ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

Abstract

Abstract The introduction of spacers in coating steroid protein complex and / or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers between enzyme and prednisolone (PSL) moiety having atomic length 3 to 10 and studied their influence on functional parameters such as sensitivity and specificity of PSL enzyme immunoassays. In this study, four enzyme conjugates of PSL-21-HS and HRP were prepared by N-Hydroxy Succinimide mediated carbodiimide reaction, these were PSL-21-HS-adipic acid dihydrazide (ADH)-HRP, PSL-21-HS- carbohydrazide (CH)-HRP, PSL-21-HS-ehylenediamine (EDA)-HRP and PSL-21-HS- urea (U)-HRP. The assays were developed using these enzymes conjugate with antibody raised against PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of bridge in enzyme conjugate were 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL and 0.018 ng/mL with ADH, CH, EDA and urea as spacer respectively. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity and showed cross-reaction with less number of steroids. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32-102.50 %. The intra and inter assay CV % was < 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study, suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.