Production Of Monoclonal Antibody Against Canine Coronavirus And Cellular Receptor And Establishment Of Immunoperoxidase Plaque Stainiing Test

Canine Coronavirus (CCV) , a member of group I Coronaviruses of family Coronaviridae, have four structure proteins, including spike protein (S), membrane protein (M) , nuclear protein (N) and small membrane protein (SM) . By monoclonal antibody method, McAbs, against CCV and cellular receptor respectively, were prepared to investigate some characterization of CCV and the interaction between CCV and the receptor. Immunoperoxidase (IP) Plaque Staining test was introduced to detect the sera after CCV infection.Positive clones fusing with spleen cells from BALB/C mouse immunized by purified coronavirus (CCV) 1-71 strain and SP2/0, were screened by indirect ELISA and NT and were subclonized with limited dilution. The false positive clones were rejected by A72 cell coating ELISA plates. Five McAbs, of which two McAbs with ELISA positive only and three McAbs with ELISA and NT positive, which were recognized 45 kD, 100 kD, 200 kD and 45 kD, 60 kD, 100 kD, respectively, were prepared. The McAbs with NT activity showed positivity with indirect Fluorescent Assay.Compared with neutralization test (NT) and indirect enzyme-linked immunosorbent assay (ELISA), Immunoperoxidase (IP) Plaque Staining test was introduced to detect canine coronavirus(CCV) infection, and serum operating dilution was designated 40. When an IP titre of 1:40 or more was regarded as IP-positive. To compare the IP test with the NT, sera from 20 healthy dogs and from 43 diarrhoeic dogs was examined. The result of 20 sera from healthy dogs showed consistence between IP and NT. However, 33 of 43 sera from diarrhoeic dogd were negative for NT, 10 positive, and 28 were negative for IP antibody, 15 positive, with 5 unagreement (15.2% ) . This indicates that the IP test is more able to detect anti-CCV antibody than NT.Cellular receptor facilitated canine coronavirus entering into cells. A monoclonal antibody(MAb) R-H-2, which blocks infection of A₇2 cells by CCV1.71 strain as well as TN449 strain with inhabitation rate of 98.8% and 95.65% respectively by plaque staining,was obtained by immunizing Balb/c mice with A72 cells which are highly susceptible to CCV. MAb R-H-2 recognized a polypeptide of relative molecular 150kD by Western-blot.