Development Of A Colloidal Gold Immunochromatographic Assay For Detection Of Wild-type Classical Swine Fever Virus Antigen

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a highly contagious and often fatal disease of domestic pigs. It is characterized by fever and hemorrhages and can run an acute, chronic, or even subclinical course. CSFV, together with Border disease virus (BDV), Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2) and a tentative species Pestivirus of giraffe, is classified within the genus Pestivirus in the family Flaviviridae .CSF outbreaks can result in large economic losses in pig production and affect international trade and therefore CSF is a notifiable disease to The World Organization for Animal Health (OIE). Although effective live-attenuated vaccines are available, vaccinated and infected pigs are serologically indistinguishable. Current methods for detection CSFV include virus isolation, antigen-capture ELISA, reverse transcription polymerase chain reaction and others. These methods, however, take a few hours to several days to accomplish, and require skilled personnel and specialized equipment. There is, therefore, an urgent need for rapid, reliable, and inexpensive tests for detection of CSFV. In this study, an immunochromatographic test strip using anti-E2 monoclonal antibodies for rapid diagnosis of CSFV was developed. This strip makes discrimination between wild-type virus and C-strain vaccine of classical swine fever virus. The results were as follows:1. A colloidal gold immunochromatographic strip was developed for the rapid detection of the wild-type classical swine fever virus (CSFV) antigen using two monoclonal antibodies (mAbs), 6E10 and HQ06, against the E2 protein. The wild-type CSFV-specific 6E10 mAb was labeled with colloidal gold prepared by reduction of sodium citrate as capture antibody and the HQ06 mAb was immobilized on the test line as detection antibody, while a rabbit anti-mouse IgG antibody was blotted on the control line of the nitrocellulose membrane. PK15 cell cultures infected with CSFV and non-infected were detected by the strips. The results showed that two red lines appeared in the test line and control line region only when detecting the CSFV-infected cells, while only a red line appeared in the control line region when detecting the non-infected cells. The detection limit of the immunochromatographic strip was 103.5 TCID50 CSFV, and the results of different batches were reproducible. The test strip did not react with hog cholera lapinized virus (HCLV), bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2). Taken together, the test strip has good specificity, sensitivity and reproducibility, and can be used to differentiate cells infected with wild-type CSFV from those infected with HCLV.2. Six methods, including the virus isolation, colloidal gold immunochromatographic assay (CGIA), antigen-capture ELISA (AC-ELISA), reverse transcription polymerase chain reaction(RT-PCR),TaqMan real-time RT-PCR(RT-qPCR) and reverse transcription loop mediated isothermal amplification assay (RT-LAMP), were used to detect CSFV in 50 samples in parallel. The results showed that 13 samples were detected positive by RT-qPCR and RT-LAMP, 11 by PCR, 10 by virus isolation, 9 by AC-ELISA and 8 by CGIA, and 8 samples were detected positive and 37 samples negative by the six methods. These results indicated that the 3 RNA-amplification assays could be used as the first choice for detection of CSFV due to the high sensitivity, while they were vulnerable to false positive results arising from sample to sample contaminations or from other contaminated sources. Although the virus isolation was time-consuming, it was still considered the"gold standard"and was indispensable for confirming CSF outbreaks. The rest two methods, AC-ELISA and CGIA, yielded the results in a short time yet their performance was hampered by a low sensitivity. Therefore, they were mainly used for herd diagnosis.In this study, the wild-type CSFV-specific mAb was used to develop a colloidal gold immunochromatographic strip. This strip, which makes discrimination between wild-type virus and C-strain vaccine of classical swine fever virus, might contribute to diagnosis of CSFV.