Carbodiimide or Periodate Method to Prepare Peroxidase Hydrazide for Its Use in Immunoassay

Abstract

Peroxidase hydrazides were prepared by conjugating horseradish peroxidase (HRP) to adipic acid dihydrazide (ADH) by carbodiimide or periodate oxidation method. The resulting HRP hydrazides (ADH–HRP) were conjugated to cortisol‐21‐hemisuccinate (cortisol‐21‐HS) by forming diimide bonds using the N‐hydroxysuccinimide (NHS) carbodiimide mediated reaction. The prepared cortisol‐21‐HS–ADH–HRP enzyme conjugates were utilized 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 sample (50 µL), along with 100 µL of cortisol‐21‐HS–ADH–HRP enzyme conjugate (ADH–HRP used is prepared by either carbodiimide or periodate oxidation method), was incubated for 1 hr at 37°C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H₂O₂) as substrate. The sensitivity, specificity, and recovery of the assays were found to be identical when ELISAs were employed with cortisol enzyme conjugates prepared by conjugating cortisol‐21‐HS to HRP hydrazide, made either by the carbodiimide method or periodate oxidation method.

Keywords:

• HRP‐hydrazide
• Direct ELISA
• Immunoassay

Acknowledgments

This study was supported by the National Institute of Health and Family Welfare, New Delhi, India. We are 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.