Preparation Of A Colloidal Gold Immunochromatography For Detection Of Porcine Epidemic Diarrhea Virus Using Monoclonal Antibodies

Porcine epidemic diarrhea(PED), caused by porcine epidemic diarrhea virus(PEDV), is a highly contagious intestinal infectious diseases characterized by watery diarrhea, womiting, dehydration, and high motality in sucking piglets. PED are easily confused with transmissible gastroenteritis and rotavirus infection while clinical diagnosis. And the preparation of specific diagnositic reagents and development a rapid detection method for PEDV are very important. Nucleoprotein(N), a highly conserved protein, is a major structural protein of PEDV and the main target for early diagonosis and disease mornitoring. In order to provide a necessary technical platform for development of specific and convenient diagnostic tool, PEDV N protein monoclonal antibodies(Mc Ab) were prepared by hybridoma technique and colloidal gold immunochromatography method was established using the Mc Ab.Four BALB/c mice were immunized with the purified recombinant N protein of PEDV. By hybridoma technique, four strains of hybridoma cells, named A7, E10, F7 and C1, secreting Mc Ab against N protein were obtained through 3~5 times of subcloning and screening. Mc Abs secreted by 4 hybridoma cells, owned 97~103 chromosomes, belonged to isotype of Ig M with kappa(?) light chain, and they targeted four different antigen epitops of N protein. Four Mc Abs combined specifically with N protein by western blot and with PEDV in intestinal epithelial cells from PEDV-infected piglets by immunohistochemistry assay, respectively. The antibody titers of strain A7, E10, F7 and C1 in cultural supernatant of hybridomas and in mouse scites were 1:1600, 1:100, 1:100, 1:100, and 1:10~6, 1 :10~5, 1:10~3, 1:10~2, respectively. All of four hydbridomas were able to keep stably secreting Mc Abs at least 30 generations by continuous passage.Colloial gold with about 20 nm particle diameter were prepared using trisodium citratec reduction. Mc Ab of A7 strain was labeled using colloidal gold. Colloidal gold-labeled Mc Ab pad with 40 ?g/m L of Mc Ab A7(p H 8.0) were made by absorbing colloidal gold-labeled Mc Ab to the glass cellulose membrane. Mc Ab F7 and goat anti-mouse Ig M of 5 ?Lwere respectively sprayed on the nitrocellulose membrane each cm at a concentration of 1mg/m L as a test line and a quality control line. Then the colloidal gold-labeled Mc Ab pad and the nitolcellulose membrane with the test line and the quality control line were assembled into a colloidal gold test strip. PEDV in porcine intestinal content could be identified specifically using the test strip, and meanwhile a red line at the test line and at the quality control line was present respectively. Furthermore, 13 samples of intestinal content from various pig farms were checked using the test strip and RT-PCR simutaneously. The positive percents of detection were 38.46 % and 46.15 % using test strip and RT-PCR, respectively, and the coincidence rate of two methods was 83.3 %. The results of sample detection indicated that the colloidal gold test strip was able to specifically and rapidly detect PEDV.