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丝状霉形体山羊亚种血清抗体间接ELISA
检测方法的建立
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赵萍，逯忠新，储岳峰，高鹏程，贺英，石琴
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（中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 农业部畜禽病毒学
重点开放实验室，甘肃 兰州730046）
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摘要：
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利用丝状霉形体山羊亚种标准株PG3制备抗原,经纯化后作为包被抗原建立了间接ELISA方法，用于检测丝状霉形体山羊亚种血清抗体。经方阵滴定确定最佳抗原包被浓度为1.09μg/mL,血清样品最佳稀释度为1∶64，兔抗羊IgG辣根过氧化物酶结合物的最佳稀释度为1∶40000，抗原抗体最佳结合时间为1h。判定标准为样品D490nm值与标准阴性血清D490nm值之比(P/N)≥3为阳性,≤2.5为阴性，介于二者之间的为可疑。重复性和稳定性试验证明，建立的间接ELISA的稳定性和重复性良好，与间接血凝试验相比，其敏感性较高。 |
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关键词：
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间接ELISA;丝状霉形体山羊亚种;血清抗体 |
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中图分类号：S 852.62：R 446.61
文献标识码：
A 文章编号：1673-4696(2007)10-0859-04 |
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Development of an indirect ELISA for detection of serum antibodies
against Mycoplasma mycoides subsp.capri |
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ZHAO Ping,LU Zhong-xin,CHU Yue-feng,GAO Peng-cheng,HE Ying,SHI Qin
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(Key Laboratory of Animal Virology of the Ministry of Agriculture/State Key Laboratory of Veterinary Etiological
Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China) |
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Abstract: Antigen against Mycoplasma mycoides subsp. capri was prepared and purified from stan-dard strain PG3 to develop an indirect ELISA for detection of serum antibodies against M.mycoides subsp.capri. Results of chessboard titration test showed that the optimal concentration of coating antigen was 109μg/mL,the optimal dilution of serum was 1∶64,the optimal dilution of HRP-IgG was 1∶40000,and the optimal combination of antigen with antibody was for one hour.The criteria for evaluation was as follows:the serum was positive if P/N ≥3,the serum was negative if P/N≤2.5,and the serum was suspicious if P/ N is between 2.5 and 3.Results of reproducibility and stability tests confirmed that the developed assay had good stability and reproducibility.Compared with indirect haemagglutination（IHA）,the indirect ELISA was more sensitive. |
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Key words: indirect ELISA;Mycoplasma mycoides subsp. capri;serum antibody |
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