Steric hindrance enzyme immunoassay (SHEIA); a novel method in enzyme immunoassay.

We have developed a new method for separation of antibody bound and unbound enzyme conjugates. The technique as applied to the assay of choriomammotropin involves the use of beta-D-galactosylamine bound to agarose to separate the unbound choriomammotropin-beta-galactosidase conjugates for antibody bound conjugates. When beta-galactosidase was conjugated with choriomammotropin using the N-hydroxy-succinamide ester of m-maleimidobenzoic acid the affinity of the enzyme conjugate to beta-D-galactosylamine attached to agarose diminished markedly following incubation with antibody. In a typical enzyme immunoassay of choriomammotropin, 5 microliter of swelled affinity gel per tube was required to precipitate unbound enzyme following one hour gentle shaking at room temperature. Choriomammotropin antibody was used at titer of 1:1,000. The standard curve for the assay was adjusted to cover a range of 0-10 mg/l with maximum sensitivity between 1-4 mg/l.