The Development of a Sensitive Enzymeimmunoassay for the Determination of Estrone and Estradiol-17.BETA. in Bovine Blood Plasma based on the Same Homologous Combination with Antiserum and Steroid-Enzyme Conjugate

Abstract

ABSTRACT. The development of a sensitive enzymeimmunoassay (EIA) for the determination of estrone (E1) and estradiol-17β (E2β) in bovine plasma is described. The assay is a homologous double-antibody EIA with E2β 17hemisuccinate (HS) as hapten for the immunoreactive reagent. The antiserum was raised against E2β 17HS bovine serum albumin conjugate in the rabbit, and E2β 17HS-horseradish peroxidase was used as steroid-enzyme conjugate. Each estrogen EIA was distinguished only by using the each working stan dard and sample for the EIA. Bovine plasma E1 and E2β were extracted and purified before EIA. The antiserum was used at 1:1,750,000 dilutions for EIA. Estrone and E2β showed high cross-reactivity with the antiserum (E1: 350.7%, E2β:100%). The sensitivities were <0.03 pg/well for E1 and <0.12 pg/well for E2 β. Recovery rates of E1 and E2β added to bovine blood plasma were 94.5% and 93.9%, respectively. The precision for EIA of estrogens was below 9.7%. The profiles of either estrogen as determined by EIA corresponded closely well with follicle dynamics in the cow during the estrous cycles and with placental function in pregnant animals. In co nclusion, our new EIA can be applied with sufficient sensitivities, recovery and precision for the routine analysis of E1 and E2β concentrations in bovine plasma. KEY WORDS: cattle, enzymeimmunoassay, estrone, estradiol-17β.

Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET

“…Plasma P 4 concentrations were determined by using a double-antibody enzyme immunoassay as described previously [35]. Briefly, 100 μl of plasma sample was extracted twice with 2 ml diethyl ether in a glass tube.…”

Multilevel regulation of steroid synthesis and metabolism in the bovine placenta

“…The dynamic changes in the concentration of oestradiol follow similar patterns to that of oestrone for most of pregnancy, although the levels are only about one-tenth to oneeighth of the concentration of oestrone (Patel et al, 1999;Takenouchi et al, 2004). The difference is at the beginning of pregnancy, when the concentration of oestradiol is initially high and exceeds that of oestrone; after the establishment of pregnancy, it decreases to a minimum at about day 25-30 (Patel et al, 1999).…”

“…From day 250 to 2-5 days pre-partum, the concentration of oestrone can increase up to 10-fold, and at a more rapid rate than in the previous period (Hoffmann et al, 1997;Patel et al, 1999). Oestrone concentrations reach a peak of around 2,500 pg/ml or 10 nmol/L between 2 and 5 days pre-partum (Smith et al, 1973;Hoffmann et al, 1997;Patel et al, 1999;Takenouchi et al, 2004).…”

“…Progesterone and oestrogen profiles in bovine pregnancy. Schematic representation of the dynamic changes in the concentrations of progesterone, oestrone, and oestrone-sulphate in maternal blood during bovine pregnancy (adapted with data from Stabenfeldt et al, 1970;Edqvist et al, 1973;Smith et al, 1973;Echternkamp, 1992;Dobson et al, 1993;Hoffmann et al, 1997;Patel et al, 1999;Takenouchi et al, 2004). pregnancies with two or more foetuses than in pregnancies with a single foetus (Echternkamp, 1992;Dobson et al, 1993;Patel et al, 1999;Hikaro et al, 2002). The profiles represented in Figure 1 and described below are for singleton pregnancies.…”

Aspectos morfológicos, vasculares e endócrinos de prenhezes produzidas por técnicas de reprodução assistida em bovinos

“…Progesterone, which is produced by the placenta and the corpus luteum (6-10), decreases abruptly from 2.5 days before parturition caused mainly by prepartal luteolysis (11). Corticosteroids, and particularly cortisol, are primarily regulated by the mature fetal hypothalamus-pituitary-adrenal axis and gradually increase over the last month of gestation (12,13), enhancing the conversion of androgens to estrogens via aromatase within the placenta (14)(15)(16). The estrogens that are synthesized in the placental membranes include 17β-estradiol and estrone (17), which play important roles in the onset of parturition by regulating uterine contractions (18), cervical dilation (19), and the subsequent opening of the birth canal.…”