CAJ | 학술논문

犬细小病毒病是危害养犬业的重要传染病之一,患病犬难以治愈。单克隆抗体治疗此病效果明显,本文介绍了制备抗CPV-2a单克隆抗体的方法。用纯化的犬细小病毒（canine parvovirus,CPV）2a型分离株免疫新西兰大白兔和Balb/c小鼠制备抗CPV-2a多克隆抗体及单克隆抗体。经亚克隆得到1H9、2B5、2B7和2C7共4株单抗,Western blotting鉴定单抗的免疫反应性;间接ELISA方法检测单抗的特异性。为了快速对犬细小病毒病作出诊断,建立了CPV-2a双抗夹心ELISA方法。兔多抗作为捕获抗体,鼠单抗作为示踪抗体,辣根过氧化物酶标记羊抗鼠IgG作为检测系统;捕获抗体和示踪抗体最佳稀释度分别为1：800和1：2000;检测系统最佳稀释度为1：4000。结果表明：所得4株单抗与pET-32a-VP2蛋白发生特异性反应,且与狂犬病毒（RV）、犬温热病毒（CDV）不交叉反应;建立的双抗夹心ELISA方法对病毒的最低检出量为4.375μg/mL,与美国RB试剂盒相比,符合率为95%。单抗制备为犬细小病毒病的治疗奠定了基础;双抗夹心ELISA方法的建立为疑似粪便样本提供了简单、快速和可靠的检测手段。
견세소병독병시위해양견업적중요전염병지일,환병견난이치유。단극륭항체치료차병효과명현,본문개소료제비항CPV-2a단극륭항체적방법。용순화적견세소병독（canine parvovirus,CPV）2a형분리주면역신서란대백토화Balb/c소서제비항CPV-2a다극륭항체급단극륭항체。경아극륭득도1H9、2B5、2B7화2C7공4주단항,Western blotting감정단항적면역반응성;간접ELISA방법검측단항적특이성。위료쾌속대견세소병독병작출진단,건립료CPV-2a쌍항협심ELISA방법。토다항작위포획항체,서단항작위시종항체,랄근과양화물매표기양항서IgG작위검측계통;포획항체화시종항체최가희석도분별위1：800화1：2000;검측계통최가희석도위1：4000。결과표명：소득4주단항여pET-32a-VP2단백발생특이성반응,차여광견병독（RV）、견온열병독（CDV）불교차반응;건립적쌍항협심ELISA방법대병독적최저검출량위4.375μg/mL,여미국RB시제합상비,부합솔위95%。단항제비위견세소병독병적치료전정료기출;쌍항협심ELISA방법적건립위의사분편양본제공료간단、쾌속화가고적검측수단。
The canine parvovirus（CPV）,which is hard to get cured,is one of the most severe diseases in dogs.However,an effective therapeutic method for the CPV is the monoclonal antibody.This article described the approach to the preparation of anti-CPV-2a monoclonal antibody.The canine parvovirus antibodies were prepared from New Zealand rabbit and Balb/c mouse with purified CPV-2a antigen.After subcloned,4 strains of positive hybridoma cells were obtained,which were named 1H9,2B5,2B7 and 2C7 respectively.Their immunoreactivity were identified by Western blotting and their specificity were done by indirect ELISA.In order to detect the canine parvovirus,a double antibody sandwich ELISA method was established.The assay used rabbit anti-CPV-2a polyclonal antibody as the capture antibody,monoclonal antibody as the trace antibody and goat anti-mouse IgG HRP conjugation as the detection system.The optimal dilution of the capture antibody and the trace antibody capable of detecting the CPV-2a antigens was found to be 1：800 and 1：2 000 respectively in the check-board titration,and that of the detection system was 1：4 000.The result showed that the immunoreaction of the monoclonal antibody with pET-32a-VP2 protein was positive and its reaction with RV or CDV antigen was negative.The detective sensitivity of sandwich ELISA was 4.375 μg/mL.Compared with RB of USA ELISA kit,the coincidence rate was 95%.The preparation of monoclonal antibody would provide the basis to treat the canine parvovirus.The establishment of double antibody sandwich ELISA method would be a simple,quick and reliable method for screening large numbers of fecal samples of dogs suspected of CPV infection.