Quantitation of serum amyloid P component by an enzyme-linked immunoassay

A solid phase enzyme-linked immunoassay (ELISA) for the murine acute-phase reactant, serum amyloid P component (SAP), was developed. The assay is based on our finding of a calcium-dependent binding of SAP to trinitrophenyl-conjugated proteins. The wells of polystyrene microtiter plates are coated with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), then incubated with SAP-containing samples. The amount of SAP is determined by indirect ELISA, wells being sequentially incubated with rabbit anti-SAP antiserum and horseradish peroxidase-linked donkey anti-rabbit IgG conjugate. The limit of sensitivity of the assay is 0.6 ng/ml SAP. Comparison with data on sera obtained by rocket immunoelectrophoresis assay yielded a correlation coefficient of 0.89. The binding of SAP to TNP-KLH was inhibited by calcium chelators and low concentrations of non-ionic detergents. Chromatography of serum on TNP-Sepharose provided a efficient and simple way of purifying SAP. The assay was also adapted for the quantitation of human SAP. The use of the assay in studying the binding specificities of SAP and its physiological role is discussed.