Anti-chikungunya fever fully human monoclonal antibody targeting specific epitopes and its application
A technology of monoclonal antibody and chikungunya virus, which is applied in the field of microbiology and immunology, can solve the problems of non-specific therapeutic drugs, achieve low immunogenicity, improve curative effect, and good stability
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Embodiment 1
[0031] Example 1 Screening and Preparation of Human Anti-Chikungunya Virus Monoclonal Antibody
[0032] 1.1 Blood sample collection
[0033] After obtaining informed consent, Guangzhou Eighth People's Hospital provided peripheral blood from a chikungunya infected patient 11 days after recovery for subsequent experiments.
[0034] 1.2 Sorting single cells by flow cytometry
[0035] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:
[0036] 1.2.1 Take fresh anticoagulated whole blood (EDTA anticoagulated) and dilute the whole blood with an equal volume of PBS.
[0037] 1.2.2 Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1.
[0038]...
Embodiment 2
[0105] Example 2 Cell neutralization experiment
[0106] 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, inoculation volume 200 μL / well, cultured in 37°C 5% CO2 cell incubator;
[0107] 2.2 On the day of the experiment, use medium MEM+2% FBS for the purified monoclonal antibody from the initial concentration (12H9 monoclonal antibody initial concentration 100 μg / ml, Ab2 control antibody 200 μg / ml, refer to the 8B10F8 heavy chain and light chain variable 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 CHO-S cells, obtained and preserved by Protein A affinity chromatography purification, named by the laboratory For 4-fold dilution of Ab2), add to a 96-well culture plate with a volume of 120 μL / well; then add 120 μL of chikung...
Embodiment 3
[0112] Example 3 Monoclonal Antibody Recognition Epitope Research
[0113]3.1 Screen specific clones by ELISA: Coat anti-chikungunya virus monoclonal antibody (100 μg / mL, 150 μL / well) on a 96-well enzyme-linked plate, overnight at 4°C; discard the coating liquid, add 5 mg / mL BSA to block Buffer (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); 100μL of the phage loop 7 peptide library was added (the original library used TBST According to 1:10 dilution, containing about phage 2×10 11 pfu / 100μL), incubated at room temperature for 30min with gentle shaking; discarded liquid, washed 10 times with TBST to wash off unbound phage; added 100μL eluent (1mg / mL BSA, 0.2mol / L Glycine-HCl, pH2.2 ), shake gently at room temperature for 10 minutes, quickly suck out the liquid and add 15 μL of neutralizing solution (1mol / L Tris-HCl, pH9.1) to neutralize; take 1 μL of the eluted phage to measure the titer, and the r...
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