Development of a Homologous Radioimmunoassay for Rat Relaxin*

Abstract

Highly purified rat relaxin has been radioiodinated to specific activities of approximately 100 μCi/μg with the Bolton and Hunter reagent [N-succinimidyl 3-(4-hydroxy-5-[¹²⁵I] iodophenyl) propionate]. A rabbit antirat relaxin serum, applicable in a final dilution of 1:100,000, was developed in a rabbit using unconjugated highly purified rat relaxin. A specific and precise double antibody RIA for rat relaxin sufficiently sensitive to routinely measure from 32–2000 pg rat relaxin was developed. Using this RIA, relaxin immunoactivity levels in extracts of pregnant rat ovaries were found to rise from 0.8 μg/geQ ovarian fresh tissue on day 8 of pregnancy to 723 μg/geq ovarian fresh tissue on day 20 of pregnancy and then to drop precipitously to 6 μg/geq ovarian fresh tissue on day 1 of lactation. Consistent with the occurrence and relative levels of relaxin in the ovarian extracts, levels of relaxin in pregnant rat serum were less than 2 ng/ml on day 10 of pregnancy, approximately 150 ng/ml on days 20 and 22 of pregnancy, and 12 ng/ml on day 2 of lactation.