A sensitive and simplified receptor assay for 1,25(OH)₂ vitamin D

Abstract

A sensitive receptor assay for 1,25(OH)₂ vitamin D using 2.5–5 ml plasma and a small incubation volume (<150 μl) is described. The standard curve is plotted as free/total counts vs in-assay ligand concentration and is fitted to a law of mass action model. The conditions in the assay are: pH 7.4 (0.05 mol/l phosphate buffer), 11 % v/v ethanol concentration, and 1 g/l total protein concentration. The reaction of the receptor protein and ligand is satisfactorily complete after 2.5 h at 25°C. The bound fraction is precipitated by 40% polyethylene glycol in ethanolic phosphate buffer, separated by centrifugation, and the supernatant drawn off and counted. The radioactivity used to trace the binding reaction is part of that originally added to the plasma.

There were no interferences in the assay from vitamin D₃, 25(OH) vitamin D₃ or 24,25(OH)₂ vitamin D₃. After further purification by high pressure liquid chromatography there was no reduction in the 1,25(OH)₂ vitamin D values measured directly after LH-20 chromatography.

The plasma levels in 20 normal men and 19 women were 106 ± 36 pmol/1 (mean ± SD) and 105 ± 40 pmol/l, respectively. These results compare well with those reported from other centres. The intra-assay coefficient of variation in the useful range of measurement is about 11%.

Key Words:

• 1,25(OH)₂ vitamin D
• man
• plasma
• receptor assay