猴B病毒抗体ELISA检测试剂盒的制备及准确性评价
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摘要
目的:以金标准试剂盒为参照,对新研制的猴B病毒抗体ELISA检测试剂盒进行准确性评价,以期为我国实验灵长类动物产业提供优质廉价的检测试剂。方法:利用3个批次共表达猴B病毒gD和g B蛋白的细胞上清分别制备ELISA检测试剂盒,与金标准试剂盒(VRL的ELISA试剂盒)同时对667只食蟹猴血清进行检测,考察新研制剂盒的准确性;然后,挑选强阳性、弱阳性、阴性样品,用另外2个批次ELISA试剂盒检测,考察不同批次试剂盒检测结果的稳定性。结果:金标准试剂盒对667只食蟹猴血清抗体检测结果为阳性280份、阴性387份,而新研制的ELISA试剂盒结果为阳性282份、阴性385份;与金标准相比,阳性样品的符合率为98.93%(277/280),阴性样品的符合率为98.97%(383/387),总体符合率为98.95%(660/667)。对100份强阳性样品、70份弱阳性样品、108份阴性样品及7份差异样品的重复检测表明,除1份弱阳性(金标准为阴性)检测为阴性外,其余样品与前期一致。结论:与金标准相比,新研制的试剂盒具有较高的准确性,可用于猴B病毒抗体的检测。
Objective: In order to provide a kind of high quality and low-cost detection reagents for experimental primates industry, the accuracy of the newly developed B virus antibody ELISA test kits was evaluated with the gold standard kit as the reference. Methods: Three batches of cell supernatants co-expressing monkey B virus g D and g B protein were used to prepare ELISA kits for B virus antibodies test. Then, 667 cynomolgus monkeys sera were detected simultaneously by gold standard kits(VRL ELISA kits) and new kits to investigate its accuracy. Finally, strong positive, weak positive and negative samples were selected and tested by the other two batches of ELISA kits to examine the stability in different kits. Results: In 667 cynomolgus monkey sera samples, positive samples and negative samples were 280 and 385 respectively, detected in gold standard kits. The corresponding results were 282 and 387 in new ELISA kits. In positive samples, negative samples and all the samples, the coincidence rate was 98.93%(277/280), 98.97%(383/387) and 98.95%(660/667), respectively, compared with gold standard kits. Then, 100 strong positive samples, 70 weakly positive samples, 108 negative samples and 7 differential samples, according to the OD values, were screened and tested with the other two batches of ELISA kits. Except for one weakly positive sample(negative in gold standard kits) tuned into negative, other samples were consistent with the previously tested. Conclusion: Compared with the gold standard ELISA kits, the newly developed kits have high accuracy, could be used for the detection of monkey B virus antibody.
Objective: In order to provide a kind of high quality and low-cost detection reagents for experimental primates industry, the accuracy of the newly developed B virus antibody ELISA test kits was evaluated with the gold standard kit as the reference. Methods: Three batches of cell supernatants co-expressing monkey B virus g D and g B protein were used to prepare ELISA kits for B virus antibodies test. Then, 667 cynomolgus monkeys sera were detected simultaneously by gold standard kits(VRL ELISA kits) and new kits to investigate its accuracy. Finally, strong positive, weak positive and negative samples were selected and tested by the other two batches of ELISA kits to examine the stability in different kits. Results: In 667 cynomolgus monkey sera samples, positive samples and negative samples were 280 and 385 respectively, detected in gold standard kits. The corresponding results were 282 and 387 in new ELISA kits. In positive samples, negative samples and all the samples, the coincidence rate was 98.93%(277/280), 98.97%(383/387) and 98.95%(660/667), respectively, compared with gold standard kits. Then, 100 strong positive samples, 70 weakly positive samples, 108 negative samples and 7 differential samples, according to the OD values, were screened and tested with the other two batches of ELISA kits. Except for one weakly positive sample(negative in gold standard kits) tuned into negative, other samples were consistent with the previously tested. Conclusion: Compared with the gold standard ELISA kits, the newly developed kits have high accuracy, could be used for the detection of monkey B virus antibody.
引文
[1]Dugan M A,Courtney C,Howerth E W.Pathology in practice.B virus infection in a rhesus macaque[J].J Am Vet Med Assoc,2013,242(9):1233-1235.
[2]Estep R D,Messaoudi I,Wong S W.Simian herpesviruses and their risk to humans[J].Vaccine,2010,28(Suppl 2):B78-84.
[3]Elmore D,Eberle R.Monkey B virus(Cercopithecine herpesvirus 1)[J].Comp Med,2008,58(1):11-21.
[4]Morton W R,Agy M B,Capuano S V,et al.Specific pathogen-free macaques:definition,history,and current production[J].ILAR J,2008,49(2):137-144.
[5]Mitsunaga F,Nakamura S,Hayashi T,et al.Changes in the titer of anti-B virus antibody in captive macaques(Macaca fuscata,M.mulatta,M.fascicularis)[J].Comp Med,2007,57(1):120-124.
[6]Katz D,Shi W,Patrusheva I,et al.An automated ELISA using recombinant antigens for serologic diagnosis of B virus infections in macaques[J].Comp Med,2012,62(6):527-534.
[7]Katz D,Shi W,Wildes M J,et al.Reassessing the detection of B-virus-specific serum antibodies[J].Comp Med,2012,62(6):516-526.
[8]杨燕飞,周洁,高诚.猴B病毒研究进展[J].动物医学进展,2015,36(7):94-99.
[9]贺争鸣,卫礼,巩薇,等.猴B病毒抗体ELISA检测方法的建立和应用研究[J].中国实验动物学报,2003,11(3):161-164.
[10]韦毅,符明泰,石寒,等.三种不同抗原对猴B病毒抗体检测结果的比较研究[J].实验动物科学与管理,2001,18(2):1-3.
[11]徐刚,周小军,宋禾,等.利用自裂解肽T2A共表达猴B病毒糖蛋白g B和g D[J].生物技术通讯,2016,27(2):163-167.
[12]O'Shea D J,Trautmann E,Chandrasekaran C,et al.The need for calcium imaging in nonhuman primates:New motor neuroscience and brain-machine interfaces[J].Exp Neurol,2017,287(Pt 4):437-451.
[13]Gunnar M R,Hostinar C E,Sanchez M M,et al.Parental buffering of fear and stress neurobiology:reviewing parallels across rodent,monkey,and human models[J].Soc Neurosci,2015,10(5):474-478.
[14]Tanaka S,Mannen K,Sato H.Use of herpesvirus papio 2 as an alternative antigen in immunoblotting assay for B virus diagnosis[J].J Vet Med Sci,2004,66(5):529-532.
[15]Tyler S D,Peters G A,Severini A.Complete genome sequence of cercopithecine herpesvirus 2(SA8)and comparison with other simplexviruses[J].Virology,2005,331(2):429-440.
[16]Perelygina L,Patrusheva I,Hombaiah S,et al.Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential[J].J Clin Microbiol,2005,43(2):620-628.
[17]Fujima A,Ochiai Y,Saito A,et al.Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera[J].J Clin Microbiol,2008,46(1):56-61.
[2]Estep R D,Messaoudi I,Wong S W.Simian herpesviruses and their risk to humans[J].Vaccine,2010,28(Suppl 2):B78-84.
[3]Elmore D,Eberle R.Monkey B virus(Cercopithecine herpesvirus 1)[J].Comp Med,2008,58(1):11-21.
[4]Morton W R,Agy M B,Capuano S V,et al.Specific pathogen-free macaques:definition,history,and current production[J].ILAR J,2008,49(2):137-144.
[5]Mitsunaga F,Nakamura S,Hayashi T,et al.Changes in the titer of anti-B virus antibody in captive macaques(Macaca fuscata,M.mulatta,M.fascicularis)[J].Comp Med,2007,57(1):120-124.
[6]Katz D,Shi W,Patrusheva I,et al.An automated ELISA using recombinant antigens for serologic diagnosis of B virus infections in macaques[J].Comp Med,2012,62(6):527-534.
[7]Katz D,Shi W,Wildes M J,et al.Reassessing the detection of B-virus-specific serum antibodies[J].Comp Med,2012,62(6):516-526.
[8]杨燕飞,周洁,高诚.猴B病毒研究进展[J].动物医学进展,2015,36(7):94-99.
[9]贺争鸣,卫礼,巩薇,等.猴B病毒抗体ELISA检测方法的建立和应用研究[J].中国实验动物学报,2003,11(3):161-164.
[10]韦毅,符明泰,石寒,等.三种不同抗原对猴B病毒抗体检测结果的比较研究[J].实验动物科学与管理,2001,18(2):1-3.
[11]徐刚,周小军,宋禾,等.利用自裂解肽T2A共表达猴B病毒糖蛋白g B和g D[J].生物技术通讯,2016,27(2):163-167.
[12]O'Shea D J,Trautmann E,Chandrasekaran C,et al.The need for calcium imaging in nonhuman primates:New motor neuroscience and brain-machine interfaces[J].Exp Neurol,2017,287(Pt 4):437-451.
[13]Gunnar M R,Hostinar C E,Sanchez M M,et al.Parental buffering of fear and stress neurobiology:reviewing parallels across rodent,monkey,and human models[J].Soc Neurosci,2015,10(5):474-478.
[14]Tanaka S,Mannen K,Sato H.Use of herpesvirus papio 2 as an alternative antigen in immunoblotting assay for B virus diagnosis[J].J Vet Med Sci,2004,66(5):529-532.
[15]Tyler S D,Peters G A,Severini A.Complete genome sequence of cercopithecine herpesvirus 2(SA8)and comparison with other simplexviruses[J].Virology,2005,331(2):429-440.
[16]Perelygina L,Patrusheva I,Hombaiah S,et al.Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential[J].J Clin Microbiol,2005,43(2):620-628.
[17]Fujima A,Ochiai Y,Saito A,et al.Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera[J].J Clin Microbiol,2008,46(1):56-61.