Henderson K M, Camberis M, Simmons M H, Starrs W J, Hardie A H
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
J Steroid Biochem Mol Biol. 1994 Aug;50(3-4):189-96. doi: 10.1016/0960-0760(94)90028-0.
The characteristics of antigen- and antibody-coated enzymeimmunoassay (EIA) formats to measure oestrone sulphate (OS) were studied using a murine monoclonal antibody as the primary antibody. In an antigen-coated format the most sensitive EIA (9 fmol/well) was achieved using 6-ketoestrone-6-O-carboxymethyloxime (OCMO) coupled to bovine serum albumin (BSA), as the coating antigen, and horseradish peroxidase (HRP), as the enzyme label. In an antibody-coated format, comparable sensitivity could be achieved using HRP conjugated to either OCMO, oestrone-3-glucuronide (OG) or oestrone-3-hemisuccinate (OHS) as the steroid 'tracer'. In both the antigen- and antibody-coated formats replacing HRP with alkaline phosphatase (AP) markedly aggravated the assay sensitivity. The antigen-coated EIA format was used to measure OS concentrations in cow's milk directly without an initial defatting step, and a progressive increase in OS concentrations in milk as pregnancy progressed was observed. Median OS concentrations rose from 1.1 nmol/l at days 70-99 of pregnancy (n = 44) to 3.2 nmol/l at days 140-160 (n = 92). Oestrone sulphate concentrations in milk from non-pregnant cows (n = 51) ranged from non-detectable to 1.3 nmol/l with a median value of 0.4 nmol/l. Only 5% of cows 120 or more days pregnant had milk OS concentrations within the range of values found in milk from non-pregnant cows. Accurate discrimination of non-pregnant and pregnant cows can thus be achieved on the basis of OS concentrations in milk samples taken at least 120 days after mating/insemination. This EIA for OS may have a role in the dairy industry as an alternative non-invasive means of determining pregnancy status in cows.
使用鼠单克隆抗体作为一抗，研究了抗原包被和抗体包被的酶免疫分析（EIA）形式用于测定硫酸雌酮（OS）的特性。在抗原包被形式中，使用与牛血清白蛋白（BSA）偶联的6-酮雌酮-6-O-羧甲基肟（OCMO）作为包被抗原，辣根过氧化物酶（HRP）作为酶标记，可实现最灵敏的EIA（9 fmol/孔）。在抗体包被形式中，使用与OCMO、雌酮-3-葡萄糖醛酸苷（OG）或雌酮-3-半琥珀酸酯（OHS）偶联的HRP作为类固醇“示踪剂”，可实现相当的灵敏度。在抗原包被和抗体包被形式中，用碱性磷酸酶（AP）替代HRP均显著降低了分析灵敏度。抗原包被的EIA形式用于直接测量牛奶中的OS浓度，无需预先脱脂步骤，并且观察到随着妊娠进展牛奶中OS浓度逐渐升高。妊娠70 - 99天（n = 44）时，牛奶中OS浓度中位数从1.1 nmol/l升至妊娠140 - 160天（n = 92）时的3.2 nmol/l。未怀孕奶牛（n = 51）的牛奶中硫酸雌酮浓度范围为不可检测至1.3 nmol/l，中位数为0.4 nmol/l。怀孕120天或更长时间的奶牛中，只有5%的牛奶OS浓度在未怀孕奶牛牛奶中发现的值范围内。因此，根据配种/授精后至少120天采集的牛奶样本中的OS浓度，可以准确区分未怀孕和怀孕奶牛。这种用于OS的EIA作为一种替代的非侵入性方法来确定奶牛的妊娠状态，可能在乳制品行业中发挥作用。
