Antibody, its preparation method and application
An antibody and dimer technology, applied in biochemical equipment and methods, chemical instruments and methods, carriers, etc., can solve problems such as false positives, reduce sensitivity and specificity of clinical applications, affect clinical diagnosis, etc., and achieve production costs low effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0035] The preparation of embodiment one anti-human D-dimer hybridoma cell line
[0036] 1. Animal immunization
[0037] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with natural human D-dimer (purchased from Meridian) according to the general immunization procedure. For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.
[0038] 2. Cell Fusion
[0039] (1) Preparation of spleen cells
[0040] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, take out their spleens in a sterile operating table, place them in a cell mesh, and grind the cells fully , pas...
Embodiment 2
[0048] Embodiment two hybridoma cell line antibody variable region sequence determination
[0049] The sequence of the variable region of the antibody of the hybridoma cell line LJ002 was determined.
[0050] (1) Extraction of RNA: According to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company), the total RNA of the above-mentioned hybridoma cell line LJ002 was extracted and immediately reverse-transcribed;
[0051] (2) Reverse transcription of RNA into DNA: refer to Thermo Scientific Reverted First strand cDNA Synthesis Kit (purchased from Thermo Company) to reverse transcribe the total RNA extracted in the previous step to obtain cDNA, and freeze it at -20°C for later use ;
[0052] (3) PCR amplification and recovery of the variable region sequence: the cDNA obtained in the above step was used as a template, and the variable region sequence of the mouse IgG subtype monoclonal antibody was used as a primer for the variable region of the heav...
Embodiment 3
[0059] Example 3 The expression vector of anti-D-dimer humanized chimeric antibody was transfected into CHO cells for expression
[0060] Carry out routine DNA recombination operations as described in "Molecular Cloning", clone the variable region of the light chain into pSRNC-Cκ, clone the variable region of the heavy chain into the corresponding site in pSRDC-Cγ1, and construct a complete anti-D-dimerization Eukaryotic expression vector of humanized chimeric antibody gene. CHO cells (preserved in this laboratory) were prepared with 10% FBS, 0.03mmol / l hypoxanthine (H), 0.003mmol / l thymidine (T), 0.1mmol / l proline (Pro), 0.1mmol Glycine (Gly), 100u / ml penicillin, streptomycin 100u / l, 2mmol / l glutamine DMEM complete culture solution in 5% CO2, 37 ° C conditions, routinely according to 1:10 passage, about 3-4 days passed down from generation to generation. The above cell culture reagents were purchased from Gibco. Using the method of gene transfection, Lipofect AMINE reagent...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More