Binding protein comprising antigen binding domain and production method and application thereof
A technology of binding domains and binding proteins, applied in the field of antibodies, can solve the problems of difficulty, difficulty in anti-phospholipid syndrome kits, and few natural anti-β2-glycoprotein I antibodies, etc., and achieve the effect of good affinity
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Embodiment 1
[0091] A method for preparing a binding protein of an antigen-binding domain, comprising the following steps:
[0092] (1) Recovery and culture of hybridoma cells:
[0093] Hybridoma cells secreting mouse-derived anti-β2 glycoprotein I antibody were prepared by hybridoma technology, and the cells were cryopreserved in liquid nitrogen. After the hybridoma cells were recovered in a water bath at 37°C, 1640 containing 10% fetal bovine serum and 1% double antibody was used. Medium at 37°C with 5% CO 2 in an incubator for expanded culture.
[0094] (2) Identification of antibody subtypes:
[0095] The hybridoma cell supernatant was detected by mouse monoclonal antibody typing reagent (sigma), and the subtype of the antibody was IgG2a, and the light chain was Kappa.
[0096]Table 1 Identification of antibody subclasses
[0097] IgG1 IgG2a IgG2b IgG3 IgA IgM mouse antibody 0 1.318 0.015 0.01 0.01 0 negative control 0 0.02 0.023 0.01 0.01...
Embodiment 2
[0126] The antibody (binding protein) prepared in Example 1 and its affinity and activity were identified.
[0127] The antibody obtained in Example 1 has the heavy chain shown in SEQ ID NO. 11 and 12 and the light chain shown in 13 and 14 according to the analysis of the binding protein (WT). The sequence information is shown in the following table.
[0128]
[0129]
[0130] After analysis, the complementarity determining region of the heavy chain is:
[0131] CDR-VH1 is G-X1-S-I-T-S-X2-Y-V;
[0132] CDR-VH2 is I-X1-Y-S-X2-S-T;
[0133] CDR-VH3 is A-X1-G-R-D-D-G-V-T-W-X2-A-Y;
[0134] The complementarity determining regions of the light chain are:
[0135] CDR-VL1 is Q-S-X1-S-T-S-S-Y-S-Y;
[0136] CDR-VL2 is Y-X1-S;
[0137] CDR-VL3 is Q-H-S-X1-E-I-P-X2-T;
[0138] The above complementarity determining region was used as the backbone, and X1 and X2 were used as mutation sites to carry out mutation. The combination of mutations is shown in Table 2.
[0139] Table...
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