| Porcine epidemic diarrhea virus(PEDV)mainly infects piglets,these animals present with diarrhea,vomiting,and death.PEDV infection has caused economic losses to the swine industry.Though PEDV vaccines have been used to protect piglets,many piglets still died for the blindness of vaccination.To overcome the problem,it is necessary to establish diagnostic methods to evaluate maternal antibody levels.And test strips for rapid diagnosis of PEDV can confirm PEDV infection quickly and decided whether or not take emergency measures.However,after infection PEDV it takes 7 days to produce antibody from inoculation.It is necessary to develop antibody drugs for PEDV treatment.Secretory IgA(SIgA)is a vital molecule in mucosal immune system.In order to detect the PEDV specific SIgA,an ELISA method based on PEDV particles was established.The HRP labeled mAb of mouse anti-pig secretory component of SIgA was used as the second antibody.The specificity,repeatability and sensitivity of the method were tested.The method didn't react with TGEV or PoRV positive colostrum.As compare with IFA,the ELISA has good sensitivity when antibody titer was between 1:200 and 1:6400.In-batch and in-batch experiments,the ELISA has good repeatability.This method provides an effective technical for studying the SIgA level.In order to detect whether diarrhea was caused by PEDV,a rapid lateral flow immunoassay detection strip using Eu chelate microparticle was developed to identify PEDV.One mAb 15B 12 against PEDVN protein and IgG antibody of rabbit were labeled with Eu chelate microparticles,and these labeled antibodies were dispensed onto the combination pad while another mAb 4H7 against PEDV N protein was dispensed onto NC membrane using dispenser to forming the test line and goat antirabbit IgG antibody was also dispensed onto the NC membrane to forming the control line.The fluorescence peak heights of the test line(HT)and the control line(HC)were measured using a fluorescence reader.The cutoff value of HT/HC ratio was 0.05.When the value was>0.05,suggest the sample has PEDV N protein or PEDV.When detect other virus,the values were less than 0.05.Sensitive test shows that the LFTS was ten times better than that of RT-PCR.The results indicate that the Eu chelate microparticle-based LFTS is a rapid,sensitive,and reliable method for the detection of PEDV,indicating its suitability for epidemiological surveillance of PEDV infection.To obtain mAb that neutralize PEDV,we inoculated SPF BALB/c mice with purified PEDV particles.After immunizations,cells from the spleen were fused with SP2/0 cells.Following culturing and subcloning,1B9,secreting neutralizing antibody,was obtained.The 1B9 mAb neutralized PEDV G2 strains,but it did not neutralize G1 strain,in vitro.To elucidate the molecular mechanism of virus neutralization of mAb 1B9,a polyclonal antibody(pAb)against PEDV and the mAb 1B9 were used to isolate escape mutants of PEDV.Finally,we isolated an escape mutant strain,mutant-1B9,but still neutralized by the pAb.Analysis showed two regions deleted in the S protein allowed mutant-1B9 to escape neutralization by mAb 1B9.These results suggest the deleted regions participate in the formation of conformational epitope and provides valuable information for mapping conformational epitopes.Importantly,no PEDV escape mutants were generated by treatment with pAbs,which suggests the potential utility of pAbs or combination therapies based on several mAbs in curing PEDV infections.In summary,we established an ELISA method to detect PEDV specific SIgA and an Eu chelate microparticle-based LFTS to detect PEDV.Also,we obtained a mAb that can neutralize PEDV and the mAb epitope is conformational.The 55IGENQ59 and 609F are key sites for the epitope.These results provide important techniques to control PEDV and can be used in the pig industry. |
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