Preparation of horseradish peroxidase hydrazide and its use in immunoassay.
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%.
Shrivastav
Tulsidas G
TG
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India. nihfw@delnet.ren.nic.in
D000272:Adipates / Q000302:isolation & purification*; D000818:Animals; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D006735:Horseradish Peroxidase / Q000302:isolation & purification*; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification / Q000592:standards; D007202:Indicators and Reagents; D011817:Rabbits; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000272:Adipates; D007202:Indicators and Reagents; D006735:Horseradish Peroxidase; C007133:hydrocortisone hemisuccinate; C010011:adipic dihydrazide; D006854:Hydrocortisone