Monoclonal antibody for recognizing bevacizumab and application of monoclonal antibody
A monoclonal antibody, bevacizumab technology, applied in the biological field, can solve the problems of unable to reflect drug content, unable to detect free drugs at the same time, unable to detect bevacizumab, etc., to achieve huge market value, The effect of broad application prospects
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Embodiment 1
[0049] Example 1 Preparation process of monoclonal antibody 5A9
[0050] The main steps of this example include: animal immunization, cell fusion, screening of hybridoma cells, cloning of hybridoma cells and preparation of monoclonal antibodies, etc.
[0051] (1) Animal immunization: 6-week-old BALB / C female mice were used, and the antigen was commercialized bevacizumab Avastin. Conventional immunization method was adopted, and the immunization route was subcutaneous injection. Mix equal volumes of doses, and inject subcutaneously at 3 to 4 points on the back of the mouse; after two weeks, carry out two immunizations with the same dose, with Freund's incomplete adjuvant; two weeks later, perform three immunizations, with Freund's incomplete adjuvant ; Boost immunization two weeks later, with Freund's incomplete adjuvant; 3 days later, take splenocytes for fusion;
[0052] (2) Cell fusion: prepared on the third day after the last immunization;
[0053]Preparation of splenocyt...
Embodiment 2
[0082] Example 2 Evaluating the Specificity of Monoclonal Antibody 5A9 Binding to Bevacizumab
[0083] Experimental procedure
[0084] (1) Coating: Dilute Avastin and the control antibody Erbitux to 5 μg / mL with coating buffer, respectively add to different 96-well plates, 100 μL / well, overnight at 2-8°C;
[0085] (2) Plate washing: Wash the plate 3 times with washing solution, 300 μL / well;
[0086] (3) Blocking: 200 μL / well 0.1% BSA solution, room temperature for 2.5 hours;
[0087] (4) Plate washing: wash the plate 3 times with washing solution, 300 μL / well;
[0088] (5) Dilute samples: Dilute antibody 5A9 with buffer, and then serially dilute 2 times to obtain 12 concentration samples, in order: 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, 1.56 ng / mL, 0.78 ng / mL, 0.39 ng / mL and 0.19 ng / mL;
[0089] (6) Transfer the diluted sample to a 96-well plate, 100 μL / well;
[0090] (7) Incubation: overnight incubation at 2-8°C;
[009...
Embodiment 3
[0099] Example 3 Evaluation of Neutralizing Activity of Monoclonal Antibody 5A9
[0100] Experimental procedure
[0101] (1) Coating plate: Dilute the recombinant human VEGF antigen to 50 ng / mL with coating solution, add it to the ELISA plate, 100 μL per well, and incubate overnight at 4°C;
[0102] (2) Plate washing: On the second day, take out the plate, empty the liquid, wash three times with 300 μL washing solution and pat dry the residual liquid;
[0103] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 2 h;
[0104] (4) Plate washing: empty the liquid, wash three times with 300 μL washing solution and pat dry the residual liquid;
[0105] (5) Dilution and loading of samples: dilute antibody Avastin to 6 μg / mL with buffer, and then serially dilute by 2 times to obtain 12 concentrations: 3000 ng / mL, 1500 ng / mL, 750 ng / mL, 375 ng / mL, 187.5 ng / mL, 93.8 ng / mL, 46.88 ng / mL, 23.4 ng / mL, 11.72 ng / mL, 5.86 ng / mL, 2.93 ng / mL, 1.96...
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