The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique.

The use of covalently bound peroxidase-immunoglobulin conjugate has been compared with soluble peroxidase-antiperoxidase complex as antigen in the third layer of the unlabeled antibody enzyme method. The conjugate conferred no staining. The low sensitivity of the conjugate appeared to be due in part to interference by unconjugated immunoglobulin and in part to diminution of antigenic reactivity of immunoglobulin as a result of conjugation. The triple specificity amplification inherent in the unlabeled antibody enzyme method with peroxidase-antiperoxidase complex may be lost if a peroxidase-immunoglobulin conjugate could be prepared in such a manner that staining occurs.