Screening Of Monoclonal Antibody Combinations Suitable For Detecting All Existing Isolates Of Potato Virus Y

Potato virus Y(PVY)is a member of the genus Potyvirus,which mainly infects solanaceous crops including potatoes,tobacco,and pepper,which seriously affect the yield and quality of crops,and can lead to crop yield reduction of 80%in serious cases.PVY can be transmitted by aphids in a non-persistent manner,by juice friction,and by seed potatoes on potatoes.The timely and accurate identification of PVY is helpful to understand the occurrence of PVY and provide theoretical basis for the monitoring,early warning,comprehensive prevention,and control of PVY.The commercial monoclonal antibodies Mab 1128,Mab 1129,and Mab 1130 of PVY can recognize the antigenic determinants of coat protein(CP)25NLNKEK30,16RPEQGSIQSNP26 and 5IDAGGS10.However,there are many PVY isolates that do not contain these three antigenic determinants.Therefore,using these three monoclonal antibodies for detection will result in missed detection.The polyclonal antibody of PVY has cross reaction with other viruses in the genus Potyvirus,therefore,the detection of polyclonal antibody will cause false detection.This study provides a combination of monoclonal antibodies that can theoretically detect all or most of the currently reported potato virus Y isolates.The main results are as follows:1.Four monoclonal antibodies that recognize different regions were screened,and the specific amino acid sites of each antibody antigenic determinant were identified.Dot-ELISA was performed with 54 monoclonal antibodies purified and 10 deletion mutants obtained by deletion mutation in pEHISTEV-PVY CP.According to the mutants that could not be recognized by each monoclonal antibody,5 monoclonal antibodies N1,M1,M2 and C1 were screened to recognize different regions.In order to further identify the specific amino acid sites of the antigenic determinant recognized by four monoclonal antibodies,we constructed deletion mutants in four regions,and conducted dot-ELISA with the deletion mutants in their respective regions.According to the mutants that each monoclonal antibody could not recognize,we identified the antigenic determinant recognized by N1 antibody as TIDAGGSTK,the 4-12 amino acid of PVY CP;The antigenic determinant recognized by M1 antibody is GTSGTHTVP,the 37-45 amino acid of PVY CP;The antigenic determinant recognized by M2 antibody is the amino acid QFDTWYE at the 89-95 position of PVY CP;The antigenic determinant recognized by C1 antibody is LLGVKN at the 261-266 amino acids of PVY CP.2.Determine the subtype,titer,specificity and sensitivity of monoclonal antibody.The subtypes and subclasses of MAb N1,M1,M2,and C1 monoclonal antibodies were identified as IgGl,IgG1,IgGland IgG2b respectively,and the light chain was kappa.The same concentration of diseased juice was detected by indirect ELISA using 5 monoclonal antibodies diluted by multiple ratio.The results showed that the diseased juice samples could still be detected by 5 PVY monoclonal antibodies diluted by 243000 times.Western blot results showed that four monoclonal antibodies specifically recognized the coat protein of PVY.Indirect ELISA and dot-ELISA analysis showed that four monoclonal antibodies reacted specifically with PVY,but did not cross react with ChiRSV CP、PepMoV CP、PVA CP、TVBMV CP and coat protein of other viruses.The sensitivity of four monoclonal antibodies to detect the disease sap by indirect ELISA and dot-ELISA can reach 1:10240,1:2560,1:640,1:2560 respectively.3.Monoclonal antibody combinations M2,M1,and M2,C1 theoretically recognize all 1885 PVY isolates.The antigenic determinants identified by four monoclonal antibodies were compared with the corresponding amino acid sequences of 1885 isolates.Through sequence analysis,it was found that the PVY isolates detected by monoclonal antibodies M1,M2and C1 were 11.35%,10.82%and 11.25%more than those detected by MAb1130,respectively.The 1885 PVY isolates were numbered according to 1-1885.Through the analysis of the PVY isolate numbers that did not contain the antigenic determinants identified by MAb M1,M2 or C1,it was found that the combination of monoclonal antibody M1 and C1 could recognize 1883 PVY isolates except 1107(AMW64585.1)and 1344(AYA57928.1),while the combination of monoclonal antibody M2 and C1 could recognize 1876 PVY isolates except 380(AEF59038.1),381(AEF59039.1),788(AGX27987.1),789(AGX27988.1),790(AGX27989.1),991(AIL50122.1),992(AIL50123.1),993(AIL50124.1)and 994(AIL50125.1).The combination of monoclonal antibodies M1 and M2 can recognize all 1885 PVY isolates.We compared the CP amino acid sequences of nine PVY isolates that do not contain M2 or C1 recognition antigenic determinants from 89-95 to the antigenic determinant sequence recognized by M2,and found that the 95th amino acid sequence of these nine PVY isolates was different from the antigenic determinant sequence recognized by M2.We used the unit point mutation method to introduce mutations on pEHISTEV-PVY CP and obtained the mutant PVY CPE95D,The result of dot-ELISA using monoclonal antibody M2 showed that monoclonal antibody M2 could recognize the mutant,so the combination of monoclonal antibody M2 and C1 could also recognize all 1885 PVY isolates.