Specific anti-ceftiofur monoclonal antibody hybridoma cell strain 2E5 and application thereof
A hybridoma cell line, cloned antibody technology, applied in specific peptides, biochemical equipment and methods, instruments, etc., can solve the problems of complex operation and high instrument cost, achieve low cross-reaction rate, good detection sensitivity and affinity, good Effects of specific monoclonal antibody cell lines
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Embodiment 1
[0021] The preparation of embodiment 1 hybridoma cell line 2E5
[0022] (1) Synthesis of complete antigen: Take 4.5 mg of EFT, add 2.0 mg of EDC and 1.0 mg of NHS, dissolve in DMF, stir at room temperature, and activate for 4 hours; another 5 mg of BSA is dissolved in 2 mL, 0.05 M, pH 9.6 CB solution, and the above The activated ceftiofur solution was slowly added dropwise to the CB solution of BSA, stirred and reacted at room temperature overnight, and dialyzed at 4°C for three days to obtain the immunogen, which was subpackaged and stored at -20°C.
[0023] (2) Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. After the complete antigen of ceftiofur (1 mg / mL) was emulsified with the same amount of Freund's adjuvant, BALB / c mice were immunized by subcutaneous multi-point injection, 100 μL each. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunization. The immun...
Embodiment 2
[0029] Example 2 Preparation and Identification of Monoclonal Antibody
[0030] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma cell 2E5, ascites fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.
[0031] The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody obtained from ascites purification. As shown in Table 1, the subtype was IgG2b.
[0032] Table 1
[0033]
[0034] Determination of IC of monoclonal antibodies against EFT, CEX and CEF using indirect competition ELISA and indirect ELISA 50 They were 2.4ng / mL, 76.9ng / mL and 45.28ng / mL respectively, and the cross-reactivity rates were all less than 10%. It can be used for specific rapid detection of ceftiofur.
Embodiment 3
[0035] Example 3 Antibody Application
[0036]The monoclonal antibody prepared by the hybridoma cell line 2E5 through in vivo ascites was applied to the addition and recovery test of ceftiofur ELISA, and the specific steps were as follows:
[0037] a. 0.5μg / mLEFT-EDC-OVA diluted with carbonate buffer solution (CBS) was used as the original coating material to coat the 96-well ELISA plate, 100μL per well, after coating at 37℃ for 2h, wash with PBST washing solution Plate three times, each time with 250 μL per well, each time for 3 minutes, and pat dry;
[0038] b. Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 hours, wash the plate three times with PBST washing solution, 250 μL per well for 3 minutes each time, and pat dry;
[0039] c. Prepare ceftiofur standard solutions of 0, 0.2, 0.5, 1, 2, 5, 10, and 20 ng / mL with phosphate buffered saline (PBS). Add the standard solution and the extract of the sample to be tested to the sealed microtiter pla...
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