Antibody, conjugate and detection kit for thioredoxin
An anti-thioredoxin, antibody technology, applied in immunoglobulin, anti-enzyme immunoglobulin, biochemical equipment and methods, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0090] Anti-Trx Hybridoma Monoclonal Antibody Screening
[0091] 1. Mice Immunization
[0092] The immunization antigen was thioredoxin (Trx), the immunization dose was 100 μg / mouse, and the immunization period was 14 days. The first subcutaneous multi-point immunization was performed on the back, the subsequent 3 immunizations were intraperitoneal injection, and the last injection was boosted by tail vein; The immune adjuvants were Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma.
[0093] 2. Mouse Fusion
[0094] Take out the mouse spleen, grind it on a 400-mesh filter to obtain dispersed single splenocytes, mix the splenocytes and myeloma cells with PEG, reconstitute by centrifugation after the termination of the culture medium, spread a 96-well plate, and place it in a carbon dioxide medium to stand still Cultured for 7 days. The medium in the examples was purchased from Gibco's RPMI 1640 medium, the fetal bovine serum was purchased ...
Embodiment 2
[0098] Expression plasmid construction
[0099] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen. Take the anti-Trx hybridoma monoclonal 5E18 cell line and resuscitate for use.
[0100] 1. Antibody gene preparation
[0101] The mRNA was extracted from the anti-Trx hybridoma cell line 5E18, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transformed into DH5α competent cells. After colonization, four heavy chain and light chain gene clones were respectively taken and sent to a gene sequencing comp...
Embodiment 3
[0108] Stable cell line screening
[0109] 1. The recombinant antibody expression plasmid was transiently transfected into CHO cells, and the activity of the expression plasmid was determined.
[0110] The plasmid was diluted to 400 μg / ml with ultrapure water, and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100 μl plasmid with 700 μl cells, transfer to electroporation cup, electroporation, take samples and count on the 3rd, 5th, and 7th day, and collect the sample for detection on the 7th day.
[0111] Coating solution (main component NaHCO 3 ) Dilute Trx to 3 μg / ml, 100 μl per well, overnight at 4°C; the next day, wash solution (main component Na 2 HPO 4 +NaCl) and washed twice, pat dry; add blocking solution (20%BSA+80%PBS), 120μl per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100μl / well, 37°C, 30min; wash 5 times with washing solution, pat dry; add goat anti-mouse-HRP, 100μl per well, 37°C, 30min; wash 5 times with washing sol...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More