Monoclonal antibody for paralichthys olivaceus resistant T cell surface marker molecule CD4-1 as well as preparation method and application thereof
A monoclonal antibody, cell surface technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, microorganism-based methods, biochemical equipment and methods, etc., to achieve novel design effects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0039] Example 1: Preparation of CD4-1 antigenic peptide and monoclonal antibody of flounder T cell surface marker
[0040] 1. Preparation of Antigenic Peptides
[0041] (1) The amino acid sequence of flounder CD4-1 was downloaded from NCBI GenBank database, the transmembrane sequence of flounder CD4-1 was analyzed by TMHMM software, and its structural domain was analyzed by SMART software. The purpose of predicting the transmembrane region and domain is to ensure that the designed antigenic peptide is located on the extracellular region and Ig-like domain of the flounder CD4-1 protein. The results showed that the transmembrane region of flounder CD4-1 was in the 415-437 region, in which the 1-414 region was the extracellular region, and the 438-464 region was the intracellular region ( figure 1 A). There are four extracellular Ig-like domains in flounder CD4-1 molecule, of which amino acids 19-118, 200-320 are variable domains, and amino acids 122-193, 325-408 are constant...
Embodiment 2
[0082] Example 2: Indirect enzyme-linked immunoassay identification of monoclonal antibody of the present invention
[0083] (1) Coating antigen: Dilute the unconjugated KLH CD4-1 polypeptide with carbonate coating solution (pH 9.6) to a concentration of 50µg / ml, add it to a 96-well microtiter plate (100µl / well), 4 ℃ coated overnight;
[0084] (2) Aspirate the coating solution, wash with PBST, 5min each time, three times;
[0085] (3) Add 200µl 3% bovine serum albumin (in PBS) to each well and block at 37°C for 1 hour;
[0086] (4) Wash three times with the method (2);
[0087] (5) Add the culture supernatant of the hybridoma cells screened and cloned above as the primary antibody to the microtiter plate at 50 μl per well, and incubate at 37°C for 1 hour;
[0088] (6) Wash three times with method (2);
[0089] (7) Add alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1:4000) as the secondary antibody to the microtiter plate at 50 µl per well, and incubate at 37°C fo...
Embodiment 3
[0093] Example 3: Identification of monoclonal antibodies of the present invention by indirect immunofluorescence method
[0094] (1) Percoll discontinuous density gradient centrifugation of flounder peripheral blood leukocytes was resuspended in 0.01M PBS, and the concentration was adjusted to 1×10 7 pieces / ml.
[0095] (2) Drop the blood cell suspension on a clean glass slide, 10 µl per drop, settle in a wet box at room temperature for 1 hour, take it out and fix it in acetone for 20 minutes, take it out and air dry.
[0096] (3) Use the hybridoma supernatant (hybridoma culture medium) as the primary antibody, and the negative control as the myeloma cell supernatant, add it to the blood cell sample on the glass slide, and incubate in a 37°C humid box for 45 minutes.
[0097] (4) Take out the slide and wash it three times with 0.01M PBS for 5 minutes each time.
[0098] (5) Use fluorescein isothiocyanate-labeled goat anti-mouse antibody as the secondary antibody, add it to ...
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com