The development of a radioimmunoassay for Tamm--Horsfall glycoprotein in serum

Abstract

The Tamm-Horsfall glycoprotein prepared by salt precipitation from urine was found to comprise a heterogeneous collection of aggregates. These could be disaggregated with 8m-urea, following which chromatography on a column of Bio-Gel A.15m yielded a homogeneous glycoprotein of mol.wt. 73000 together with several unidentified impurities. Gel filtration of normal plasma showed the glycoprotein to exist predominantly in a form that is eluted identically with the purified preparation. In one case, material of higher molecular weight was also detected. The purified glycoprotein was used to develop a rapid specific radioimmunoassay for its measurement in human serum or plasma by the use of the Tamm-Horsfall glycoprotein, labelled with (125)I by the chloramine-t method as the tracer, an antiserum raised in rabbits, and separation of the bound and free fractions by a second antibody covalently linked to magnetizable particles. Parallelism was demonstrated between the standard preparation and samples. Recovery of added standard to serum varied between 99 and 109%. Total assay time was less than 4h with an intra-assay and inter-assay coefficient of variation of less than 10%. There were no significant differences in the ranges covered with regard to either age or sex, and no circadian rhythm was observed in normal subjects. A physiological range of 70-540ng/ml was established based on serum samples from 95 subjects with normal renal function, as defined by their serum creatinine and urea concentrations. No Tamm-Horsfall glycoprotein was detected in the serum of six anephric patients.