities thereof. Linear determinants present a very restricted set of epitopes to the immune system. Unlike IgGs targeting proteins, those that bind hapten targets virtually always attack the same or overlapping linear epitopes to the extent that binding is mutually exclusive. Thus anti-hapten IgGs produced by polyclonal methods very often have monoclonal properties as far as molecular selectivity is concerned. This is quite important, as the limiting property of a specific anti-hapten IgG is typically its affinity and polyclonal techniques actually provide a better, faster opportunity for finding a high-affinity, selective anti-hapten IgG than monoclonal methods. This may be surprising to some, but has great practical importance. Furthermore, although components of linear determinants may have some mobility, this is motion is limited. A linear determinant might exist in a few configurations that constitute different epitopes, but the same locale will pass through configurations with different probabilities and IgG binding will follow correspondingly.

Titer and Affinity

Titer is the amount of antibody produced in a host and is a rough measure of quality. Several features of an immunogen impact titer - the novelty of the epitopes on the immunogen, immunogen dose, immunogen lifetime and immunization schedule among other things. Several of these can be manipulated to produce high titers and these often results in high dilution antisera (1:30,000 and higher). The key advantages to such sera are (1) that the IgG concentration required for antigen detection can be had with very little original serum, obviating affinity purification, and (2) they produce very low background signals. However, high titer is often confused with sensitivity, which is primarily a function of a single molecule and is more properly expressed as apparent affinity. Polyclonal IgG affinities for nonlinear determinant immunogens are complex, but in the case of hapten immunogens with very small, linear antigenic determinants there is a convergence of virtually all useful IgGs on a very narrow range of affinities. That is why, in spite of differences in reported titers, anti-hapten immunocytochemistry with very different IgG sources show almost exactly the same ranges of antigen detection. The affinity of an IgG is determined by the dual molecular structures of epitope and paratope, and since the physicochemical properties of epitopes from linear haptens arise from a small number of nearly fixed residues, useful IgGs (even from polyclonal sources) all likely converge on the same narrow range of affinities. Titer employed tells little about the levels of antigen in a sample, but much about the process of immunization. After having made the IgG, the latter is not of much concern.


The practice of immunocytochemistry requires some estimates of the selectivity of an IgG in the presence of mixed targets. Cross-reactivity is the propensity of one serum to bind to different but somehow related targets, usually with very different affinities. There are two classes of cross-reactivity. Type I cross-reactivity is simply due to the presence of multiple antibodies in the serum targeting different but similar antigens, likely different affinities and often targeting different epitopes. A variety of affinity purification tactics can be used to resolve this problem. Often a simple hapten affinity column will produce very clean IgG preparations. Type II cross-reactivity is a property of a single IgG, where sterically similar, but truly different molecular targets are bound by the same paratope. The solutions to this problem are (1) search for the host or clone that gives the required specificity or (2) map the relative affinities for different targets. This is actually one of the fundamental features of IgGs against haptens - that closely related molecules can sometime be bound by the same paratope because they differ only in fine detail - such as the placement of a single hydrogen residue. This is an asset in immunochemical analysis of stereoisomers and a nuisance in immunocytochemistry. However, the severity of type II cross-reactivity can assessed several ways. (1) Analysis of whole tissue amino acids or other compounds by HPLC can often define the spectrum of possible cross-reactive targets for an amino acid. (2) Comparing dilution curves on serial sections with standards of known primary and cross-reactive molecules can identify whether the apparent affinity of the IgG for a tissue target matches the expected standard. (3) Competitive inhibition with model antigens of different types on serial sections can differentiate targets by apparent affinities. This is roughly the inverse of (2). Of course the simplest solution is to have a panel of good IgGs against diverse targets.

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