Antigen leakage from immunosorbents: Implications for the detection of site-directed auto-anti-idiotypic antibodies

The detection of site-directed anti-idiotypic antibodies is usually based on their ability to inhibit the binding of antigen to idiotype, in either solid- or fluid-phase radioimmunoassays. Passage of serum over antigen-coupled immunosorbents for the purpose of removing the idiotypes from complexes with putative anti-idiotypic antibodies resulted in the release of significant amounts of antigen into the effluents. Normal sera or even isotonic buffers were similarly contaminated with antigen. The amount of antigen released ranged between 200–400 ng/ml, well in excess of the minimal amount required in the inhibition assay. Antigen was detected in effluents passed over a number of antigen coupled-matrices and even in affinity-purified antibody preparations obtained by elution from immunosorbents coupled with dinitrophenyl (DNP)-protein conjugates. Attempts to stabilize the antigen-coupled matrices with glutaraldehyde resulted in a perceptible but insufficient decrease in the amount of antigen released. In the case of anti-hapten antibodies, antigen interference was circumvented by utilizing monovalent haptens such as DNP-lysine coupled to the immunosorbent either directly or through a spacer arm. In the case of protein antigens, the leakage was almost completely prevented by preparing glutaraldehyde-polymerized immunosorbents directly from solution.