At The Same Time, The Monoclonal Antibody And Colloidal Gold Test Strips Of Type 1 And Type 3 Duck Hepatitis A Virus Were Identified

Duck viral hepatitis is a kind of highly contagious and acute infectious disease that was caused by duck hepatitis virus A. And this disease mainly infected young ducklings under 3 weeks and led liver swell and bleed, which caused economic loss to duck industry for its high mortality and infection rate. In this article, we obtained and purified monoclonal antibodies based on DHAV-1. Besides, we conjugated monoclonal antibodies to colloidal gold and developed an immunochromatographic strip assay to detect DHAV. There are two parts in this research.1. DHAV monoclonal antibody preparation and preliminary identificationWe inoculated duck embryo allantoic fluid with DHAV-1 X in the duck embryos.And embryos dead within 24-72 h were collected, we obtain allantoic fluid after they were placed at 4? overnight. Purifying DHAV-1 antigen with three steps:Centrifugating allantoic fluid, Mixing allantoic fluid with chloroform, Ultracentrifugating allantoic fluid.We used PBS to dissolve the precipitation that contained DHAV-1 antigen content is 0.5mg/mL. and then used this purified virus to immune after BALB/c female mice of 6 weeks old. Then splenocytes from the immunized mice were fused with SP2/0, then the the hybridoma cells were harvested by limited dilution through three times of cloning. End up with five strains can stable,secretion specificity hybridoma, and respectively named 1#,2#,3#,4#,5#; 1# monoclonal antibody was IgM,2# monoclonal antibody was IgG1,3# belongs was IgG2b monoclonal antibody,4,5# belongs to IgG2a monoclonal antibody. The ascites titer of McAbs that purified is more than 2×105.The purified McAbs showed strong reactivity only with the DHAV, confirmed that the newly developed McAbs does not show any cross-reactivity. After passaged in vitro and cells cryopreserved recovery that remain stable can secrete antibody; affinity constant of strains of monoclonal antibodies are orders of magnitude more than 109, that has a good ability of the antigen. by comparing the five strains of monoclonal antibody affinity constant value, can get 3# monoclonal antibody affinity is higher.2. The development and application of immunochromatographic stripWe developed an immunochromatographic strip test based on 2# monoclonal antibodies. In this assay, monoclonal antibodies were conjugated to colloidal gold, and on the nitrocellulose membrane, test line were coated with rabbit anti-DHAV-1 polyclonal antibodies and control line was coated with goat anti-mouse IgG. What's more, we obtain the optimal pH and amount of monoclonal antibodies. The optimal pH was that 12ul 0.1mol/L K2CO3 should be added to 1mL colloidal gold solution, and the optimal amount was that 34ug monoclonal antibodies should be added into 1mL colloidal gold solution. Besides, the optimal concentration of rabbit anti-DHAV-1 polyclonal antibodies on test line and goat anti-mouse IgG on control line were lmg/mL and lmg/mL. The specificity analysis of ICS indicated that ICS can detect the four strains of DHAV-1 and six strains of DHAV-3, and the results of the detection of other duck pathogenic agents(Duck enteritis virus, Duck Tembusu virus, Escherichia coli, Salmonella, Riemerella anatipestifer and so on) were negative. And the detection limit were 0.9?g of DHAV-1 and 1.8?g of DHAV-3. The stability analysis of ICS suggested that strips can be stored in the 4? and 37? and at room temperature at least 3 months. Repeatability analysis indicated that the repeatability of strips was good. In terms of the detection of clinical samples, the positive and negative coincidence rate between ICS and RT-PCR were 96.5%(111/115) and 97.9%(46/47). The sensitivity, specificity, repeatability and stability were reliable.