Abstract
Precipitation profiles of spot immunoprecipitate assay (SIA) reactions in gel are shown to have characteristics analogous to classic precipitin curves representing such immune reactions in solution. Thus SIA profiles of precipitated human albumin (HSA) and anti-HSA as checkerboard analysis permit: (1) determination of optimal proportions of antibody and antigen, (2) titration of antiserum as to antigen-binding capacity and (3) estimation of the concentration of specific antibody in an antiserum. The validity of the method was confirmed by similar results already reported for precipitin analysis of anti-HSA in solution. The stained protein assay method for determination of total protein used for the calculations had a reliable ( r = 0.9979) working range for quantification between 0.1 and 0.83 μg protein in 3 μl samples ( ≥ 0.03 g/l ). Study of SIA profiles in the extreme antibody excess region confirmed the validity of SIA quantification. The high sensitivity of SIA as compared with radial immunodiffusion is related to high ratios (8–9) of antibody to antigen; the reliable detection limit for SIA is 10 mg/l in a 3 μl sample. SIA is easy to perform with simple laboratory equipment, allows measurement of any precipitable antigen with very small amounts of reactants, and gives results within an hour.
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