Fluorescent microsphere marking method and detection kit
A technology of fluorescent microspheres and detection test strips, which is applied in biological detection and biological fields, can solve the problems that there are not many or not enough detection methods of alpha-fetoprotein fluorescent microspheres, and achieve the effect of good specificity and good application prospects
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Embodiment 1
[0021] Example 1 Preparation of alpha-fetoprotein AFP monoclonal antibody
[0022] The purchased alpha-fetoprotein AFP was used as the immunogen (Cat. No. M101336T, WOLCAVIBIOTECH). 100 μg of AFP protein was mixed with an equal volume of Freund's complete adjuvant, fully emulsified, and BALB / c mice were immunized by intraperitoneal injection. Afterwards, immunization was performed once every 2 weeks, and 100 μg AFP protein was mixed with an equal volume of incomplete Freund's adjuvant, emulsified and immunized by intraperitoneal injection, for a total of 4 immunizations.
[0023] The peritoneal macrophages of normal BALB / c mice were prepared in advance as trophoblasts for cell fusion: 3 days after the last immunization, the spleen was taken, and blood was taken from the eyes at the same time, and the serum was separated by centrifugation. Mice splenocytes and Sp2 / 0 cells in logarithmic growth phase were mixed in a 50mL centrifuge tube at a ratio of 10:1, placed in a 37°C wate...
Embodiment 2
[0026] Example 2 Titer determination of 2D5 monoclonal antibody
[0027] The AFP protein was coated by ELISA, AFP was diluted to 1 μg / mL with carbonate buffer, 100 μL per well was added to a 96-well plate, and coated for 4 h at 37°C. The plate was washed once with PBST and blocked with PBS containing 3% BSA for 2 h at 37°C. Monoclonal antibodies with different dilution ratios were used as primary antibodies (the initial concentration was 1 mg / mL, and the gradient was diluted from 1:10 dilution ratio to 1:10000000), 100 μL per well, incubated at 37 °C for 1 h; washed with PBST for 5 times, Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse (GAM) antibody as secondary antibody, incubate at 37 °C for 30 min; wash with PBST 5 times, add 100 μL of TMB chromogenic solution to each well, and incubate at 37 °C Incubate for 15min; finally add 50μL of stop solution (2mol / L sulfuric acid), and read the A450nm value with a microplate reader. The titer of the antibody is ...
Embodiment 3
[0033] Example 3 Affinity identification, isotype identification and sequence analysis of 2D5 monoclonal antibody
[0034] Affinity constants of monoclonal antibodies were determined using the biosensor IAsysPlus produced by AffinitySensors. The sample pool was pretreated with carboxymethyl dextran (CMD), and AFP protein of different concentrations was added to the sample pool. After 5 min, the free carboxyl groups were blocked with ethanolamine for 3 min. Then, free and non-specifically bound protein molecules were washed away with 1 mol / L formic acid. Put the AFP-coated sample cell into the biosensor and equilibrate for 10 minutes. ①Baseline: Add pH7.2, 0.01mol / L PBS50μl, wait for 5 minutes, and make a stable baseline. ②asso-ciation: Aspirate PBS, add 45 μl PBS and 5 μl mAb, and aspirate the mAb solution when the mAb binds to saturation. ③Dissociation: Change 50μl PBS, wait until the mAb binding and dissociation reach equilibrium. ④Regeneration: change 20mmol / L HCl50μl fo...
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