A CHEMIFLUORESCENT IMMUNOASSAY FOR THE DETERMINATION OF MARINOBUFAGENIN IN BODY FLUIDS

Abstract

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG–bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Keywords:

• bufodienolides
• cardiac glycosides
• diagnostic
• hypertension
• immunoassay
• marinobufagenin

ACKNOWLEDGMENTS

This work was supported (in part) by grants-in-aid from the Joe W. and Dorothy Dorsett Brown Foundation, New Orleans, LA, Dialysis Clinic, Inc., Nashville, TN, from Scott & White Healthcare, Inc., Temple, TX, the Texas A&M Health Science Center College of Medicine, College Station, TX (to JBP), and by grants from the USDA (grant 2008-35204-04554 to LRB), from the National Institutes of Health (RO1 GM086307 to DR), grants R01 CA 90441-01-04, 5R01, and CA090441-07-08 from the Division of Cancer Treatment and Diagnosis, NCI, DHHS (GRP), and the Welch Foundation (A-1280 to DR). In addition, funding from the College of Science, Texas A&M University (for JL) is gratefully acknowledged. The authors thank Lonnie Doyle for the production of this article.