Development Of Time-resolved Fluorescence Immunochromatographic Assay For Detection Of PEDV IgG And IgA Antibody

Porcine epidemic diarrhea(PED)is an acute and highly contact infectious disease caused by porcine epidemic diarrhea virus(PEDV)in pigs.Pigs can be infected at all ages.Clinical symptoms manifested as fever,vomiting,diarrhea,and dehydration.The lethal of piglets under 7 days of age reached 100%.At present,the prevention of PED in China ’s pig farms mainly depends on measures such as feeding management and vaccine immunization,but the disease still occurs frequently.PED is still one of the major diseases of the swine industry in our country,causing huge economic losses to the swine industry in China.Obtaining maternal antibodies from milk is an important way for piglets to be protected from PEDV.The detection of PED antibody in serum can indirectly reflect the potential protection level of the vaccine.Therefore,it is very extremely important to develop a method to detect PED antibodies in serum or milk.At present,the detection methods for Porcine epidemic diarrhea virus(PEDV)antibodies include enzyme-linked immunosorbent assay(ELISA),indirect immunofluorescence(IFA),and serum neutralization test.The above-mentioned detection methods are time cosuming,require complicative laboratory equipment,complicated operations,and require professionals skills to perform.Therefore,it is necessary to establish a quick,convenient and sensitive detection method for PEDV antibodies.In this study,we used PEDV recombinant S1 protein-coupled carboxyl fluorescent microspheres to prepare time-resolved fluorescent immunochromatographic test strips.By comparing the difference in fluorescence intensity between the test line(T line)and the control line(C line),the PEDV IgA or IgG antibody levels in serum or milk can be quantitatively detected,and evaluation of vaccine effects.The contents of this study are as follows: 1.Development of time-resolved fluorescence immunochromatographic(TRFIA)method for PEDV IgG antibodiesIn this study,competitive method and fluorescence immunochromatography technique were used,in which carboxy fluorescent microspheres were coupled with PEDV recombinant S1 protein,anti-IgG Fc fragment monoclonal antibody and PEDV S1 monoclonal antibody were respectively used as test strip test line(T-line)and control line(C line)to preparae for dectection of PEDV IgG antibody fluorescence quantitative immunochromatographic test strip(PEDV IgG-TRFIA).The results showed that the lowest detection limit of PEDV IgG reference was 23.772 mg/L,the optimal reaction time was 15 minutes,and the average recovery rate ranges from 100.88% to 102.70%,and the CV in intra-assay and inter-assay were less than 15%,and there was no false positive results in detecting PEDV IgA antibody,Pseudorabies virus(PRV)positive serum sample,Porcine circovirus type 3(PCV3)positive serum samples,Seneca Valley virus(SVV)positive serum samples and Haemophilus parasuis(HPS)antibodies positive serum sample.The coincidence rate in detection of 90 clinical serum samples and 60 milk samples between indirect immunofluorescence assay(IFA)and our fluorescence quantitative immunochromatographic test strip were 95.6%,93.3%,respectively.In summary,the time-resolved fluorescence immunochromatographic detection method of PEDV IgG antibody developed in this study has high sensitivity,high specificity,high accuracy,simple operation,and rapid dectection,can be potential used for the clinical detection of PEDV IgG antibodies.2.Development of time-resolved fluorescence immunochromatographic method for PEDV IgA antibodiesIn this study,competitive method and fluorescence immunochromatography technique were used,in which carboxy fluorescent microspheres were coupled with PEDV S1 protein,anti-pig IgA antibody and PEDV S1 monoclonal antibody were respectively used as test strip test line(T-line)and control line(C line)to preparae for dectection of PEDV IgA antibody fluorescence quantitative immunochromatographic test strip(PEDV IgA-TRFIA).The results showed that the lowest detection limit ofPEDV IgA reference was 42.763 mg/L,the optimal reaction time was 15 minutes,and average recovery rate ranges from 100.66%to 101.32%,and the CV in intra-assay and inter-assay were less than 15%,and there was no false positive results in detecting IgG antibody,PCV3 positive serum sample,HPS antibodies positive serum sample and Streptococcus suis(SS)antibodies positive serum sample.The coincidence rate in detection of 90 clinical serum samples and 60 milk samples between indirect immunofluorescence assay(IFA)and our fluorescence immunochromatographic test strips were 96.7%,96.7%,respectively.In summary,the time-resolved fluorescence immunochromatographic detection method of PEDV IgA antibody developed in this study has high sensitivity,high specificity,high accuracy,simple operation,and rapid dectection,and be potential used for the clinical detection of PEDV IgA antibodies.In summary,in this study we established time-resolved fluorescence immunochromatographic test strips that can quantitatively detect PEDV IgG or IgA antibodies in pig serum or milk,It meets the requirements for the rapid clinical detection method,and has potential clinical application significance.It can provide technical support for evaluating the immune effect of PED vaccine or the correlation between PEDV IgG and IgA antibody in milk and serum.