EVALUATION OF A KINETIC APPROACH TO IMMUNOASSAY WITH APPLICATION TO IMMUNOPRECIPITIN REACTIONS
Abstract
A new kinetic approach is described for the quantitation of antigens that react with antibodies to form precipitates. The proposed approach is evaluated using the reactions of the immunoglobulins (IgG, IgA and IgM) with their respective antibodies as model systems. The approach is based on differences in kinetic behavior of the antigen/antibody interaction, as monitored by either nephelometric or turbidimetric measurements, and the use of empirical mathematical models with nonlinear least-squares data processing methods to compute kinetic parameters that can be used to distinguish among the three regions (excess antibody, equivalence, and excess antigen) of the calibration curve. Results obtained with stopped-flow mixing and nephelometric detection for the reaction of purified IgG with antibody show that the sign of the zero-order rate coefficient computed from fits of the data to a first-order/zero-order model can be used to distinguish among the three regions of the calibration curve and to quantify IgG in the region of equivalence. A variety of measurement objectives (computed intensity change, maximum rate, and the difference in intensity at fixed time intervals) can be used to quantify IgG in regions of excess antibody or excess antigen. For IgG concentrations in excess of the expected physiological range and for data with the combined rate and regression-kinetic options, a least-squares fit of determined (y) vs prepared (x) concentrations yielded y = 1.04 x - 0.54 mg/dL, with S$\sb{\rm yx}$ = 1.44 mg/dL and r = 0.998. A comparison of nephelometric and turbidimetric responses revealed significant differences in the shapes of the kinetic profiles in the region of excess antigen. The effects of concentrations of antibody, PEG and NaCl on rates and total changes in the nephelometric and turbidimetric responses were most pronounced in this region and these results were used in subsequent optimization experiments to adjust reaction conditions to a centrifugal mixing system with turbidimetric detection. Results for turbidimetric measurements showed that a ratio of rate coefficients could be used to distinguish among the regions of the calibration curve and the first-order rate coefficient could be used for the quantitation of IgG, IgA and IgM in the region of equivalence. There was good correlation between computed concentrations by the proposed methods with reported concentrations from local hospital laboratories.
Degree
Ph.D.
Subject Area
Analytical chemistry
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