Analysis of Parameters Affecting the Solid Phase Radioimmunoassay Quantitation of Antibody to Meningococcal Antigens

Abstract

An indirect solid phase radioimmunoassay (SPRIA) performed in flexible polyvinyl microtiter plates was modified for quantitative determination of antibodies to various bacterial antigens. Parameters affecting quantitation of the assay were investigated by using meningococcal serotype antigens and rabbit antisera to whole meningococci as a model system. Binding of purified antigens to the plate was essentially complete after 1 hr at 37°C, and only 4 to 8% of the bound antigen was released during the course of the assay. About 90% of the specific primary antibody (PAb) added to the antigen-coated wells was bound after overnight incubation at room temperature, whereas, 10 to 25% of the bound PAb was released before completion of the assay. Under conditions of limiting PAb the time required for saturation binding of ¹²⁵I-anti-immunoglobulin (secondary antibody = SAb) to the PAb was dependent on the concentration of SAb. At a concentration of 20 ng SAb/25 µl, maximum binding occurred in 12 to 16 hr at 22°C. Under conditions of extreme SAb excess the cpm of ¹²⁵I-SAb bound was directly proportional to the amount of PAb bound. Under these conditions the cpm of ¹²⁵I-SAb bound per well can be related to the amount of PAb added per well by a time-dependent calibration coefficient K(t) which is the product of three parameters: 1) the specific activity of the ¹²⁵I-SAb, 2) the ratio of PAb bound to SAb bound and, 3) the fraction of added PAb remaining bound to the plate. Experimentally determined values for these parameters were used to calculate K(t) and quantitate the specific antibody in nine rabbit antisera with the SPRIA. A close correlation (r = 0.985) was found between these results and the results of quantitative precipitin tests performed by using the same antisera and antigens. Although the SPRIA results were an average of 33% lower, a more accurate value for K(t) can easily be determined by performing the SPRIA on several sera calibrated by the quantitative test. Reproducibility of the SPRIA for 10 replicate determinations was ± 6.7%. The assay described is capable of measuring a minimum of about 0.5 µg antibody/ml of serum and appears to be applicable to many different antigens.