单抗I-ELISA和TAS-ELISA检测百合无症病毒的研究

 以抗百合无症病毒(Lily symptom less virus,LSV)的单克隆抗体为核心,建立了间接ELISA (I-ELISA)和三抗体夹心ELISA (TAS-ELISA)的检测方法。I-ELISA检测体系中,病叶汁液和单克隆抗体腹水的工作浓度分别为1:20和1:6 000,对病叶汁液的检测灵敏度达到了1:2 560,可检测到提纯病毒绝对量为1.35 ng。TAS-ELISA检测体系中捕获抗体和单克隆抗体腹水的工作浓度分别为1:200和1:6 000,检测病叶汁液的灵敏度达到了1:5 120,对于提纯病毒可检测到0.68 ng。使用美国Agdia公司双抗体夹心ELISA (DAS-ELISA)检测试剂盒对病叶汁液的检测灵敏度为1:2 560,提纯病毒检出量1.35 ng。用3种ELISA方法检测了采自浙江省丽水市的46个田间样品,I-ELISA、TAS-ELISA和DAS-ELISA测出的阳性样品数分别为19、21和18个,阳性率为41%、46%和39%。灵敏度检测和田间样品检测结果显示,TAS-ELISA的灵敏度高于DAS-ELISA和I-ELISA。相同样品I-ELISA所测出的OD₄₀₅值和P/N值普遍高于DAS-ELISA,表明LSV单抗比多抗具有更强的特异性和更高的灵敏度。

Detection of Lily symptomless virus by I-ELISA and TAS-ELISA with monoclonal antibody

On the basis of monoclonal antibody(MAb) against Lily symptomless virus(LSV), indirect enzyme-linked immunosorbent assay(I-ELISA) and three antibodies sandwich-ELISA(TAS-ELISA) were established. In IELISA, the appropriate titer of infected leaf sap and MAb was 1:20 and 1:6 000 respectively. It could successfully detect virus in plant sap at 1:2 560 dilution or 1.35 ng purified LSV. In TAS-ELISA, the appropriate titer of capture antibody and MAb was 1:200 and 1:6 000 respectively. The method could successfully detect virus in plant sap at 1:5 120 dilution or 0.68 ng purified LSV. The kit of double antibodies sandwich-ELISA(DAS-ELISA) from Agdia Inc. of USA could detect virus in plant sap at 1:2 560 dilution or 1.35 ng purified LSV. All these means were used to detect the samples of lily from Lishui, Zhejiang pro-(vince). Among the 46 samples, 19, 21 and 18 samples were positive tested by I-ELISA, TAS-ELISA and DAS-ELISA. The data showed that TAS-ELISA was more sensitive than DAS-ELISA and I-ELISA. It also could illustrate that the MAb against LSV prepared was more specific and sensitive than the polyclonal antibodies(PAbs) from Agdia, OD₄₀₅ values and P/N values of I-ELISA were normally higher than those of DAS-ELISA.