Abstract: An enzyme immunoassay for detecting an antigen in a biologic fluid or tissue which comprises contacting the fluid or tissue with an antibody specific for the antigen under binding conditions, at least one of the fluid or tissue and antibody having a solid component, contacting the resulting solid with a conjugate bindable with the antibody under binding conditions and determining the enzyme activity of the resulting solid phase is described. The conjugate is of peroxidase and an allergen, non-immunoglobulin protein or primary amino group containing drug having an average of 2-3 molecules of peroxidase per molecule of substance with an average molecular weight of about 30,000 daltons, prepared by reacting peroxidase previously treated with phenyl isothiocyanate and oxidized to form aldehyde groups with the substance to form a Schiff's base which is titrated with a reducing agent to form a stable conjugate.

Abstract: Horse radish peroxidase (HRP) is treated with phenyl isothiocyanate (PITC) to block the free amino groups on the enzyme. The PITC derivative of HRP is treated with periodate to oxidize the carbohydrate moiety on the enzyme, thus generating aldehyde groups. Gamma G globulin fraction (IgG) purified from an anti-human IgE serum is conjugated to the peroxidase-aldehyde by formation of a Schiff's base between the aldehyde group on the enzyme and the amino groups on the IgG. The Schiff's base is stabilized by reduction using the optimal amounts of sodium borohydride determined by tiration. A stable HRP-anti IgE IgG conjugate prepared thus is employed in a solid phase enzyme immunoassay for the detection of allergen specific IgE. The results of this assay can be used to determine a safe initial hypersensitization dosage level.