A Sensitive and Specific Radioimmunoassay for Oxytocin

Publisher Website
Google Scholar
Abstract
Recently, measurements of oxytocin content in serum have been reported from several institutes using radioimmunoassay (RIA) techniques. But these assay values showed significant differences, and the plasma levels of this hormone related to pregnancy and labor are still unclear. In order to clarify those problems, a sensitive and specific RIA system has been developed, and moreover, in this RIA system, any purification or extraction procedures of plasma samples were unnecessary. Antiserum against oxytocin was produced in New Zealand white rabbits with multiple intradermal and intrasplenal injections of oxytocin-bovine serum albumin (BSA) complex with complete Freund's adjuvant. For the coupling of oxytocin with BSA, water soluble carbodiimide was used. After twelve weeks from the first immunization, antiserum capable of binding with 45-50% of125 I-oxytocin at a final dilution of 1 : 72000 was obtained. Iodination of synthetic oxytocin was done with the chloramine T method with some modifications, and the purification of iodinated oxytocin was achieved by using a Sephadex G-25 fine gel filtration (0.8 X 10 cm) eluted with 0.1M acetic acid. Further purification with a Dowex ion exchange column chromatography gave no benefit on this assay system. In the RIA procedure, 125I-oxytocin was added after a 24 hour-preincubation, and a double antibody method was used for the separation of B/F hormones. All measurements were done in duplicate, and 0.05M phosphate buffer pH 7.5 containing 0.01M EDTA-2Na, 0.015M NaN3, 2.5 X 10-5M o-phenanthlorine and 0.1% human serum albumin was used. A linear dose response curve was obtained at an oxytocin concentration between 250 μU/ml and 16 μU/ml, and the minimal detectable dose was 2 μU/ml. Cross-reactivities with Arg-vasopressin and Lys-vasopressin were less than 0.2%. The values of inter-assay coefficient of variation obtained from five different assays with two pooled plasma were 18.6% and 15.8%. The values of intra-assay coefficient of variation obtained from five different concentrations of pooled plasma were less than 4.9%. The recovery rate was improved with the immobilization of plasma samples, and in this RIA system, anti-oxytocin serum, normal rabbit serum and anti-rabbit gamma globulin goat serum as well as plasma samples were used after immobilization. The serial dilution of pregnant women's plasma gave reasonable assay values. From these results, this RIA system is thought to be satisfactory for measuring oxytocin in plasma. Blood samples were obtained from normal pregnant women at Osaka University hospital at 2-3 pm. Sampling was done from the antecubital vein with a previously cooled and heparinized syringe, and the blood was immediately mixed with an ice cold solution, containing 0.01M EDTA and 2.5 X 10-5M o-phenanthlorine for the inhibition of oxytocinase activity. After immobilization, the plasma samples were kept in a deep freezer (-20°C) and in this condition no change was observed in the assay values at least within one month. The plasma oxytocin levels during normal pregnancy, evaluated by this RIA system, showed a gradual rise towards the estimated delivery date, and the mean value at the 10th month of pregnancy was 12.5 μU/ml.

This publication has 0 references indexed in Scilit: