Development of Isotopic and Non‐Isotopic Microwell Based Immunoassays for hCG Using ¹²⁵I and Biotin Labeled hCG

Abstract

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 µL of standard or samples along with 100 µL of biotinylated hCG were incubated for 1 hour at 37°C. After incubation, wells were washed and 100 µL of streptavidin‐HRP conjugate was added to each well and incubated again for a half hour at 37°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 µL of standard or samples along with 100 µL of ¹²⁵I‐hCG were incubated for 1 hour at 37°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).

Keywords:

• hCG
• Immunoassay
• Micro‐well
• Isotopic
• Non‐isotopic
• Avidin
• Biotin
• ELISA
• RIA

Acknowledgments

The authors are grateful to HOD, RBM, and the Director of NIHFW for their support to conduct the work. The authors also express sincere gratitude to Prof. Somnath Roy for his valuable suggestions throughout the study. Technical support from Mrs. S. Bala is also gratefully acknowledged.