An anti-avilamycin a monoclonal antibody hybridoma cell line and its application
A hybridoma cell line and monoclonal antibody technology, applied in the field of (food safety) immunological detection, can solve the problems of cumbersome operation, unsuitable for rapid screening, and easy to be interfered by subjective conditions for result determination, and achieves a simple preparation method. Effect
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Embodiment 1
[0022] Embodiment 1: the preparation of avelamycin artificial antigen and hybridoma cell line
[0023] 1. Preparation of Avelamycin A-hemisuccinic acid (AVI-A-HS): Weigh 200 mg of Avelamycin A in mass ratios of 5:1, 10:1, 20:1, and 40: 1 (mass ratio) ratio to add succinic anhydride, were divided into 4 groups. Dissolve avelamycin A in 2 mL of pyridine, then add succinic anhydride in proportion, and stir for 48 h; until the precipitate is completely precipitated, and blow dry. It was monitored and detected by thin-layer TLC (the developer was chloroform: isopropanol = 94:6).
[0024] 2. Preparation of the immunogen Avelamycin A-BSA: Weigh 50 mg of the above hapten, dissolve it in 1 ml of dimethylformamide, and then add dicyclohexyl carbon disulfide in a ratio of 5:2:4 (mass ratio). Imine and N-hydroxysuccinimide were stirred and reacted for 8 hours in the dark, as liquid A. Using 15mg / mL bovine serum albumin (BSA) solution as solution B, slowly add solution A to 5ml solutio...
Embodiment 2
[0033] Embodiment 2: Preparation and identification of anti-avilamycin A monoclonal antibody
[0034] 1. Ascites preparation and purification: intraperitoneally inject 0.5 mL of sterile paraffin oil into BALB / c mice, and intraperitoneally inject 7×10 5 0.5 mL of hybridoma cells per mL, ascites was collected around 7 days. Purified by octanoic acid-ammonium sulfate method, followed by protein G affinity chromatography, and stored at -20°C.
[0035] 2. Antibody subtype identification: use the monoclonal antibody subtype detection kit to identify the antibody obtained in step 1, and dilute the original coating to 7 μg / mL with carbonate buffer solution with a concentration of 0.01mol / L pH9.6 Then coat the ELISA plate, overnight at 4°C; discard the coating solution, wash the plate with PBST, 5 times, 30S each time. Add 0.1 mL of CBS blocking solution containing 5% skimmed milk powder per well, incubate at 37°C for 1 h, discard the blocking solution, and wash the plate 5 times wit...
Embodiment 3
[0045] Embodiment 3: the determination of the IC50 value of hybridoma cell line monoclonal antibody to avelamycin A
[0046] 1. Take 1 μg / mL avelamycin-A-OVA as the coating material and coat a 96-well ELISA plate with 100 μL per well. After overnight at 4°C, wash the plate 5 times with PBST for 30 seconds each time, and pat it with filter paper. Dry.
[0047] 2. Block with CBS containing 5% skimmed milk powder, 200 μL per well, block at 37°C for 1 hour, wash the plate 5 times with PBST, 30 seconds each time, and pat dry with filter paper.
[0048] 3. Prepare 0.00246, 0.00449, 00898, 0.01797, 0.03594, 0.07187, 0.14375, 0.2875, 0.575, 1.15 μg / mL avilamycin standard solutions, 50 μL / well, add to the sealed microplate, every Each sample was replicated in 3 parallels, followed by adding 50 μL of 1:4000 diluted monoclonal antibody to each well as a positive control, reacted at 37°C for 1 hour, washed the plate and patted dry.
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