Anti-VEGF (Vascular Endothelial Growth Factor) and anti-EGFR (Epidermal Growth Factor Receptor) bispecific antibody fusion protein and application thereof
A technology of fusion protein and single-chain antibody, which is applied in the field of antibody protein, can solve the problems of low adaptability of antibody and indicator coupling, limited imaging effect, and single target, so as to broaden the application mode in vivo and reduce the false positive rate , the effect of improving the diagnosis rate
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Embodiment 1
[0036] Example 1 Preparation of anti-VEGF and anti-EGFR bispecific antibody
[0037] 1.1 Establishment of high-capacity natural antibody library.
[0038] Isolation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10ml of peripheral blood was extracted from each person. Dilute 1:1 with RPMI-1640 culture medium containing 10% heparin, add to the centrifuge tube containing lymphocyte separation medium (the volume ratio of diluted venous blood to lymphocyte separation medium is 2:1), 2,000×g, Centrifuge for 17 minutes. Aspirate the milky white mononuclear cell layer at the interface of the lymphocyte separation solution, and wash twice with PBS buffer.
[0039] 1.2 Extraction of total cellular RNA
[0040] Press every 5×10 6 Add Trizol reagent at the ratio of cells / ml, and lyse the cells by pipetting. Incubate at room temperature for 5 minutes, transfer to a DEPC-treated EP tube, add 1 / 5 volume of chloroform, shake vigorously ...
Embodiment 2
[0097] Example 2. Purification of bispecific antibodies
[0098] The sequences of the two ScFvs were re-genetically synthesized in the order that the heavy chain variable region and the light chain variable region of the VEGF single-chain antibody were connected to the light chain variable region and the heavy chain variable region of the EGFR single-chain antibody. Composition of new bispecific antibodies. Synthesized by Beijing Nuosai Biotechnology Co., Ltd.
[0099] Pick a single colony and inoculate it in 5ml LB medium, and cultivate overnight at 37°C with vigorous shaking.
[0100] Inoculate the above bacterial solution into a Erlenmeyer flask containing 400ml LB (antibiotics) at a ratio of 1:100, and incubate vigorously at 37°C for 2 hours.
[0101] The final concentration was 1 mmol / LIPTG, and the expression was induced at 37°C for 3-4 hours.
[0102] Collect the culture medium, centrifuge at 5,000 rpm for 10 minutes, and discard the supernatant. Wash the bacterial ...
Embodiment 3
[0109] Example 3. FITC-labeled VEGF ScFv, EGFR ScFv and VEGF ScFv+EGFR ScFv on the immunofluorescence results of pancreatic cancer tumor cell panc-1 (confocal)
[0110] The pancreatic cancer cell line panc-1 was used to verify the binding of VEGF ScFv, EGFR ScFv and VEGF ScFv+EGFR ScFv to tumor cells.
[0111] First, FITC (fluorescein isothiocyanate) or irdye800 labeled VEGF ScFv, EGFR ScFv and VEGFScFv+EGFR ScFv, the steps are as follows:
[0112] (1) Dialyze the protein to be cross-linked (concentration ≥ 1 mg / ml) against the cross-linking reaction solution three times (4° C.) until pH = 9.0. Preparation method of cross-linking reaction solution: 7.56g NaHCO 3 , 1.06g Na2CO 3 , 7.36g NaCl, add water to make up to 1L.
[0113] (2) FITC was dissolved in DMSO at a concentration of 1 mg / ml. The FITC used for each cross-linking should be freshly prepared and protected from light.
[0114] (3) Slowly add FITC to the antibody solution according to the ratio of P:F (protein: FI...
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