Antibody excess

The detection and accurate quantitation of any protein in an immunoassay requires that a condition of antibody excess exist. This is required for each protein in the reference impurity preparation. The acquisition and characterization of broad spectrum antisera against complex protein mixtures, therefore, is a fundamental goal in the development of these assay systems. 

Precipitates were prepared under conditions of extreme antibody excess. These values represent a minimal estimate of the antigen valence. 

With certain horse antitoxins and antiprotein sera and in some patients with Hashimoto s thyroiditis who have antibodies to thyro-globulin, one finds a different type of quantitative precipitation curve, termed a flocculation curve.Precipitation occurs only over a narrow range, and soluble antigen-antibody complexes are formed in the region of antibody excess as well as of antigen excess. Flocculation curves have... 

Agglutination occurs only over a certain range of serum dilutions because of either antigen or antibody excess. In the prozone, a large excess of antibody is present, so that each antibody behaves univalently and cross-linking does not occur (Eq. 5.5) ... 

An agglutination test for a bacterium was performed on serial dilutions of a freshwater sample. Using dilution factors of 1 1 to 1 512, the results showed the appearance of an agglutination zone and a zone of antibody excess, but no prozone was observed. Was the bacterium present Why was no prozone observed ... 

Antibody excess All antigenic sites are covered with antibody, and lattice formation is inhibited. 

Figure 9-3 Schematic diagram of precipitin curve illustrating zones of antibody excess (A), equivalence (B), and antigen excess (C). The parameter measured may be quantity of protein precipitated, light scattering, or another measurable parameter. Antibody concentration is held constant in this example.

Loss of end point detection is matched by a loss in OD at high concentrations of IgG. For example, in rows F CH at 5 pg/mL of IgG, there is substantial and increasing loss in color, as compared to where maximal color (in antibody excess row A) is observed. Note that row H barely titrates the IgG very low color is obtained. 

Assess the best antigen concentration for use in the competition assay, and select the IgG concentration that gives a plateau maximum (in antibody excess) of around r Cl. 5 OD. (curves 4 and 5). 

Experimental data bearing on the valence of the antibody molecules are given in Tables III and IV. The observed antibody/antigen molecular ratio of about 0.7 in both the equivalence zone and the region of antibody excess (Table III) corresponds, with antigen known to be bivalent, to 2/0.7 = 2.8 for the average valence of the antibody molecules in the precipitate. 

Effect of Time of Standing on Amount of Precipitate Obtained in the Regions of Antibody Excess and Optimum Precipitation... 

Solubility of the precipitate in excess of antiserum has been reported for a few systems, such as diphtheria toxin and horse antitoxin, but not for antigen-antibody systems in general. Theoretical considerations indicate that solubility in antibody excess should occur for antigens wi small valence (such as the dihaptenic substances of Table IV) but not for antigens with large valence, which would only witii difificulty be saturated with antibody to form a soluble complex. 

A number of experiments other than those reported in Table II were carried out in which varying amounts of purified antibody were added to a constant amount of antigen in a constant volume of solution. A 5 to 10% decrease in precipitated protein was noted at the largest amounts of antibody in every experiment. This effect of inhibition of precipitation by antibody excess is similar to but not nearly so marked as the effect in serum, in which a four-fold increase in amount of antiserum above the optimum was found to decrease the amount of precipitate to a third of the maximum value. 

A fluorescent phenytoin derivative was applied to the column at time r = 0 to saturate the antibodies. Excess phenytoin was washed off with pH 7.4 buffer. 

There are innumerable formats for immimoassays, which, however, are not very different in their fundamental steps. The most important classes are the competitive (antibody limited) and the non-competitive (antibody excess) assays. 

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