An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein

Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research
A Uhland-SmithH F DeLuca

Abstract

In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated. The receptor-antibody complex is detected with avidin-alkaline phosphatase and rho-nitrophenyl phosphate. The amount of receptor in each sample is determined by comparison with a standard curve made from purified receptor protein. This assay is highly sensitive, measuring as little as 2 fmol of receptor, and has an intra-assay coefficient of variation of 6.6% and an interassay coefficient of variation of 13.8%. The assay can be used to measure the receptor from mammalian and avian species and is independent of the presence of hormone. By eliminating the need for a radio-iodinated monoclonal antibody and incorporating the ease of a plate assay, we have a significantly improved method for measuring the vitamin D receptor protein. This paper also presents Western analysis of the antibodies used to demonstrate that they do not recognize other steroid hormone receptors.

References

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Citations

Jan 24, 2007·The Journal of Steroid Biochemistry and Molecular Biology·Hector F DeLucaMargaret Clagett-Dame
May 14, 2003·Domestic Animal Endocrinology·M A TryfonidouH A W Hazewinkel
Mar 17, 2005·Proceedings of the National Academy of Sciences of the United States of America·Kevin D HealyHector F DeLuca
Aug 6, 2003·Proceedings of the National Academy of Sciences of the United States of America·Kevin D HealyHector F DeLuca
Jan 20, 1999·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·E M ColinJ P Van Leeuwen
Sep 11, 2002·The Journal of Nutrition·M A TryfonidouH A W Hazewinkel
Mar 5, 2005·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Alexander J KarnauskasMurray J Favus
Oct 12, 2000·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·E M GardinerJ A Eisman
Jun 14, 2000·Archives of Biochemistry and Biophysics·F Jehan, H F DeLuca
Jun 11, 2002·Archives of Biochemistry and Biophysics·Matthew J Beckman, Hector F DeLuca
May 4, 2001·Prostaglandins, Leukotrienes, and Essential Fatty Acids·F LeonardM C Kruger

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