Preparation of horseradish peroxidase hydrazide and its use in immunoassay

Journal of Immunoassay & Immunochemistry
T G Shrivastav


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%.


Oct 1, 1979·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·H G Rennke, M A Venkatachalam
Jan 1, 1990·Methods in Enzymology·E A Bayer, M Wilchek
Dec 1, 1974·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·P K Nakane, A Kawaoi
May 11, 2000·Journal of Immunoassay·A Basu, T G Shrivastav
Jan 1, 1948·Helvetica Chimica Acta·A STOLL, E SEEBECK


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

Related Concepts

Adipic dihydrazide
Antibody Specificity
Enzyme-Linked Immunosorbent Assay
Hydrocortisone, (9 beta,10 alpha,11 alpha)-Isomer
Chinchilla Rabbits

Related Feeds

Antibody Specificity

Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.