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Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.

The irreversible binding of penicillin to serum protein was investigated by incubating 14C-benzyl-penicillin with reconstituted lyophilized human serum and dialyzing away the non-bound penicillin. Binding appeared in each protein fraction as was observed by polyacrylamide gel electrophoresis and liquid scintillation counting.The nature of penicillin binding was investigated by preincubating reconstituted human serum with specicific blocking reagents.Ball State UniversityMuncie, IN 47306