Time-resolved fluoroimmunoassay (TR-FIA) of porcine relaxin.

Abstract

We developed and validated a new assay system for porcine relaxin that overcame the drawbacks of RIA by adapting time-resolved fluoroimmunoassay (TR-FIA), which was recently introduced as a non-RIA format. The assay system was a solid-phase TR-FIA based on competition for a polyclonal anti-porcine relaxin antibody between europium (Eu)-labeled porcine relaxin and test samples. Antibody-relaxin complexes were then bound to the second antibody coated on the solid phase, achieving rapid and complete separation of bound and free antigen. A standard curve was produced over the range of 1 pg/well to 1000 pg/well. Serum and corpus luteum extracts from pigs in late pregnancy exhibited inhibition curves parallel to that of the relaxin standard, whereas male pig serum caused no displacement of the labeled hormone. No cross-reactivity was seen with other hormones, such as insulin, LH, and FSH, indicating a high specificity of the assay. The sensitivity was 4 pg/well (80 pg/ml), which was high and equivalent to that of the porcine relaxin RIA. The intra-assay and inter-assay coefficients of variation were less than 3.8% and 6.7%, respectively. Recovery of porcine relaxin added to male pig serum sample averaged 103%. The advantages of this TR-FIA were that addition of tyrosine was not necessary for labeling, unlike the RIA, Eu-labeled relaxin was stable enough to allow long-term storage for more than one year, the assay was completed in only 5 h versus two to seven days for the RIA, and no special safety precautions were needed. To validate this TR-FIA, the serum relaxin concentrations during late pregnancy, parturition and early lactation were investigated in pigs. Serum relaxin levels determined by this assay were similar to those obtained previously by RIA. In conclusion, this TR-FIA could replace RIA as the method of choice for assay of relaxin.