Immunoradiometric Assay (IRMA) for Human Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) Using Common Avidin Solid Phase

Abstract

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with ¹²⁵I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0–200 mIU/mL) and analytical recovery (87–110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).

Keywords:

• Avidin-Biotin interaction
• Biotinylation
• FSH
• LH bicarbonate buffer
• pH 8.4

Acknowledgments

The authors wish to thank Dr. N. Ramamoorthy, Chief Executive, Board of Radiation and Isotope Technology (BRIT), for continuous encouragement and helpful discussions throughout this work and Dr. M. R. A. Pillai, Senior General Manager (MBPP), BRIT for his enthusiastic support.