The pivotal reagent common to all immunohisto-chemical* techniques is the antibody. The availability ofantisera, immunoglobulin fractions and monoclonal anti-bodies to an ever-increasing number of clinically usefultissue antigens has enormously expanded the quantityand quality of the immunohistologic repertoire. To bettercomprehend the potential of immunohistochemicalstaining methods as well as any latent problems that maybe associated with the same, it is necessary to have abasic knowledge of antibodies and their potentials, aswell as their limitations.
Antibodies belong to a group of proteins calledimmunoglobulins (Ig). Listed in order of decreasingquantityfound in plasma or serum, the immunoglobulinscomprise ﬁve major classes: immunoglobulin G (IgG), IgA,IgM, IgD and IgE. Each immunoglobulin is composed of twoidentical heavy chains (H) and two identical light chains (L).The H chains differ in antigenic and structural propertiesand determine the class and subclass of the molecule. Thetwo L chains are either type kappa or lambda. The distribu-tion of kappa and lambda chains differs in all Ig clases andsubclasses, as well as between different species. Covalentinterchain disulﬁde bridges join L to H and H to H chains. Byparticipating in the tertiary structure, they confer greaterstability to the immunoglobulin molecule.Of the ﬁve classes of immunoglobulins, IgG and IgM will beconsidered in more detail here, as they are by far the mostfrequently utilized antibodies in immunohistochemistry.Unless otherwise noted, most of what is described of the IgGstructure in this text was learned from studies with humanIgG of subclass IgG
IgG has the general formula of gamma
, which denotes that one molecule ofIgG (MW = 150 kD) is composed of two gamma heavychains, and two light chains of either type kappa or typelambda (Figure 1). The structure of the IgG molecule hasbeen determined in part by proteolytic digestions andreductive dissociation of the molecule (Figure 2). Digestionby papain results in the cleavage of a susceptible bond onthe N-terminal side of the inter-heavy chain disulfidebridges. This yields two monovalent antigen-binding frag-ments (Fab) and one crystalline fragment (Fc). Pepsincleaves the gamma chains on the C-terminal side of theinter-heavy chain disulﬁde bridges, resulting in one biva-lent antigen-binding fragment, F(ab')
. In this case, the Fcfragments are destroyed. Reductive dissociation of an IgGmolecule splits the interchain disulﬁde bridges and, if thefree sulfhydryl groups are blocked, results in the formationof two H chains (molecular weight 50 kD each) and two Lchains (25 kD each).The IgG molecule can be further divided into so-calleddomains, namely the variable domains (V) and the constant
Diagram showing the structure of an immunoglobulin molecule.It comprises two identical heavy (H) chains and two identical light (L)chains. Inter- and intrachain disulﬁde bonds ( ) contribute to thestructure and stability of the molecule.
Diagram showing the structure of rabbit IgG (which exists as asingle major subclass). The heavy (H) and light (L) chains are composed ofvariable (V) and constant (C) domains and are linked by inter- and intra-chain disulﬁde bonds ( ). Proteolytic digestion with papain ( )yields two antigen-binding fragments (Fab) and one crystalline fragment(Fc), whereas digestion with pepsin ( ) yields one F(ab')