Development Of Monoclonal Antibodies Against Potato Virus M And Potato Virus A And Their Serological Detection Techniques

Potato is the fourth largest food crop after wheat,rice and maize in the whole world.However,potato viral diseases seriously harm potato crop and cause huge yields and economic losses.Among them,Potato virus M?PVM?and potato virus A?PVA?are two important potato viruses.At present,potato virus diseases are mainly controlled by planting virus-free seedlings and virus-free seed potatoes.Production of virus-free seedlings and virus-free seed potatoes requires the establishment of fast,simple,efficient,and sensitive virus detection methods.For this purpose,in this paper,monoclonal antibodies?MAbs?against PVM and PVA were separately prepared,and three serological detection assays based on the prepared monoclonal antibodies were established for PVM and PVA detection.The prepared MAbs and the developed serological assays will provide technical support for the detection of PVM and PVA,production of virus-free seed potato seedlings and potato seed tubers,and the establishment of a scientific prevention and control systems of those two potato viral diseases.?1?Preparation and detection application of MAbs against PVMBALB/c mice were immunized with purified PVM virions.Four hybridoma cell lines?1E1,2A5,8A1 and 17G8?capable of secreting anti-PVM MAbs were obtained by the hybridoma technology.Indirect ELISA titers of ascites containing MAb secreted by these four hybridoma cells reached 10^-7.Western blot analysis showed that all four MAbs reacted specifically with PVM coat protein.Using the prepared MAbs,three serological methods i.e.ACP-ELISA,dot-ELISA and Tissue print-ELISA for specifically and sensitively detecting PVM in potato plants and tubers were established.The specificity analysis results indicated that ACP-ELISA and dot-ELISA assays could specifically detect different isolates of PVM in potato leaves,while negative results were obtained when to detect healthy potato leaves or potato leaves infected with other viruses.The dot-ELISA for detecting potato tubers and the Tissue print-ELISA for detecting potato tubers and potato plant stems have the same specific results as described above.The results of sensitivity analyses demonstrated that the sensitivities of ACP-ELISA and dot-ELISA for monitoring tissue crude extracts of PVM-infected potato leaves were up to 1:163,840 and1:10,240 dilution?w/v,g/mL?,respectively.The established serological methods were used to detect 50 potato plant samples showing virus-like symptoms collected from potato fields in Yunnan and Heilongjiang provinces in 2017-2018.A total of 12 field samples were found to be PVM-positive by all three serological assays.The accuracy of test results of the three serological methods was verified by RT-PCR detection,DNA sequencing and nucleic acid sequence alignment.?2?Preparation and detection application of MAbs against PVAUsing purified PVA virions as the immunogen,four hybridoma cell lines?2D4,8E11,14A6,and 16H10?secreting anti-PVA MAbs were obtained by the hybridoma technology.Titers of MAb ascites secreted by these four hybridoma cell lines reached up to 10^-9 through an indirect ELISA.Western blot analysis demonstrated that all MAbs reacted specifically with PVA coat protein.Using the prepared MAbs,three serological methods?i.e.ACP-ELISA,dot-ELISA and Tissue print-ELISA?for specifically and sensitively detecting PVA were established.The results of specificity analyses indicated that the ACP-ELISA and dot-ELISA could specifically detect different isolates of PVA in potato leaves,while no positive results were observed when to detect healthy potato leaves or potato leaves infected with other viruses.Moreover,the dot-ELISA for detecting potato tubers and the Tissue print-ELISA for detecting potato tubers and potato plant stems had the same specific results as described above.The results of the sensitivity analyses showed that the sensitivities of ACP-ELISA and dot-ELISA for testing tissue crude extracts from PVM-infected potato leaves were 1:327,680 and 1:10,240 dilution?w/v,g/mL?,respectively.The established serological methods were used to detect 22 potato plant samples showing virus-like symptoms collected from potato fields in Yunnan and Zhejiang provinces in2019.A total of 3 field samples were found to be PVA-positive by all three serological assays.The accuracy of test results of the three serological assays was verified by RT-PCR detection,DNA sequencing and nucleic acid sequence alignment.