118 DEVELOPMENT AND EVALUATION OF A TIME-RESOLVED FLUORESCENCE IMMUNOASSAY FOR ESTRONE-1-SULFATE IN URINE AS A TOOL FOR DIAGNOSIS OF EARLY PREGNANCY IN SWINE

Abstract

Early identification of pregnancy or non-pregnancy in sows is considered very important, as the management of sows during the post service period is crucial if the breeding efficiency of a herd is to be maximized. Studies of steroid hormones in pregnant sows showed that there was a significant increase in plasma estrone-1-sulfate concentration by the 16th day of gestation, which reaches peak values between Days 23 and 30 of gestation. Since plasma estrone-1-sulfate concentrations are high between Days 23 and 30 of pregnancy, its determination has been used as a means for early pregnancy diagnosis and monitoring fertility in sows. However, the application of the method in pig farms on a routine basis remains restricted because blood sampling is difficult and disturbs the animals. The present study describes the development of a simple and reliable time-resolved fluorescence immunoassay (TR-FIA) method for the estimation of estrone-1-sulfate in swine urine, which was assessed as a means for early diagnosis of pregnancy and monitoring fertility in sows. We demonstrated cross activity between Anti-estrone-1-glucuronide antibody (Clone 155) and estrone-1-sulfate. The method is based on a direct competitive heterogeneous immunoassay by the typical procedure of competitive immunocomplex formation. For detection of estrone-1-sulfate, anti-estrone-1-glucuronide antibody (Clone 155) was first coated on polystyrene microplates, and estrone-1-sulfate was captured by the primary antibody with estrone-1-glucuronide labeled with europium. The immunocomplex was subsequently dissociated by the enhancement solution containing Triton X-100, acetic acid, and chelators. The free europium was detection by DELFIA 1420 detector (Perkin-Elmer Life Sciences, Waltham, MA, USA). The fluorescence intensity of free europium at 613 nm was proportional to the logarithm of the concentration of estron-1-sulfate in a dynamic range of 0.078~10 ng mL^–1. Intra-assay variation for estrone-1-sulfate was 4%. The limit of quantification was 100 pg mL^–1. The mean estrone-1-sulfate concentration was significantly higher in pregnant sows (15.6 ± 5.3 ng mL^–1) than in non-pregnant sows and in sows in estrus (0.74 ± 0.44 ng mL^–1). Taking the concentration of 20 pg mL^–1 as a cut-off, all cases of non-pregnant sows and sows in estrus were negative. Urine estrone-1-sulfate concentrations in pregnant sows at 23-day intervals post-service were 14~16 ng mL^–1. According to the results of our field trial, urine estrone-1-sulfate concentrations are very low during estrus and remain low in non-pregnant sows at different stages of the estrous cycle, whereas the concentration increases significantly during specific stages of pregnancy at 23-day intervals. It is concluded that the satisfactory sensitivity of the present assay in combination with the good correlation for pregnancy from the present field trial makes this method a very useful technique for early pregnancy diagnosis in swine; the simplicity of urine sampling makes also it suitable for practical use.