A method for testing the efficacy of chicken infectious bronchitis bivalent vaccine
A technology for bronchitis and chicken infectivity, applied in the field of vaccines, can solve the problems of inaccuracy and time-consuming, and achieve the effects of accurately reflecting experimental results, avoiding cross-influence, and reducing detection time and implementation costs.
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Embodiment 1
[0028] Embodiment 1 (preparation of two hybridoma cells)
[0029] 1. Antigen preparation
[0030] 1. Preparation of monoclonal antibody 6G8 strain antigen
[0031] Chicken infectious bronchitis virus K136 strain was inoculated into 10-day-old SPF chicken embryos, and the allantoic fluid of chicken embryos was aseptically harvested 6 days later. The specific method of differential centrifugation of the allantoic fluid is as follows: After melting the virus liquid frozen at -20°C, centrifuge at 5000g at 4°C for 30min, discard the precipitate and take the supernatant, then centrifuge at 14000g at 4°C for 20min, discard the impurity protein, and take supernatant. Then perform ultracentrifugation: centrifuge the supernatant obtained in the previous step at 66100g (TYPE60Ti, BeckmanL8-M) at 4°C for 1.5h, discard the supernatant, add 2mL TEN buffer solution (50mM Tris-HCL, 50mM NaCl, 5mM Na2EDTA, pH7 .4), and drop the precipitate under sterile conditions to disperse and dissolve i...
Embodiment 2
[0041] Embodiment 2 (specific identification of antibodies produced by the two hybridoma cells prepared in embodiment 1)
[0042] The K136 strain virus and the H120 strain virus were respectively coated in a 96-well microtiter plate, and the supernatants of two hybridoma cell lines were used to react with them respectively. The results are shown in Table 1:
[0043] Table 1 Antigen-antibody reaction results
[0044] K136 strain H120 strain NDV blank control 6G8 1.964* 0.106 0.089 0.043 5D5 0.096 1.725 0.091 0.052
[0045] *OD value of ELISA experiment
[0046] The above results show that the hybridoma cells 6G8 and 5D5 prepared in Example 1 have good specificity to chicken infectious bronchitis K136 strain and H120 strain respectively.
Embodiment 3
[0047] Example 3 (Using the two hybridoma cells prepared in Example 1 to prepare and purify antibodies respectively)
[0048] 1. Preparation and purification of two monoclonal antibody ascites
[0049] Take 8-week-old BALB / c mice, intraperitoneally inject sterilized liquid paraffin, 0.5mL / mouse; 1 week later, intraperitoneally inject two cell lines in logarithmic growth phase diluted with PBS, 1×10 6 CFU / mouse; when the abdomen of the mouse was obviously raised, ascites was collected from the abdominal cavity with a 16-gauge needle, centrifuged at 2500rpm for 10min, the adipose tissue was removed, the supernatant was taken out, and stored at -70°C for later use.
[0050] 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 6G8 strain was 1:6.4*10 4 ; 5D5 monoclonal ant...
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