Preparation of Horseradish Peroxidase Hydrazide and Its Use in Immunoassay

Abstract

Preparation of horseradish peroxidase (HRP) hydrazide that is HRP linked to adipic acid dihydrazide (HRP-ADH) and its use in enzyme immunoassay (EIA) is described. In this new strategy, horseradish peroxidase was conjugated to adipic acid dihydrazide using a carbodiimide coupling method. The resulting HRP-ADH was then coupled to cortisol-21-hemisuccinate (Cortisol-21-HS) to prepare enzyme conjugate. The prepared cortisol-21-HS coupled ADH-HRP (Cortisol-21-HS-ADH-HRP) enzyme conjugate was used for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum samples (50 µL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 µL) were incubated for 1 h at 37°C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H₂O₂) as substrate. The sensitivity of the assay was 0.05 µg/dL and the analytical recovery ranged from 92.9 to 101.7%.

Keywords:

• HRP-hydrazide
• Direct ELISA
• Immunoassay

Acknowledgment

This Study was supported by National Institute of Health and Family Welfare, New Delhi, India. The author is grateful to Prof. M. C. Kapilashrami and Prof. Somnath Roy for their keen interest and encouragement throughout the study. Secretarial support from Shri K. K. Bhalla and technical support from Mrs. S. Bala are also gratefully acknowledged.