Preparation method of monoclonal antibodies for IC (infectious coryza) of chickens

[0028] 1.1 Antigen preparation

[0029] Avian Bacillus paragallinarum type B was cultured, and its culture fluid was used as antigen.

[0030] 1.2 Animal immunity

[0031] Take an appropriate amount of bacterial solution and fully emulsify it with an equal volume of Freund's complete adjuvant, and inject subcutaneously into 6-week-old BALB / c mice, 0.2ml each; 2 and 4 weeks later, use Freund's incomplete adjuvant in the same way. The antigens emulsified with adjuvant were immunized once again; 6 weeks later, the same dose of antigen was injected intraperitoneally without adjuvant; 3 days later, they were fused.

[0032] 1.3 Fusion

[0033] Three days after the booster immunization, blood was collected from the orbit of the mice as positive serum, the mice were killed by cervical dislocation, the abdominal cavity was aseptically opened, the spleen was taken out, squeezed with a grinding rod to fully release the spleen cells, and a spleen cell suspension was made. The selected

[0041] Example 2 (identification of the monoclonal antibody prepared in Example 1)

[0042] 2.1 Determination of monoclonal antibody ascites titer

[0043]Using the method of indirect ELISA, the prepared ascites was diluted 1:1000 first, and then diluted by 2 times, and then added to the enzyme-labeled wells coated with antigens, and the other steps were the same as before. Results The monoclonal antibody titer of this strain was 1:6.4*10 4 .

[0044] 2.2 Identification of monoclonal antibody subclasses

[0045] Follow the method introduced in the monoclonal antibody subclass kit instructions. The specific method is as follows: Add four kinds of monoclonal antibody ascitic fluid 1:5000 to dilute 100uL / well in the microtiter plate, incubate at 37°C for 1 hour, wash with PBST 3 times, each time for 5min; add working concentration of goat anti-mouse IgG respectively 1 , IgG 2a , IgG 2b , IgG 3 , IgM, IgA subclass serum 100 μL / well, add two wells of each subclass to each m

[0057] Embodiment 3 (preparation of B-type Avian bacillus paragallinarum antigen diagnostic kit)

[0058] Using the monoclonal antibody prepared in Example 1 as a raw material, a diagnostic kit for Avibacterium paragallinarum type B antigen was prepared by using known techniques in the field of antigen detection kit preparation technology. The diagnostic principle of the kit is ELISA method.

[0059] 1.1 Specificity test

[0060] Using the established ELISA method, add the same number of bacteria (10 9 CFU / ml) Type A Avian bacteria,

[0061] Paragallinarum type B, type C, Pasteurella, Salmonella and Escherichia coli, and a blank control was set at the same time. The results are shown in Table 2:

[0062] The specificity experimental result of monoclonal antibody prepared in table 2 embodiment 1

[0063]

[0064] 1.2 Sensitivity test

[0065] With the counted good B type Avian bacteria paragallily, its concentration is 10 9 CFU / ml, serial 10-fold dilution, determined