Fully-humanized monoclonal antibody against chikungunya fever and with high neutralizing activity, and application thereof
A monoclonal antibody, chikungunya virus technology, applied in the field of microbiology and immunology, can solve problems such as non-specific therapeutic drugs, achieve low immunogenicity, improve efficacy, and avoid the generation of drug-resistant strains Effect
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Embodiment 1
[0029] Example 1. Screening and Preparation of Human Anti-Chikungunya Virus Monoclonal Antibody
[0030] 1.1 Blood sample collection
[0031] After obtaining informed consent, the Guangzhou Eighth People's Hospital provided the peripheral blood of a chikungunya infected patient 15 days after recovery for subsequent experiments.
[0032] 1.2 Sorting single cells by flow cytometry
[0033] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation. The process is as follows:
[0034] 1.2.1 Take fresh anticoagulated whole blood (EDTA anticoagulation) and dilute the whole blood with an equal volume of PBS.
[0035] 1.2.2 Add a certain volume of separation liquid to the centrifuge tube, and spread the diluted blood sample above the liquid surface of the separation liquid to keep the interface between the two liquid surfaces clear. Separation solution, anticoagulated undiluted whole blood, PBS (or normal saline) volume is 1:1:1.
[0036] 1.2.3...
Embodiment 2
[0118] Example 2. Cell Neutralization Experiment
[0119] 2.1 The day before the experiment, the Vero cells were diluted to 1.5×10 with medium (MEM+10%FBS) 5 cells / mL concentration, inoculated into 96-well cell culture plate, inoculated volume 200μL / well, placed in 37°C 5% CO2 cell incubator;
[0120] 2.2 On the day of the experiment, the purified monoclonal antibody was used in the medium MEM+2% FBS from the initial concentration (the initial concentration of 8D1 monoclonal antibody was 100ug / ml, and the Ab2 control antibody was 200ug / ml (refer to the variable heavy chain and light chain of 8B10F8 disclosed in U.S. Patent US9738704). Region sequence, chemically synthesized variable region gene and obtained full-length antibody gene by fusion PCR technology, cloned into the expression vector pMH vector, transfected into CHO-S cells, purified by Protein A affinity chromatography, obtained and stored, named after the laboratory Ab2), diluted 4 times, added to a 96-well culture ...
Embodiment 3
[0125] Example 3. Monoclonal antibody recognition epitope research
[0126] 3.1 ELISA to screen specific clones: Coat 96-well enzyme-linked plate with anti-chikungunya virus monoclonal antibody (100 μg / mL, 150 μL / well), overnight at 4°C; discard the coating liquid and add 5 mg / mL BSA to block The buffer solution (0.1mol / L NaHCO3, pH8.6) was blocked at 4°C for 2h; the liquid was poured off, washed 6 times with TBST (1mL / L Tween-20, TBS); 100 μL of the phage loop 7 peptide library was added (the original library used TBST Diluted at 1:10, containing about 2 × 10 bacteriophages 11 pfu / 100 μL), incubate with gentle shaking at room temperature for 30 min; discard the liquid, wash 10 times with TBST, and wash away unbound phage; add 100 μL eluate (1 mg / mL BSA, 0.2 mol / L Glycine-HCl, pH 2.2 ) elution, shake gently for 10 min at room temperature, quickly aspirate the liquid and add 15 μL of neutralizing solution (1 mol / L Tris-HCl, pH 9.1) to neutralize; take 1 μL of the eluted phage ...
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