摘要
应用孕马血清促性腺激素(PMSG)纯品主动免疫家兔制备抗血清,用辣根过氧化物酶标记葡萄球菌A蛋白(SPA-HRP)代替酶标IgG抗体,建立了PMSG抗原-抗体-SPA-HRP酶联免疫吸附测定方法。方法学鉴定证明,该法灵敏度高(1.8miu/孔),重复性好(板内和板间变异系数分别为4.7％和8.6％,样本数均为27);PMSG抗血清的特异性和方法的准确性均达到放射免疫测定中世界卫生组织所要求的质量控制指标。应用该法测定水牛注射PMSG2000iu后每天的PMSG残留量,证明第一天最高,平均为69.4±1.8miu/ml,以后逐渐降低到第11天的9.5±0.7miu/ml。
An Antigen-Antibody-SPA-HRP Enzyme-Linked Immunosorbent Assay ( ELISA ) for measurement of PMSG in blood serum of swamp buffalo cows treated with PMSG .using a horseradish peroxidase staphy-lococcal protein A conjugate ( SPA-HRP ) in stead of enzyme labeled antibody against rabbit IgG and an antiserum raised against pure PMSG has been developed. PMSG was, firstly, coated on a 96 well microtiter-plate. Secondly, the mixture prepared beforehand with PMSG standard references ( or blood samples ) and PMSG antiserum was added into the microtiter-plate coated with PMSG. The plate was incubated at 37℃ for one hour and was washed 5 times. To each well, 100μl of SPA-HRP ( 1:50) was added. The plate was incubated for one hour ( 37℃ ) and washed 5 times. Finally, the substrate of enzyme ( ortho-phenylendiamine) was added and incubated for 0.5 hour in the dark. The substrate reaction was stoped by an addition of 25 μl of 3 mol/l H2SO4 to each well. After mixing the optical density value each wvell was measured at 492 nm with an ELISA reader. This method was specific and had the sensitivity of 1,8 miu/well. The coefficients of variation among and between plates in an assay were 4.7% and 8.6% respectively. By the method, PMSG level was still measurable in blood serum of buffalo cows about 11 days after an injection of 2 000 iu PMSG.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
1991年第1期9-14,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金
