Neil S. Greenspan

Size-exclusion HPLC analysis of epitopes.

A size-exclusion chromatography method based on competition between intact antibodies and antigen-binding fragments (Fabs) is used to evaluate relative spatial location of epitopes recognized by monoclonal antibodies. If epitopes are spatially distinct, the presence of a potentially competitive Fab results in formation of complexes of higher molecular weight than those formed by antigen and intact IgG. Complexes of lower molecular weight are formed if the epitopes overlap, are identical, or if, upon formation of the antigen-antibody complex, conformational change occurs that results in loss of epitope. Size-exclusion chromatography of the relevant mixtures reveals these shifts in molecular weight distribution and, therefore, provides an unambiguous determination of spatially independent epitopes. In addition, chromatographic analysis of competition between IgG and its enzymatically prepared Fab is used to evaluate the dissociation rate constant of the antigen-antibody reaction in re-equilibrating mixtures.

Topographic Analysis with Monoclonal Anti-Idiotopes: Probing the Functional Anatomy of Immunoglobulin Variable Domains

Attempts to correlate immunoglobulin variable domain functional properties with variable domain primary structure have been valuable, but these efforts have suggested that to more fully account for variable domain function in terms of structure will require knowledge of molecular relationships in three dimensions. In this review we describe generally applicable methods, using monoclonal anti-idiotopes, for the determination of spatial relationships of idiotopes relative to one another and relative to two orienting structural markers of variable domains: 1) the hapten-binding site and 2) the junction of the variable and constant domains. Using these methods it has been possible to construct an oriented idiotope map which spans the variable domain along an axis connecting the paratope and the variable domain-constant domain junction. In addition, it has been possible to correlate idiotope position with other properties of idiotope expression. This approach may contribute to the development of predictive principles of idiotope expression.

Antibody Diversity in the Response to Streptococcal Group A Carbohydrate

In the past several years, a number of laboratories have used the murine antibody response to streptococcal group A carbohydrate (GAC) as a model system in which to study the development and regulation of humoral immunity. This system displays a number of features which are common to other antigen-antibody systems as well as unique features which make it particularly suitable to the study of antibody diversity. The scope of this chapter is to review the major characteristics of this antibody response and to discuss in more detail recent experiments from our laboratory which have exploited the anti-GAC response to ask questions concerning the rules which govern the pairing of heavy- and light-chain variable regions in the generation of antibody diversity. As with most other antibody systems which have been examined in detail, it has become increasingly apparent that the antibody response to GAC is assembled from a restricted set of heavy- and light-chain variable regions and constant regions. These observations have raised questions about the extent to which combinatorial diversity functions within the immune system and suggest the possibility that coordinated regulation results in the expression of VL, VH, and CH gene products in restricted sets, irrespective of the constraints imposed by an antigen-specific system.

Involvement of Self in the Interactions of Lymphocytes and Target Cells: Some Speculations on the Nature of MHC Restriction

Presentation of antigen on the cell surface and in the context of appropriate major histocompatibility complex (MHC)-encoded antigens is apparently a necessary prerequisite for T cell stimulation. It seems likely that murine T cells (with the exception of some suppressors) are unable to respond to stimulation by soluble antigens alone, either by proliferation and secretion of helper factors or by differentiation to cytotoxic effector function. Depending on the function of the responding T cell subset, the appropriate cell surface structures may be encoded in the IA, IE/C (helper), IJ (suppressor), or K, D or L (cytotoxic) region of the MHC. Much of the debate about this MHC restriction phenomenon has been concerned with determining whether T cells recognize, for example, viral antigen with one receptor entity, and MHC antigens with a second (dual recognition) or alternatively, if there is a single receptor specific for some associative interaction between virus and H-2 antigen. The idea that the T cell may express a single, two-chain receptor which recognizes a virus-H-2 complex is claimed by proponents of both models. The concept that, if there are two components to the receptor(s) they must be closely linked, has recently been supported by the experiment of Kappler et al. (1981) who showed that different MHC restriction and antigen specificity patterns did not assort independently following fusion of two T cell clones.