PREPARATION AND PROPERTIES OF ANTIGEN- OR ANTIBODY-ENZYME CONJUGATES EMPLOYED IN ENZYME IMMUNOASSAYS.

• Main Author: SITTAMPALAM, GURUSINGAM.
• Other Authors: Vemulapalli, Krishna
• Language: en
• Published: The University of Arizona. 1982
• Subjects: Immunoenzyme technique.
• Online Access: http://hdl.handle.net/10150/185171

Human immunoglobulin G-glucose oxidase conjugates (HIgG-GO) were studied in detail to establish optimum conditions under which maximum enzyme activities and immunological reactivities can be retained. These properties determine the analytical utility of these conjugates. Reactions of four different coupling reagents (glutaraldehyde, p-benzoquinone, sodium periodate and N-succinimidyl 3-(2-pyridyldithio) propionate) with glucose oxidase (GO) and human immunoglobulin G (HIgG) were investigated. Conjugates made with p-benzoquinone were found to be far superior since they retained maximum enzyme and/or immunological reactivities. Enzyme activities were determined by spectrophotometric measurements of the active flavin content of GO. This method was simple, convenient and correlated well with the standard peroxidase procedure. Rate nephelometry was established as a reliable method to measure apparent immunological reactivities of the conjugates at μg/ml levels. Hill plot treatment of the rate nephelometric data shows that the immunoprecipitin reactions may be cooperative, with two antigenic sites or steps in operation. This may explain the need for mixtures of monoclonal antibodies to observe immunoprecipitin reactions. Angular and concentration dependence of the scattered intensity during immunoprecipitin reactions was measured for the conjugated and unconjugated HIgG. From these data the weight-average-molecular weight (Ṁ) of the transient scattering complexes were estimated using Zimm plots. The Ṁ values were in the range of 2.8 x 10⁸ - 11.0 x 10⁸ daltons and were in good agreement with the theoretically predicted values. Equilibrium binding constants of the conjugates were not significantly different from that of the unconjugated HIgG (K´ₒ = 6.0 x 10⁷), showing that under equilibrium conditions the binding properties are similar. However, heterogeneity indices obtained via Sip's transformation were higher for the conjugates (a = 0.68-1.0). Such a behavior may be due to some restriction of the number and type of antigenic sites recognized by the immobilized antibodies in the presence of an enzyme label on the antigen.