Impact of bridge heterology on functional parameters of ELISA for 17 α-methyltestosterone

In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testo...
Source: Journal of Immunological Methods - Category: Allergy & Immunology Authors: Source Type: research