Research And Development Process Of Lateral Flow Immunoassay For PEDV Antigen Detection

Objective:For purpose of developing and optimizing antigen test lateral flow immunoassay（LFA）for porcine epidemic diarrhea virus（PEDV）detection,aiming to improve its analytical performance.Further the pilot scale production and application technics of LFA were investigated to use for point-of-care detection of virus antigen,which providing a research experience for the development and of industrialized test products of PEDV detection.Methods:In this study,a visual double-antibody sandwich format immunochromatographic strip using latex beads as signal probes was established and optimized parameters for achieving ideal analytical performance.Moreover,in order to satisfy the need of sensitive and quantitative virus antigen detection,choosing europium chelate nanoparticles as signal probes,a fluorescent immunochromatographic strip for PEDV sensitive detection was established and optimized technological conditions and reaction system by iterative process.Subsequently,the LFA evaluated its analytical performances and applied to clinical fecal samples detection by comparing the results with RT-PCR.Results:Under optimal conditions,the visual limit of detection of immunochromatographic strip based on latex beads was defined as the minimum PEDV concentration of 10^3.96TCID₅₀/m L,no cross-reaction.In the test of collected pig fecal samples test（n=64）,the coincidence with the RT-PCR was 95.3%（61/64）.Additionally,the limit of detection of fluorescent immunochromatographic strip developed by europium chelate nanoparticles was 10^2.65TCID₅₀/m L and the precision and accuracy of strips met the need.Of the 159 fecal samples enrolled in the trial,the coincidence with the RT-PCR result was 97.5%（155/159）,which showed good performance.The evaluation of real-time stability showed that the storage life of latex beads-LFIA and Eu NPs-LFIA could keep at least 56 days until now and accelerated aging test results indicated that the estimated storage life could achieve at least 12 months.Conclusions:From this research,latex beads and europium chelate nanoparticles lateral flow immunoassay were successfully prepared for PEDV antigen detection,which satisfied the need of the visible light and fluorescence output results.And both of prepared LFIA showed good specificity,stability and accuracy.Meanwhile,establishing technological production process was benefit for high-volume production of PEDV detection LFIA and provided experimental basis.