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.

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

“…It probably also labeled the N-terminus of native boar INSL3, comprising the B-C-A single-chain form, which is made up of 111 amino acid residues. The TR-FIA procedure was performed according to our protocol (Ogine et al 1999). Briefly, 50 ml of standard or samples was added to goat anti-rabbit immunoglobulin G (IgG) coated microplates (Perkin-Elmer) together with 100 ml of anti-INSL3 antiserum (1:5000).…”

Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy

“…The CL starts to synthesize this hormone in the early stage of pregnancy, and the relaxin level increases progressively and slowly pregnancy, after which a marked decline occurs around the onset of parturition (Anderson et al 1973). These changes coincide with the changes of relaxin levels in the blood (Sherwood et al 1975;Anderson et al 1983;Ogine et al 1999) and with the appearance and accumulation of electron-dense cytoplasmic granules in the luteal cells (Belt et al 1971), which have been identified as the site of relaxin storage (Kendall et al 1978;Fields and Fields 1985;Kohsaka et al 1992). Since the cellular level of a particular mRNA is usually a central factor in the resultant level of the synthesis of its protein, the relaxin mRNA level in the CL might be expected to change in parallel with the relaxin level during pregnancy.…”

Application of time‐resolved fluorometry to immunoassays for bovine reproductive hormones

“…Tr‐FIA are based on a single antibody binding to a hormone, whereas Tr‐IFMA are based on a pair of antibodies binding to a specific part of a hormone molecule. Several groups have described Tr‐FIA or Tr‐IFMA for peptide hormones and growth factors that control reproductive activities in domestic animals, including relaxin (Ogine et al . 1999; Kohsaka et al .…”

“…Tr-FIA are based on a single antibody binding to a hormone, whereas Tr-IFMA are based on a pair of antibodies binding to a specific part of a hormone molecule. Several groups have described Tr-FIA or Tr-IFMA for peptide hormones and growth factors that control reproductive activities in domestic animals, including relaxin (Ogine et al 1999;Kohsaka et al 2003), insulin (Løvendahl & Purup 2002), gonadotropins (Kaneko et al 2002a), inhibins (Kaneko et al 2002a(Kaneko et al ,b, 2003, interleukin-6 (Ishikawa et al 2004), growth hormone (Sugino et al 2002;Miura et al 2004) and ghrelin (Sugino et al 2002;Miura et al 2004). However, in general, fewer studies have utilized time-resolved fluorometry in comparison with radiometry.…”