Whole-human HER2 antibodies, and coding genes and application thereof
A fully human, antibody technology, applied in applications, antibodies, genetic engineering, etc., can solve problems such as recurrence and unmet needs
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Embodiment 1
[0042] Example 1 Screening clones that specifically bind to human HER2-Fc from a fully human scFv phage library
[0043] Using the antigen-antibody binding specificity of ELISA technology, the human HER2 (extracellular domain)-Fc fusion protein (referred to as hHER2-Fc) antigen is coated on the microtiter plate, and it is specifically adhered to the coating by washing and panning. Phage on antigen. PBS (0.01M Na 2 HPO 4 12H 2 O+0.002M KH 2 PO 4 +0.14M NaCl+0.002M KCl, pH=8.6) was diluted to 5 μg / ml, added to a microtiter plate at 100 μl / well, and coated overnight at 4°C. After the plate was washed 4 times with PBST (PBS buffer containing 0.05% Tween 20), 300 μl / well of 5% BSA (purchased from Amresco, product number: 0332-100g, solution in PBS) was added, and blocked for 1 hour at 37°C. Then wash the plate twice with PBST. will contain 7×10 10 Independently cloned fully human scFv phage antibody library (this antibody library was constructed by Eureka (Beijing) Biotechn...
Embodiment 2
[0044] Example 2 Using enzyme-linked immunosorbent assay (ELISA) to identify the immunoreactivity of phages specifically binding to human HER2-Fc
[0045]The immunoreactivity of the phage specifically binding to human HER2-Fc obtained in Example 1 was further identified by enzyme-linked immunosorbent assay (ELISA). Human HER2-Fc antigen (purchased from Sino Biological Company, product number: 10004-H02H) was diluted 2 μg / ml with PBS (pH=8.6), added to the microtiter plate at 100 μl / well, and coated overnight at 4°C. After washing the plate 4 times with PBST, 300 μl / well of 5% BSA (purchased from Amresco, product number: 0332-100g, PBS solution) was added, and blocked at 37°C for 1 hour. Then wash the plate twice with PBST, add 100 μl / well phage clone suspension, and incubate at 37° C. for 2 hours. Wash the plate 4 times with PBST. Add HRP-labeled anti-M13K07 phage antibody (GE, catalog number: 27-9421-01, diluted 1:5000 in PBST, 100 μl / well), and incubate at room temperature...
Embodiment 3
[0049] Example 3 Detecting Species Cross-reactivity of 102 Human HER2-Fc-specific scFvs and Cross-reactions Between HER Family Member Molecules by ELISA
[0050] The species cross-reactivity of 102 human HER2-Fc-specific scFv and cross-reaction with HER family members were detected by ELISA. The method was the same as in Example 2, and the coated human HER2-Fc antigen was replaced with monkey HER2-Fc (purchased from Sino Biological Company, catalog number: 90295-C02H), mouse HER2-Fc (purchased from Sino Biological Company, catalog number: 50714-M02H), human HER1-Fc (purchased from Sino Biological Company, catalog number: 10001-H02H), human HER3-Fc (purchased from Sino Biological Company, catalog number: 10201-H05H) and human HER4-Fc (purchased from Sino Biological company, article number: 10363-H02H). The antigen was diluted 2 μg / ml with PBS (pH=8.6), added to the microtiter plate at 100 μl / well, and coated overnight at 4°C. After washing the plate 4 times with PBST, 300 μl / ...
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