Recombinant monoclonal antibodies H7 resisting fumonisins B1
A monoclonal antibody, fumonisin technology, applied in antibacterial immunoglobulin, tissue culture, microorganism-based methods, etc., can solve problems such as deletion frameshift and termination mutation, and achieve low production cost and simple operation. Effect
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Embodiment 1
[0033] Example 1: Antigen Preparation
[0034] Fumonisin FB1 (purchased from Sigma) was coupled to keyhole limpet cyanin (KLH, purchased from Sigma) using glutaraldehyde one-step coupling method (KLH-NH 2 +OHC-(CH 2 ) 3 -CHO+FB1-NH 2 → KLH-N=HC-(CH 2 ) 3 -CH=N-FB1) The FB1-KLH conjugate obtained after being used as an immune antigen, the fumonisin FB1 was coupled with bovine serum albumin (BSA, purchased from Sigma Company) (BSA-NH 2 +OHC-(CH 2 ) 3 -CHO+FB1-NH 2 →BSA-N=HC-(CH 2 ) 3 The FB1-BSA conjugate obtained after -CH=N-FB1) was used as the detection and screening antigen. The specific operation process is as follows: Weigh 1mg KLH and dissolve it in 1mL PBS (ingredients: NaCl at 137mM concentration, KCl at 2.7mM concentration, NaCl at 10mM concentration 2 HPO 4 , 1.8 mM concentration of KH 2 PO 4 , pH7.2~7.4) solution, add 0.6mg FB1, mix thoroughly, add an equal volume of 2% (v / v) concentration of glutaraldehyde solution dropwise, shake at 4°C for 1h, then a...
Embodiment 2
[0035] Example 2: Immunization of mice
[0036] The antigen FB1-KLH prepared in Example 1 was used to immunize Balb / c mice (purchased from the Experimental Animal Center of Wuhan Institute of Virology, Chinese Academy of Sciences) four times. The second immunization was carried out 4 weeks (28 days) after the first immunization, and the cycle of the last three booster immunizations was 3 weeks. The immunization was performed by multi-point subcutaneous injection on the back. For the first time, 200 μL of immune antigen (100 μg) was mixed with an equal volume of Freund’s complete adjuvant for immunization, and for the last three times, 200 μL of immune antigen was mixed with an equal volume of Freund’s incomplete adjuvant for immunization. After the third immunization, blood was collected from the tail vein on the 10th day, left standing at 37° C. for 1 hour, then centrifuged at 6000 r / min for 10 minutes, and the supernatant (antiserum) was collected for indirect ELISA to detec...
Embodiment 3
[0037] Embodiment 3: Detection of antiserum titer
[0038] The specific steps are as follows:
[0039] 1) The antigen FB1-BSA prepared in Example 1 was diluted at a volume ratio of 1:400, and 100 μL / well was coated on an ELISA plate, and left overnight at 4°C.
[0040] 2) Wash the wells of the ELISA plate three times with 200 μL PBS, add 150 μL of 1% (w / v) BSA solution, and block in a 37° C. water bath for 2 hours.
[0041] 3) Wash the wells of the ELISA plate three times with 200 μL of PBS, add 100 μL of antiserum diluted with PBS, the initial concentration of dilution is 1:1000, and bathe in water at 37° C. for 2 hours.
[0042] 4) Wash the ELISA wells 3 times with 200 μL PBST (PBS solution containing 0.1% (v / v) Tween-20) and PBS, add 100 μL of AP-labeled goat anti-mouse antibody diluted at a volume ratio of 1:5000 (purchased from Sigma Company), in a 37°C water bath for 1h.
[0043] 5) Wash the wells of the ELISA plate 3 times with 200 μL PBST and PBS respectively, add 1...
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