猪附红细胞体双抗体夹心ELISA检测方法的建立和应用

Development and Application of a Double Antibody Sandwich ELISA for Detection of Mycoplasma suis

为建立猪附红细胞体双抗体夹心ELISA(DAS-ELISA)检测方法,本研究以抗猪附红细胞体单克隆抗体为捕获抗体,兔抗猪附红细胞体多克隆抗体为检测抗体,优化工作条件,建立猪附红细胞体DAS-ELISA检测方法。结果显示:该方法的最佳工作条件为:捕获抗体包被浓度为5.42 g/mL,检测抗体浓度为6.84 g/mL,酶标二抗的稀释倍数为1∶2000;判定标准为:当检测样品OD492>0.290为阳性;该方法与弓形虫、猪链球菌、猪肺炎支原体和猪繁殖与呼吸综合症病毒等常见猪病原微生物均无交叉反应,特异性好,敏感度可达3.25 mg/L,其重复性变异系数均小于10%;平行检测68份猪临床样品,DAS-ELISA检测的阳性率为29.4%,比血涂片染色镜检阳性率(19.1%)高10.3%。本研究所建立的猪附红细胞体DAS-ELISA检测方法可以作为猪附红细胞体的快速检测方法。

The experiment was conducted to develop a method for Mycoplasma suis(M.suis).A double antibody sandwich ELISA(DAS-ELISA)was developed using M.suis monoclonal antibody(mAb)as captured antibody and rabbit polyclonal antibodies against M.suis as detecting antibody.The optimized reaction conditions showed that the optimal coating concentration of M.suis mAb was 5.42 mg/L,the optimal working concentration of rabbit polyclonal antibodies against M.suis was 6.84 mg/L,and the optimal working dilution of HRP-labelled goat-anti-rabbit lgG was 1:2000 with a cutoff value of 0.290(OD492).The ELISA had no cross-reaction with Toxoplasma gondii,Streptococcus suis,Mycoplasma hyopneumoniae and Porcine reproductive and respiratory syndrome virus(PRRSV),and at least3.25 mg/L antigen of M.suis was able to be detected.The coefficient of variation of reproducibility was less than 10%.A total of 68 clinical samples were detected by the DAS-ELISA and blood smear staining,and the positive rates of DAS-ELISA was 29.4%,showed a 10.3%higher than the positive rates of blood smear staining.These data demonstrated that the DAS-ELISA was low cost,specific,sensitive and convenient shortcut which provided a useful tool for diagnosis of M.suis infection.