Anti-CEACAM5 nanometer antibody
A nanobody and expression carrier technology, which is applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of low CEA production and lack of specificity in diagnosis, etc. Achieve low immunogenicity, high stability, and high throughput
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0031] Example 1: Construction of a nanobody library against CEACAM5 protein.
[0032] The recombinant CEACAM5 protein was mixed with Freund's adjuvant to immunize alpacas for 4 times. After the 4 times of immunization, 50 mL alpaca peripheral blood lymphocytes were extracted and total RNA was extracted; then the VHH region of the antibody was cloned by Nest-PCR technology, and the cloned product was inserted into phage by homologous recombination to obtain a phage display library.
Embodiment 2
[0033] Example 2: Affinity panning and library amplification after panning.
[0034] (1) Affinity panning
[0035] 1) Dilute the recombinant CEACAM5 protein with carbonate buffer solution with a pH value of 9.6 to a final concentration of 5 μg / mL, add 100 μL / well into the wells of the enzyme label, coat 8 wells per target molecule, and coat overnight at 4°C ;
[0036] 2) Discard the coating solution, wash with PBS 3 times, add 300 µL of 3% BSA-PBS blocking solution to each well, and block for 1 hour at 37°C;
[0037] 3) Wash 3 times with PBS, add 100 µL phage library, and incubate at 37°C for 1 hour;
[0038] 5) Aspirate unbound phage, wash 6 times with PBST and 2 times with PBS;
[0039] 6) Add 100 µL Gly-HCl eluate, incubate at 37°C for 8 min, and elute the specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube, and quickly neutralize the buffer with 10 µL Tris-HCl and;
[0040] 7) Take 10 µL for gradient dilution, measure the titer, and calcu...
Embodiment 3
[0049] Example 3: Identification and analysis of specific phage clones.
[0050] (1) Phagemid rescue
[0051] 1) From the plate with the eluate titer in the first round of panning, randomly pick 96 single clones with a sterilized toothpick and inoculate them in 1mL 2×YT-A, culture at 37°C and 220 r / min for 8 h with shaking;
[0052] 2) Take 200 µL of the above culture, add M13K07 phage at the ratio of cell:phage = 1:20, let stand at 37°C for 15 minutes, and shake at 220 r / min for 45 minutes;
[0053] 3) Add 800 µL of 2×YT-AK, culture at 30°C overnight with shaking;
[0054] 4) The next day, centrifuge at 12,000 rpm for 2 minutes, and take the supernatant for monoclonal ELISA identification.
[0055] (2) Identification of positive phage clones
[0056] 1) Dilute the recombinant CEACAM5 protein with carbonate buffer solution with a pH value of 9.6 to a final concentration of 2 μg / mL, add 100 μL / well to the enzyme-labeled well, and coat overnight at 4°C;
[0057] 2) Discard t...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap