单抗I-ELISA和TAS-ELISA检测百合无症病毒的研究

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

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Agd ia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。

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 I-ELISA, 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 province. 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, OD4os values and P/N values of I-ELISA were normally higher than those of DAS-ELISA.