Received on ,
Accepted on ,

Automation and Analytical Techniques

Monoclonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study

¹ Unilever Corporate Research, Sharnbrook, United Kingdom
² Folkhalsan Research Center, Institute for Preventive Medicine, Nutrition and Cancer and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland
³ Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
⁴ Department of Human Nutrition, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark
⁵ Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel
⁶ School of Medicine, Health Policy & Practice, University of East Anglia, Norwich, United Kingdom

^* To whom correspondence should be addressed. E-mail: Duncan.Talbot{at}Unilever.com.

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women.

Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction.

Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA, and gas chromatography-mass spectrometry showed correlations (r, 0.911-0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied.

Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.