Preparation of horseradish peroxidase hydrazide and its use in immunoassay

Journal of Immunoassay & Immunochemistry
T G Shrivastav

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 microL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The sensitivity of the assay was 0.05 microg/dL and the analytical recovery ranged from 92.9 to 101.7%.

References

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Citations

Mar 14, 2009·Nature Protocols·Ramadan A AbukneshaZakeya Baalawy
Oct 6, 2004·Journal of Immunoassay & Immunochemistry·T G Shrivastav

Related Concepts

Sopolkort
Adipic dihydrazide
Adipates
Metazoa
Antibody Specificity
Enzyme-Linked Immunosorbent Assay
Alpha-Peroxidase
Hydrocortisone, (9 beta,10 alpha,11 alpha)-Isomer
Indicators
Chinchilla Rabbits

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