Dot-PAA-ELISA快速检测猪丹毒抗体方法的建立与应用

Development and application of Dot-PAA-ELISA rapidly detecting antibodies against swine erysipelas

以猪丹毒CD30 0 4 (1a型 )和C4 3_12 (2型 )菌株为抗原提取菌株 ,EDTA渗透法提取的抗原作为膜载抗原 ,建立了Dot_PAA_ELISA检测猪丹毒血清抗体方法 ,经研究确定了Dot_PAA_ELISA的最佳工作条件。试验制备的诊断膜片特异性强 ,不与猪瘟、猪伪狂犬病、猪肺疫、猪链球菌病、猪大肠杆菌病、仔猪副伤寒、猪布氏杆菌病、猪衣原体病的阳性血清发生交叉反应 ;灵敏度高 ,能够最低检出猪血清中含 2 6 1× 10 -9g的猪IgG抗体 ;膜片保存期长 ,在室温可保存 4个月 ,4℃可保存 7个月其检测活性不变。用该方法检测了仔猪免疫后抗体变化 ,并通过猪丹毒三型混合强毒攻毒试验确定了猪能抗强毒攻击的抗体临界保护值为 1∶32。对 336份血清进行猪丹毒抗体检测 ,阳性检出率为 96 13%。试验结果表明 ,本试验方法操作简便、快速准确、结果可存档、重复性和稳定性好 ,所用试剂材料均可做到标准化 。

EDTA-treated antigens of Erysipelothrix.rhusiopathiae C43004 strain (serotype 1 a) and C43-12 strain (serotype 2) were selected as diaphragm-carried antigens in Dot-PAA-ELISA.Detecting antibodies to swine erysipelas by Dot-PAA-ELISA was delevoped.Optimum trial conditions of Dot-PAA-ELISA were picked out.The specificity was proved by the blocking and cross-reaction tests.The diagnostic diaphragms did not react with antibodies to Pasteurellosis.Streptococcosis,Chlamydiosis,Salmonellosis,Colibacillosis,Brucellosis,HCV and PRV.Detecting antibodies to swine erysipelas by Dot-PAA-ELISA had good sensitivity and could detect (2.61)×10^(-9)/g IgG antibodies in swine serum.Stored at 4 ℃ for 7 months or at room temperature for 4 months,the sensitivity of diagnostic diaphragms did not change.The positive titre protecting swine erysipelas was established as 1∶32 by all immunized and unvaccinated control pigs challenged exposure with three composite virulent strains of E.rhusiopathiac.A total of 336 sera were detected and (96.13 %) of them were positive.These results showed that Dot-PAA-ELISA is a convenient,rapid,more sensitive and specific diagnostic method and is fit for serological surveillance and seroepidemiological investigation of swine erysipelas.