Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine

Abstract

Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation form normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulfate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtiter plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay [RIA] for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the RIA to have a sensitivity of at least 5 ng/ml, with linearity between 30-600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 .+-. 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 .+-. 44.1 mg.