Preparation method of high-throughput whole rabbit source monoclonal antibody
A monoclonal antibody, high-throughput technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc. throughput, high-quality preparation, etc.
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[0053]The preparation method of the high-throughput whole rabbit-derived monoclonal antibody of the present invention includes several steps of immunization, sorting, amplification, recombination and expression. The method isolates naturally paired heavy chain and light chain genes from a B lymphocyte, and realizes a high-throughput method for preparing rabbit monoclonal antibodies. This method retains the natural pairing of the light chain and heavy chain variable regions, and has the advantages of good gene diversity, high titer, good antibody affinity, and strong specificity, and provides a new technical solution for the discovery of rabbit monoclonal antibodies.
[0054] In step 1 of the preparation method of the present invention, the immunized antigen is from plants, animals or microorganisms. Preferably, the microorganisms are pathogenic microorganisms. In some embodiments, the microorganisms include viruses, fungi, and bacteria. The type of antigen in the present inve...
Embodiment 1
[0095] Embodiment 1 animal immunization
[0096] For each target project, 3 healthy female New Zealand rabbits aged 4-6 months were used to immunize the neck, limbs and back with Taq enzyme, and the immunization times were 6-9 times at two weeks interval, and the immunization dose was 100 μg.
[0097] Serum was collected after the third and fifth immunizations. If the serum titer was not lower than 243K, the spleen, intestinal lymph nodes and PBMCs were collected 7 days after the sixth immunization. If the serum is unqualified, after 2 more immunizations, take the serum for verification, and take the spleen, intestinal lymph nodes and PBMC 7 days after the ninth immunization.
Embodiment 2
[0098] The preparation of embodiment 2 single cell suspension
[0099] 1. Preparation of Spleen and Enteric Lymph Node Cell Suspension
[0100] Place the extracted spleen and intestinal lymph nodes on a petri dish, add 10 mL of PBS+2% FBS, then place the spleen on a 70 μm cell mesh, and use a 2 mL syringe plunger to grind the spleen. After grinding, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 400g for 6min at 4°C, discard the supernatant, and resuspend the cells in FACS buffer for direct cell staining and flow sorting, or add cells for cryopreservation Dilute cells to 1x10^7 cells / mL with cell freezing solution, divide 1 mL into cryopreservation tubes on average, freeze in liquid nitrogen, and take it at any time.
[0101] 2. Preparation of peripheral blood mononuclear cell (PBMC) cell suspension:
[0102] The PBMC separation method is as follows: the obtained fresh blood sample is diluted 1:1 with the diluent, take a 50mL centrifuge tube, add 20mL ...
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