Development And Application Of Immunocolloidal Gold Diagnosis Strip For Deer Mucosal Disease

Deer mucosal disease(DMD) is an infectious disease caused by bovine viral diarrhea/mucosal disease virus(BVD/MDV), which can cause many clinical symptoms as fever, mucosa erosion, ulcer, abortion, stillbirth and deformities, resulting in immune tolerance, persistent infection and immunosuppression. Currently, this disease is widely distributed over the world and seriously affects the development of deer farming industry. However, there is no diagnosis on colloidal gold test strip corresponding to deer mucosal disease in China. And other methods have evident disadvantages of time-consuming, laborious performance, requiring professional and technical personnel, or special test equipments, which are not suitable for clinical detection. At present, it is necessary to develop a simple, fast and convenient method for the Deer mucosal disease diagnosis.In this study, deer BVD/MDV protective antigen E2 protein was expressed by Bac-to-Bac expression system, and the protein had good reactiongenicity identified by Western blotting. Balb/c mice were immunized with purified E2 protein in accordance with conventional procedures. The splenic mononuclear cells from immunized mice were isolated and then fused with murine myeloma cells SP2/0. The hybridoma cells were selected using indirect enzyme-linked immunosorbent assay. Two single-secreting anti-BVD/MDV Mc Abs were obtained, which were named 1F11 and 1B11, respectively. The identification of immunoglobulin subtypes showed that these monoclonal antibodies were Ig G2 a and Ig G2 b, respectively. Two monoclonal antibodies could effectively recognized E2 protein by Western blotting and specifically react with BVD/MDV by indirect immunofluorescence detection, but not the distilled water.The colloidal gold rapid diagnostic test for DMD were prepared with anti-BVD/MDV monoclonal antibodies 1F11 and 1B11. The colloidal gold particleswere processed using sodium citrate reduction method, and particles size of approximate 20 nm was observed by the electron microscopy. The optimal p H value of colloidal gold particles combining with 1F11 monoclonal antibodies was 7.0 and the optimal combining concentrations was 14.4 μg/m L. The colloidal gold-labeled Mc Ab 1F11 solution was dispensed onto glass fiber paper, 1B11(1.5mg/m L)or goat anti-mouse antibody(1mg/m L)was dispensed at the test line or the control on the NC membrane. The results showed that the lowest detectable limits was 103 TCID50/m L of the BVD/MDV. Fifty samples suspected BVD/MDV infections were detected using the colloidal gold diagnostic test paper strips for diagnosis of DMD. The result of positive rate was the same as RT-PCR.The consistency rate comparing to RT-PCR was 100%.Research results show that the prepared immune colloidal gold diagnosis test strip can be as a rapid, sensitive and specific diagnosis methods used into deer mucosal disease clinical site pathogen detection.With the development of the methods,it can enrich the field diagnosis means of the deer mucosal disease with the infectious diseases of rapid diagnosis and epidemiological investigation and eradicating BVD/MD in deer has important value in clinical application.