Sandwich immunoassay of small molecules. I. Investigation with testosterone as model hapten

A new strategy has been developed for enzyme immunoassay of small molecules. The method is based on the formation and estimation of the complex Ab.H-H.Ab* where Ab and Ab* are immobilised and labeled antibodies respectively and H-H is a synthetic bis-analogue of the hapten H. The amount of the complex formed is inversely related to the concentration of free hapten present. The estimation of the sandwich complex increases the sensitivity and the specificity of the assay compared to conventional assays. Using the N,N'-diamide of testosterone-3-(O-carboxymethyl)oxime with ethylenediamine and a polyclonal rabbit anti-testosterone antibody (for both labeling and immobilisation) a sensitivity of 100 fg/well (3.5 x 10(-16) mol) was obtained for testosterone. The cross-reactivity of 5 alpha-dihydrotestosterone was 16% compared to 27% obtained by conventional competitive assays.