Novel anti-CXCR4 antibody and its use for the detection and diagnosis of cancer
An antibody, CDR-H1 technology, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antitumor drugs, measuring devices, etc., can solve the problem of tumor growth and angiogenesis having no effect
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Embodiment 1
[0292] Example 1: Production of anti-CXCR4427aB1 monoclonal antibody (Mab) (F50067-006(5C)427aB1 cl1B, CNCM number I-4018)
[0293] To generate monoclonal antibodies against CXCR4, Balb / c mice were immunized with recombinant NIH3T3-CXCR4 cells and / or peptides corresponding to the N-terminus and loop of CXCR4 cells. In the first immunization, mice aged 6-16 weeks are immunized subcutaneously (sc) with the antigen in complete Freund’s adjuvant, and then immunized with antigen sc in incomplete Freund’s adjuvant for 2 to 6 Times. Immune response was monitored by bleeding after the orbit. The serum was screened by ELISA (as described below), and mice with higher titers of anti-CXCR4 antibodies were used for fusion. Two days before sacrifice and spleen excision, mice were boosted intravenously with antigen.
[0294] -ELISA
[0295] To select mice that produce anti-CXCR4 antibodies, sera from immunized mice were tested by ELISA. In short, a microtiter plate was coated with purified [1-...
Embodiment 2
[0298] Example 2: 427aB1 monoclonal antibody recognizes both CXCR4 monomer and homodimer in cell lysate
[0299] Wash NIH3T3-hCXCR4 transfected cells, MDA-MB-231 (breast) and U937 (AML) cancer cells twice in PBS. Use the following buffer: 20mM TrisHCl pH8.5, 100mM(NH 4 ) 2 SO 4 , 10% glycerol, 1% CHAPSO and 1% protease inhibitor mixture will be 100.10 6 Cells / ml were lysed for 30 minutes. The cell lysate was collected by centrifugation at 10000 g at +4°C for 20 minutes and analyzed by western blotting with 427aB1 monoclonal antibody as the primary antibody. figure 1 It was shown that mAb 427aB1 recognizes both CXCR4 monomer and homodimer on NIH3T3-CXCR4. The cancer cell lines MDA-MB-231 and U937 seem to express CXCR4, which is mainly homodimer.
Embodiment 3
[0300] Example 3: 427aB1 monoclonal antibody immunoprecipitation of both CXCR4 monomer and homodimer
[0301] Use pH8.5 containing 100mM (NH4) 2 SO 4 Wash the NIH3T3-CXCR4 cell pellet with 20mM Tris HCl, and then resuspend it in the lysis buffer (20mM TrisHCl, pH8.5, containing 100mM(NH4) 2 SO 4 , 10% glycerol, 1% CHAPSO and a mixture of 10 μL / mL protease inhibitor). The cells were disrupted using a Potter Elvehjem homogenizer. The dissolved membrane was collected by centrifugation at 105000g at +4°C for 1 hour, and then incubated with Sepharose 4B beads conjugated with 427aB1 monoclonal antibody at +4°C overnight, the mixture was poured into a glass column and washed with lysis buffer. The protein captured by the 427aB1 monoclonal antibody was eluted and analyzed by western blotting with the anti-427aB1 monoclonal antibody as the primary antibody. Combine and concentrate the target fractions and use them for WB analysis and preparative SDS-PAGE resolution (4-12% Bis-Tris gel). ...
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