Coated Tube Enzyme Immunoassay: Factors Affecting Sensitivity and Effects of Reversible Protein Binding to Polystyrene

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Abstract
Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) antiserum was studied to determine conditions of maximum sensitivity. The competitive binding assay utilized showed a large increase in sensitivity with immobilized antigen levels below the levels giving rise to the maximum in the coating-antigen dilution series. The effects of reversible antigen binding to the solid phase were investigated by comparison of untreated polystyrene tubes, polystyrene tubes treated with glutaraldehyde and glass tubes activated with an aminosilane. The use of glutaraldehyde treated tubes reduced, and the use of activated glass tubes prevented the time dependent release of immobilized antigen seen with the untreated polystyrene tubes. By comparison of these solid phases, it is shown that reversible antigen immobilized in a competitive binding assay gives rise to poorer conjugate binding (three-fold), and poorer sensitivity (sixfold). A noncompetitive response was found to occur at high free antibody levels and low competing antigen concentrations. This binding behavior is moderated by the minimization of the reversible antigen immobilization.