Development Of A New Gold-immunochromatographic Assay For The Detection Of CDV Antigen

Canine distemper(CD) is a highly contagious and lethal disease, which infects the predators such as dogs. CDV belongs to the genus Morbillivirus, family Paramyxoviridae, along with measles virus, rinderpest virus, peste-des-petits-ruminants virus, and cetacean Morbilliviruses. Dogs infected with CDV with clinical symptoms of depression, fever, anorexia, diarrhea, and neurological symptoms are late signs of clinical symptoms of the CDV-infection. It is one of the most serious epidemics hazards in dog keeping, fur animal industry, and wildlife conservation. The diagnosis of CDV included isolation of virus, inclusion test, serum neutralization test, FA test, ELISA and RT-PCR. But these methods need specific equipments and a certain technical training for the staff in the lab not suitable for veterinarian to make fast diagnosis. Therefore, this study aimed to develop a fast and simple gold-immunochromatographic assay for the detection of CDV antigen with two strains of monoclonal antibodies against CDV-N protein. The contents and results of this study shows as below:A total of two strains of monoclonal antibodies against CDV-N protein of 1B7 and 2F11, intraperitoneal injection of Balb/c mouse monoclonal antibody, then antibody purification and identification, respectively. McAb 1B7 labeled with colloidal gold, was dispensed on a conjugate pad as the detector. Purified McAb 2F11 and Goat anti-mouse IgG were blotted on a nitrocellulose membrane for the test(T) and control lines(C), respectively. Based on this, we developed the Gold-immunochromatographic Assay for the detection of CDV. Criterion for judgment: if the tested sample contains the CDV, antigen against the McAb 1B7, CDV will interact with the colloidal gold-1B7 and form an complex(gold-1B7-CDV). The complex will react with the immobilized McAb 2F11 on the T line and Goat anti-mouse IgG at the C line of the ICS and form two visible red band. If there is no CDV against McAb 2F11 in the sample, only the C line will be visible. We explored the best reaction condition for the immunochromatographic Assay, finally determined that McAb 2F11(600 μg/mL) and Goat anti-mouse IgG(800 μg/mL) were dispensed onto the T line and C line, the optimal conditions for conjugation between gold and McAb 2F11( 14.26 μg/mL) was pH 8.6, and samples diluted to 50 times was best for the ICS test.In this paper, we study specifically, sensitivity, repeatability and stability as well as the conformity of the ICS proved that the strip was capable of specifically detecting CDV antigen and detected negative for other virus serums such as CPV, CCV, CPIV, PRV and NDV. The strip sensitivity can reach 300 times dilution degrees. Its stability and reproducibility were quite excellent after storage at 4℃within 6 months. Compared with American commercialization of CDV Antigen Test Kit, the strip sensitivity was slightly higher using the kit as a reference, the relative specificity and sensitivity of the ICS were proved to be well. Furthermore, there was an excellent agreement between the results obtained by the commercial product and the ICS.In conclusion, we develop a new gold-immunochromatographic assay for the detection of CDV antigen.