The Development of a Radioimmunoassay for the Measurement of Urinary Tammhorsfall Glycoprotein in the Presence of Sodium Dodecyl Sulphate

Abstract

1. A specific and quantitative radioimmunoassay was developed for the measurement of low concentrations of human and rabbit Tamm—Horsfall glycoprotein in the presence of other proteins. Antibody-coated tubes were used as a solid phase in the assay and the optimum antibody concentration and duration of antibody coating were established. 2. Pure Tamm—Horsfall glycoprotein was labelled with ¹²⁵I and, because of its apparent susceptibility to radiation damage, was labelled at weekly intervals. 3. Sodium dodecyl sulphate, an ionic detergent, was included in the assay at a final concentration of 0.0005% to disaggregate the glycoprotein. An overnight preincubation step in the presence of the detergent was necessary before the disaggregated glycoprotein solutions were allowed to react with the antibody. Pretreatment of the tracer with detergent was not necessary. 4. Two glycoprotein standards were prepared fresh for each assay from freeze-dried material. The average linear range of the assay was between approx. 150 ng/ml and 2.5 μg/ml. Albumin was only shown to interfere with the assay at concentrations greater than 100 μg/ml. 5. Urines were dialysed against water for 3 days before assay to remove inhibitory material. Urines were never frozen as this was found to affect the assay. 6. A recovery experiment showed that the pure freeze-dried standard behaved in an immunologically identical way to the urinary glycoprotein. 7. Human Tamm-Horsfall glycoprotein cross-reacted with guinea-pig anti-(rabbit Tamm—Horsfall) antiserum and rabbit Tamm—Horsfall glycoprotein cross-reacted with guinea-pig anti-(human Tamm—Horsfall) antiserum, but not with rabbit anti-(human Tamm—Horsfall) antiserum. This showed a partial immunological identity between Tamm-Horsfall glycoprotein from humans and rabbits which was only evident when the antiserum was raised in a third species. 8. The excretion rate of Tamm—Horsfall glycoprotein in normal humans was found to be 48.1 ± 9.6 (SD) mg/24 h for males and 50.5 ± 14.8 (SD) mg/24 h for females. The mean excretion rate of the glycoprotein in New Zealand White rabbits was 34.8 ± 7.9 mg/24 h.