单抗捕获ELISA法检测禽多杀性巴氏杆菌特异性IgM抗体

DETECTION OF SPECIFIC IgM ANTIBODY AGAINST PASTEURELLA MULTOCIDA IN CHICKEN BY MONOCLONAL ANTIBODY CAPTURE ELISA

本试验采用纯化的鸡IgG和环磷酰胺预处理后,再以亲和层析纯化的鸡IgM免疫BALB/C小鼠,运用淋巴细胞杂交瘤技术,以间接ELISA法筛选,获得了5株能稳定地分泌抗鸡IgM(μ链)特异性单克隆抗体的杂交瘤细胞株。经ELISA交叉反应性试验和羊抗鸡IgM(μ链)抗血清阻断试验,证明这5株单克隆抗体均是抗鸡IgM(μ链)特异性的。应用该抗鸡IgM(μ链)单克隆抗体建立了检测鸡抗多杀性巴氏杆菌血清中特异性IgM抗体的抗体捕获ELISA(Mac-ELISA),本法具有很高的特异性和良好的重复性。用于检测鸡抗多杀性巴氏杆菌的特异性IgM抗体发现:鸡在注射免疫接种多杀性巴氏杆菌灭活菌苗后,第4天即可检测到IgM抗体,并且上升较快,峰值在第2周,滴度较高(1∶5120);鸡在口服免疫接种多杀性巴氏杆菌弱毒活菌苗后IgM抗体上升缓慢,峰值在第4周左右,滴度较低(9∶320);鸡由不同途径感染不同的多杀性巴氏杆菌强毒后第8天时,IgM抗体滴度均明显上升。我们认为,Mac-ELISA方法是检测鸡抗多杀性巴氏杆菌特异性IgM抗体的良好方法,本项试验方法对建立检测鸡抗其它病原微生物的特异性IgM抗体方法具有借鉴意义。

Five hybridoma cell lines were obtained which secret antibodies specific for chicken IgM (μ chain) by fusion of SP2/0 myeloma cells with spelenocytes from BALB/C mice immunized with purified chicken IgM by affinity chromatography from chicken serum,after pretreatment of adult mice with chicken IgG and cyclophosphamide.All monoclonal antibodies (McAbs) were specific for μ chain of chicken IgM by crossreactivity test with indirect ELISA and ELISA blocking test with serum of goat anti-μ chain of chicken IgM. Monoclonal antibody capture ELISA (Mac-ELISA) have been developed to detect specific IgM serum antibody against Pasteurella multocida (P.m.) in chickens with McAb specific for μ-chain of chicken IgM,and it has high specificity and good stability.The specific IgM serum antibodies in all groups of chickens were detected by Mac-ELISA.IgM antibody in the group injected inactived P.m.was able to be detected after 4 days of inoculation and reached rapidly a peak(1:5120) within 2 weeks.IgM antibody in the one administrated orally low viulence P.m.rose slowly to the highest titer (1:320) until 4 weeks or so.IgM antibody titers of chickens infected with various virulent P.m.by different ways increased obviously on day 8 It is suggested that the Mac-ELISA is an effective method to detect IgM antibody and to develop methods detecting IgM antibody against other pathogens in chickens.