The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten

BackgroundIn hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays.MethodAntibodies were generated against cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3-O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated.ResultThe incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids.ConclusionSpacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.