Monoclonal antibody of glycosylphosphatidylinositol-anchored proteins (C4.4A) and application of monoclonal antibody
A technology of glycosylphosphatidylinositol and glycoprotein, which is applied in the field of biomedicine and can solve problems such as the lack of antibody tools
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[0128] Preparation of monoclonal antibodies
[0129]Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).
[0130] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (a...
Embodiment 1
[0150] Example 1 Elisa (immunoenzyme-linked) pairing verification of antibody and antigenic polypeptide
[0151] Coat the 96-well ELISA plate with ascites antibody to be paired, incubate, wash and block overnight with skim milk, wash with PBS, and store at 4°C until use. Antigen peptides were incubated, washed with PBS, and controls were set at the same time. HRP-labeled detection antibody was added to the previously incubated ELISA plate. TMB color reaction, microplate reader reading. The titers of the 8 cell lines obtained by screening are shown in Table 2:
[0152] Table 2 Titers of 8 cell lines
[0153] Antibody name clone number 3.125K 6.25K 12.5K 25K 50K 100K 200K 400K 800K 1600K PC NC whether it is passed potency anti-C4.4A 2A1 2.866 2.791 2.73 2.566 1.846 1.284 0.682 0.304 0.121 0.064 3.016 0.238 qualified 400K Anti-C4.5A 3B6 2.652 2.641 2.243 1.561 1.084 0.583 0.339 0.152 0.081 0.04 3.188 0.151 ...
Embodiment 2
[0154] Example 2 Flow Cytometry Verification of Antibodies
[0155] PC9 cells were washed with PBS, digested with EDTA, transferred to a centrifuge tube, centrifuged to discard the supernatant and resuspended with PBS, blocked with goat serum at room temperature for 1 hour, incubated with primary antibody, working concentration 50ug / ml, incubated with fluorescent secondary antibody, working concentration 1 : 300. result( figure 2 ) shows that this antibody can be applied to the flow cytometry experiment of C4.4A.
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