Development of isotopic and non-isotopic microwell based immunoassays for hCG using 125I and biotin labeled hCG.
Isotopic and non-isotopic immunoassays of hCG, based on the principle of competitive inhibition, using micro-well as solid support and 125I and biotin as labels for hCG, have been developed. In both the assays, rabbit polyclonal antibody was immobilized onto micro-wells. In the non-isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of biotinylated hCG were incubated for 1 hour at 37 degrees C. After incubation, wells were washed and 100 microL of streptavidin-HRP conjugate was added to each well and incubated again for a half hour at 37 degrees C. Bound enzyme activity was measured using tertramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. In the isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of 125I-hCG were incubated for 1 hour at 37 degrees C. The bound radioactivity was measured using a gamma counter. The sensitivities of the non-isotopic and isotopic assays were 0.12 IU/mL and 0.1 IU/mL, respectively. The intra- and inter-assay CVs for both the assays were less than 12.3%. There was a good correlation between the developed non-isotopic and isotopic immunoassays (r = 0.97, n = 20).
Basu
Anupam
A
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India.
Shrivastav
Tulsidas G
TG
Maitra
Saumen Kumar
SK
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D006063:Chorionic Gonadotropin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007457:Iodine Radioisotopes; D011817:Rabbits; D011863:Radioimmunoassay / Q000295:instrumentation / Q000379:methods
D006063:Chorionic Gonadotropin; D007457:Iodine Radioisotopes; D001710:Biotin