Application of the peroxidase anti-peroxidase system as an universal reagent for the two-site binding enzyme immunoassay

An indirect two-site binding enzyme immunoassay (EIA) is described, in which horse-radish peroxidase (HRP) is bound immunologically to anti-HRP IgG in the form of peroxidase anti-peroxidase complexes (PAP-complex). In this EIA both purified HRP and crude HRP had the same sensitivity due to the selective reaction of the monospecific anti-HRP IgG with the highly active HRP isoenzymes in both preparations. To obtain similar results the amount of crude HRP needed is 18 times higher than that of purified HRP. A comparison of differently composed PAP-complexes showed that only those formed in an excess of HRP yielded a highly sensitive EIA. Urea splitting of the PAP-complexes did not raise the specific activity of the enzyme. The PAP-complexes were used in an assay for quantification of the pregnancy-associated α 2-glycoprotein and compared with an indirect two-site binding EIA, in which purified HRP was covalently bound to IgG. Both test variants resulted in the same sensitivity and showed similar precision.