A disease detection kit comprising an antibody specifically binding to the AFP protein
A protein detection and kit technology, applied in the field of biomedicine, can solve the problems of non-specific binding and low sensitivity
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Embodiment 1
[0019] Example 1. Preparation and purification of anti-AFP antibody
[0020] After the AFP antigen (concentration of 1 µg / µl) was thoroughly mixed with an equal volume of complete Freund's adjuvant (CFA) to form a watery oil packet, BALB / C mice were initially immunized with 200 µl per mouse by intraperitoneal injection. After 3 weeks, the first boost was carried out. AFP antigen (concentration: 1 µg / µl) was mixed and emulsified with equal volumes of incomplete Freund's adjuvant (IFA), and injected into multiple points on the back, 200 µl per mouse. Thereafter, booster immunization was performed every 2 weeks, and the tail blood was collected on the 7th day after each immunization, and the antibody titer was detected by ELISA method. When the antibody titer reaches 10 -4 When left or right, mix and emulsify equal volumes of AFP antigen (concentration: 1 µg / µl) and incomplete Freund's adjuvant (IFA), and boost immunization once by multi-point injection on the back, 200 µl per m...
Embodiment 2
[0021] Example 2. Antibody and human serum albumin cross-reaction experiment
[0022] AFP protein has a high homology with human serum albumin and is prone to cross-reaction, which affects the specificity of AFP antibody. Therefore, cross-reaction experiments were used to screen out antibodies that do not cross-react with HAS. Add human serum albumin to the 96-well ELISA plate, 100 ng per well, and incubate overnight at 4°C. Remove the supernatant, add 200 μl of PBST washing solution to each well, let it stand for 2 minutes, deduct the washing solution, repeat the washing 3 times, and pat the microplate as dry as possible after each washing. Add bovine serum albumin and block for 1 h at 37°C. Remove the supernatant, add 200 μl PBST washing solution to each well, let it stand for 2 minutes, deduct the washing solution, and repeat washing 3 times. The antibodies prepared in Example 1 were added to the detection wells respectively, and incubated at 37° C. for 2 h. Remove the s...
Embodiment 3
[0025] Example 3. Competitive inhibition ELISA experiment
[0026]Since the establishment of a double-sandwich ELISA detection system requires two AFP antibodies that bind to different epitopes, the AFP antibodies that bind to different epitopes were screened by competitive inhibition ELISA experiments. The hAFP-6 antibody was labeled with horseradish peroxidase (HRP), which was used as an enzyme-labeled control antibody. Wherein the HRP labeling technique is well known in the art. hAFP-2, hAFP-3, and hAFP-5 were used as unlabeled antibodies to be tested. Add the AFP enzyme-labeled antibody to the 96-well enzyme-labeled plate coated with AFP antigen, and add different proportions of the antibody to be tested in the experimental group (antibody to be tested: enzyme-labeled antibody=1, 2, 4, 8, 16, 32 ), at the same time, set up a positive control of AFP enzyme-labeled antibody with PBS; incubate at 37°C for 1 hour; remove the supernatant, add 200 μl of PBST washing solution t...
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