Mutayoba B M, Meyer H H, Schams D, Schallenberger E
Institut für Physiologie, Südd. Versuchs- und Forschungsanstaft für Milchwirtschaft, Technische Universität München, Freising-Weihenstephan, FRG.
Acta Endocrinol (Copenh). 1990 Feb;122(2):227-32. doi: 10.1530/acta.0.1220227.
Using the biotin-streptavidin amplification technique, highly sensitive specific second-antibody enzyme immunoassays for determining LH in bovine plasma with long (48 h) and short (4 h) incubation periods were developed. Biotin was linked to bLH by the N-hydroxysuccinimide method and the product (biotinyl-bLH) used to bridge between streptavidin-peroxidase and the immobilised bLH antibody in competitive tests. The assays were validated and their performance compared with a radioimmunoassay currently in use. The sensitivities of the long and short incubation enzyme immunoassays (8 pg and 15 pg/well, respectively) were superior to that of 5-day incubation radioimmunoassay (100 pg/tube). Plasma interference in both assays were acceptable and volumes of 5 to 40 microliters gave parallel standard curves and comparable LH levels, 10-20 microliters plasma was sufficient to measure LH baseline levels by the long incubation enzyme immunoassay. The mean recovery of added standard bLH to plasma samples containing different endogenous LH was greater than 90% (range 91.7-112) in both assays. The intra- and inter-assay variations of both assays were less than 10 and 17%, respectively. When both enzyme immunoassay and radioimmunoassay were used to measure LH in cyclic cows, the basal levels measured by enzyme immunoassay were lower than that measured by radioimmunoassay. Enzyme immunoassay offers an attractive alternative to the lengthy radioimmunoassay in current usage, with an added advantage of employing non-isotopic label.
利用生物素-链霉亲和素放大技术，开发了用于测定牛血浆中促黄体生成素（LH）的高灵敏度特异性第二抗体酶免疫分析法，其孵育期分别为长（48小时）和短（4小时）。通过N-羟基琥珀酰亚胺法将生物素与牛促黄体生成素（bLH）连接，产物（生物素化-bLH）用于在竞争试验中介导链霉亲和素-过氧化物酶与固定化的bLH抗体之间的反应。对这些分析方法进行了验证，并将其性能与目前使用的放射免疫分析法进行了比较。长孵育和短孵育酶免疫分析法的灵敏度（分别为8皮克和15皮克/孔）优于5天孵育放射免疫分析法（100皮克/管）。两种分析方法中的血浆干扰均可接受，5至40微升的体积给出平行的标准曲线和相当的LH水平，10至20微升血浆足以通过长孵育酶免疫分析法测量LH基线水平。在两种分析方法中，向含有不同内源性LH的血浆样品中添加标准bLH的平均回收率均大于90%（范围为91.7-112）。两种分析方法的批内和批间变异分别小于10%和17%。当使用酶免疫分析法和放射免疫分析法同时测量发情周期奶牛的LH时，酶免疫分析法测得的基础水平低于放射免疫分析法测得的水平。酶免疫分析法为目前使用的冗长放射免疫分析法提供了一种有吸引力的替代方法，并且具有采用非同位素标记的额外优势。
