Antibody of complete human-derived anticomplement factor H
A fully human, complement technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problem of difficult to develop therapeutic reagents
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Embodiment 1
[0042] Example 1 Preparation of fully human monoclonal CFH antibody TRN1004
[0043] 1. PBMC Isolation and Single Memory B Cell Sorting
[0044] Using avidin-labeled CFH 15-mer polypeptide (SEQ ID NO: 9) as a bait, the flow cytometry sorter was used to sort out the pooled peripheral blood mononuclear cells (PBMC, collected from patients expressing CFH autoantibodies) Single memory B cells, CD20+ / CD27ALL / CFH- antigen-binding memory B cells were sorted by fluorescent-fluorescent labeling. To minimize false positives, Streptavidin Fluor647 and Brilliant Violet 421 were labeled with Alexa. Each fluorophore was labeled with streptavidin on a separate aliquot which was then mixed together and interacted with the biotinylated antigenic peptide. Cells of both fluorophores showing elevated fluorescence were sorted into wells of a single 96-well plate.
[0045] 2. Isolation of VHDJH and VL genes
[0046] VHDJH and VLJL gene segment pairs of mAb were amplified by RT / PCR from flow-sor...
Embodiment 2
[0057] Example 2 Binding site verification test
[0058] Biotin-labeled CFH 15-mer peptide antigen (SEQ ID NO:9), each amino acid was mutated separately to form 15 mutant peptides, plus the wild-type peptide as a positive control and the sequence-scrambled peptide as a As a negative control, use Elisa to detect the binding of the TRN1004 antibody to the above 18 polypeptides at different concentrations, draw a curve and calculate the Area Under Curve (AUC), and analyze the key binding sites between the antibody and the polypeptide. The result is as figure 2 It was shown that the key binding sites between the TRN1004 antibody and the CFH antigen polypeptide are the 5th, 6th, 7th, 8th, 11th, and 12th positions in the polypeptide sequence GPPPPIDNGDITSFP, which proves that it can specifically bind to the antigenic polypeptide. in, figure 2 Among them, CFH is positive control 1, its sequence is as shown in SEQ ID NO: 9, OR is positive control 2, its sequence is as shown in SEQ...
Embodiment 3
[0059] Example 3 Determination of antibody affinity
[0060] The capture molecules were coupled to the SA chip first, and then the dextran surface of the chip was activated, and the coupling amount was determined by the injection time. Finally, the SA chip was used to capture the molecular capture ligand: the prepared fully human anti-rabies virus neutralizing antibody was used as the ligand, and the signal value obtained by calculation was used to determine the injection concentration and contact time of the monoclonal antibody. Affinity and kinetics analysis of monoclonal antibody binding to CFH 15-mer polypeptide (antigen): CFH 15-mer polypeptide was diluted with HBS-EP buffer as analyte, analyte flowed through the chip in sequence with increasing concentration, and signals were obtained respectively curve. Each concentration was regarded as one cycle, and after completing one cycle, the chip was regenerated with 10 mmol / L glycine-hydrochloric acid to return to the origina...
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