Antibody Populations

As shown in Table 2, free DAS, as expected, is its own best displacing agent, whereas only DAS—HMS showed any appreciable displacing capabiUty. This can be expected because the hemisuccinate linker is also immunogenic and leads to the production of antibodies specific for the linker in the polyclonal antibody population. AH the other toxins had at least lOOx less the avidity for the antibody, illustrating the specificity of the aDAS for DAS. 

Fig. 5. Schematic representation of the domain structure of albumin, the position of the disulfide loops, and the fragments of the molecule that have been isolated. Included in the figure are the cleavage methods used to obtain the fragments plus the regions to which the restricted antibody populations used in these experiments are directed. Reprinted, with permission, from Teale and Benjamin (1976). Copyright by the American Society of Biological Chemists, Inc.

However, not all antibody detected by ELISA corresponds to the antibody molecule that blocks the biological activity or active sites of the therapeutic protein. These neutralizing (blocking) antibodies are only a small fraction of the binding antibody population. Different cell- and receptor-based neutralizing antibody assays are developed for different protein drugs. 

If some of the antibodies in the antibody population of a serum against antigen A also precipitate with another antigen B, the process is called a cross-reaction between A and B. Cross-reactions are caused by antigenic determinants that A and B have in common. 

The continuing refinement in the selection of reference materials and the production of antibodies to complex protein mixtures has resulted in immunoassay systems of remarkable sensitivity and specificity. In particular, the selection and enrichment of the antibody population by immunoaffinity purification against the reference impurities has afforded an additional level of control over the production and validation of these reagents and served to improve the assay range and sensitivity (6,17). This normalization of the antibody population to a stoichiometric relationship with the reference impurities has suggested the term Antigen Selected Immunoassay (ASIA) for these methods. 

Such plots include Michaelis Menten curves, Scatchard plots, and Sips plots. The later two plots will also give an estimate of the heterogeneity of the antibody population (4,44,46). 

The problem of antibody heterogeneity may well be complicating the precise delineation of antigenic determinants of proteins. Thus far all efforts to locate the antigenic determinants have been made with heterogeneous antibody populations. The subject must be reexplored with antiprotein antibodies obtained from hybridomas, which would provide monoclonal antibody populations. In this way one cannot have uniform reactivity with only the antigen itself, but must have it also with the vari-... 

Elution of the antibodies with a linear concentration gradient of the hapten, fractionated the antibodies according to their affinity. Elution in this manner permits the study of antibody populations selected on the basis of their affinity. 

Painter, R.H. Freedman, M.H. Isolation and characterization of electrophoretically homogenous rabbit antihapten antibody populations. II. Survey of the isoelectric properties of antihapten antibodies directed against charged and uncharged proteins. J. Biol. Chem. 1971, 246, 1742-1751. 

Monoclonal antibodies Homogeneous antibody populations directed at against a single antigenic determinant, produced by a single clone of cells... 

Polyclonal antibodies Heterogeneous antibody populations that exhibit different properties and binding characteristics Precision Extent to which certain measurements agree to each other, usually stated as coefficients of variation, standard deviation or confidence limits... 

K = the equilibrium constant for the overall reaction As would be predicted from the law of mass action, the concentration of Ab, Ag, and AbAg will be dependent on the magnitude of ki and For polyclonal antiserum, the average avidity of the antibody populations will determine K (typically 10 to 10 L/mol), and the magnitude of in comparison with L., will determine the ultimate Emit of detection attainable with a given antibody population. 

A method used frequently to express heterogeneity of the antibody population of a serum is the Sips plot. From eq. 7 (cf. eqs. 14-16) it follows that ... 

Quantitative immunoelectrophoresis (Birkmeyer et al., 1981) and reverse quantitative immunoelectrophoresis (Birkmeyer et a)., 1982) allow the determination of average affinity of different antibody populations for an antigen. Plots of rocket area vs. the amount of antibody applied yield straight lines, the slope of which is indicative of affinity (steeper if the affinity is higher). Actual values of K are not obtained. Relative avidity indices can also be obtained with the test of Farr (1958) (Griswold and Nelson, 1984). 

Information on the major interactive variables of the EMIT tests is scanty (Mule et al., 1974 Osterloh and Butrimovitz, 1982). Antibodies used for the inhibition or reactivation of the enzyme are heterogeneous and the later-bound (lower-afYinity) antibodies differ in their effect on the enzyme. The may change due to different antibody populations or to allosterism (Osterloh and Butrimovitz, 1982). The antibodies in commercial kits are often heterogeneous on purpose to obtain a wider specificity. 

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