Antibody of TrkA and application thereof
A technology of antibodies and antigens, applied in applications, antibodies, anti-inflammatory agents, etc., can solve problems such as frequent administration, poor patient compliance, and short half-life of small molecule drugs
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Embodiment 1
[0260] Embodiment 1 hybridoma technology screening anti-TrkA monoclonal antibody
[0261] Hybridoma cell lines that can produce anti-TrkA monoclonal antibodies that block the combination of NGF and TrkA at the molecular level were screened by hybridoma technology and ELISA. The NCBI database was used to design the gene encoding the extracellular region of TrkA protein, which was constructed into a mammalian eukaryotic expression system, and the extracellular region of TrkA protein was expressed for screening of anti-TrkA monoclonal antibodies. The extracellular region of the immunogen TrkA protein was obtained from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. After Balb / C mice were immunized by intraperitoneal injection three times, the serum antibody titer of the immunized mice was determined by ELISA method and reached 10 5 class. After booster immunization, the spleen was fused with myeloma cells. Use HAT selection medium and HT selection medium to selectively culture ...
Embodiment 2
[0262] Example 2 HEK-293T-TrkA cell model screening anti-TrkA monoclonal antibody
[0263]The HEK-293T-TrkA cell model was constructed by lentivirus technology, and the hybridoma cell line that could produce anti-TrkA monoclonal antibody that blocked the binding of NGF and TrkA at the cellular level was screened by flow cytometry. Biotinylate NGF, use NGF to bind to the extracellular region of TrkA protein on HEK-293T-TrkA cells, anti-TrkA monoclonal antibody can also bind to the extracellular region of TrkA protein on HEK-293T-TrkA cells, design competition experiments , by flow cytometry, detect the combination of NGF and the extracellular region of TrkA protein on HEK-293T-TrkA cells under the action of anti-TrkA monoclonal antibody, and screen the anti-TrkA monoclonal antibody that blocks the binding of NGF and TrkA at the cellular level. After screening, a total of 9 monoclonal hybridoma cell lines that can produce anti-TrkA monoclonal antibodies that block the binding of...
Embodiment 3
[0264] Example 3 Application of ELISA method to detect positive clones to produce human-mouse cross-reaction of monoclonal antibodies
[0265] The binding of the monoclonal antibody produced by the supernatant of each positive clone to the Mouse-TrKA receptor was detected by ELISA method, and the human-mouse cross-reaction of the monoclonal antibody produced by the positive clone was detected. The results are shown in image 3 , in the figure, OD 450 The value reflects the binding strength of the antibody to the protein, and the larger the reading value, the stronger the binding of the antibody to the protein. As shown in the figure, the OD of 23E12, 21E5, 15D4, 27H3 groups 450 The value is around 3.5, which is significantly different from the negative control group; OD of 20C8, 1B9, 4H4, 3A5, 22D12 groups 450 The value is almost close to 0, and there is no significant difference compared with the negative clone group. It can be seen that clones 23E12, 21E5, 15D4, and 27H3 ...
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