Preparation method of GFP (green fluorescent protein) antibody and GFP antibody
An antibody and antibody fragment technology, applied in the field of antibody preparation, can solve the problems of harsh use conditions, easy occurrence of heavy chain and light chain pollution, etc., and achieve the effect of small batch differences, simple structure, and reduced pollution.
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Embodiment 1
[0063] Embodiment 1 (alpaca immunization):
[0064] 11. Use the His-tagged GFP protein recombinantly expressed in Escherichia coli as the antigen, mix and emulsify it with GERBU-LQ (Gerbu, Cat. No. 3030.0100) as the immunogen.
[0065] 12. Use the immunogen after the above treatment to immunize two alpacas every 2 weeks, and inject 2 mg of the emulsified immunogen into the vicinity of the alpaca neck lymph node each time. 3-4 days after the 4th, 5th, and 6th immunization, 20ml of blood was drawn from the carotid artery of the alpaca into an anticoagulant tube to complete the immunization of the alpaca.
Embodiment 2
[0066] Embodiment 2 (construct phage library):
[0067] 13. Mix the blood and white blood cell separation solution extracted from the immunized alpaca at a ratio of 1:1, centrifuge at 400g for 30 minutes at room temperature, and use a pipette to suck out the immune cells in the upper layer of the cotton-like layer in the middle.
[0068] 14. After adding PBS buffer to the aspirated immune cells to 10 mL, centrifuge at 200 g for 20 minutes at room temperature to remove the supernatant, then add 5 mL of PBS buffer again, and centrifuge at 200 g for 20 minutes at room temperature to remove the supernatant.
[0069] 15. Add 1mL Trizol to the cleaned immune cells and mix well to obtain peripheral blood lymphocytes from immunized alpacas and store them in a -20°C refrigerator for later use.
[0070] 16. Transfer the extracted peripheral blood lymphocytes to a 1.5mL centrifuge tube, add 1 / 5 volume of chloroform to mix, let stand at room temperature for 5 minutes, and then centrifuge ...
Embodiment 3
[0095] Embodiment 3 (screening target fragment):
[0096] 35. Add 100ug GFP antibody and 2mL PBS to each immunotube, incubate overnight at 4°C to coat the immunotube.
[0097] 36. After amplifying and purifying the phage library, add 500 ul of phage into 1 mL of 3% BSA, and incubate with rotation at room temperature for 2 h. At the same time, add 2-3mL 3% BSA to the coated immunotube and incubate with rotation at room temperature for 2h.
[0098] 37. Wash the immunotube sealed in step 35 with PBS containing 0.01% Tween 3 times, 5 minutes each time. Then, add the blocked phage library into the blocked immunotube, add PBS until 2-3 mL, and incubate with rotation at room temperature for 1 h.
[0099] 38. Wash the immunotubes incubated with antigen and phage 20 times with PBS containing 0.01% Tween, 5 minutes each time.
[0100] 39. Add 1mL 100mM Trimethymime to the immunotube, incubate at room temperature for 10 minutes, add 1M Tris-HCl to neutralize Trimethymime, and transfer...
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