This article details a method for producing recombinant antibodies using a cationic lipid-based transfection agent. The process involves transfecting mammalian cells with plasmids carrying genes for metallopeptidase-targeting antibodies and green fluorescent protein (GFP).
Begin with a tube containing a cationic lipid-based transfection agent and add recombinant expression plasmids.
The plasmid carries a gene encoding metallopeptidase-targeting recombinant antibodies and green fluorescent protein or GFP under one promoter, while the antibiotic resistance gene is under another promoter.
Positively charged lipids interact with a negatively charged plasmid, forming a complex.
Transfer the complexes into a multi-well plate containing mammalian cells, enabling the plasmid to enter the cytoplasm.
The plasmid reaches the nucleus and gets transcribed into mRNAs for antibiotic resistance proteins and bicistronic mRNAs encoding recombinant antibodies and GFPs.
These mRNAs lead to the production of recombinant antibodies, GFPs, and antibiotic resistance proteins.
Post-transfection, replace the medium with a serum-enriched medium.
Introduce an antibiotic, selectively eliminating non-transfected cells while the antibiotic-resistant transfected cells survive.
Using the fluorescence-activated cell-sorting technique, collect the green fluorescent cells expressing recombinant antibodies.
Seed the diluted transfected cells in a serum-free medium and incubate to produce recombinant antibodies against metallopeptidase.
Grow the pET-28a(+) recombinant antibodies aminopeptidase in BL21-transformed bacteria in the presence of 0.4 millimolar beta-D-1-thiogalactopyranoside in an orbital shaker at 37 degrees Celsius for 10 hours. Seed 100 microliters 0.5 x 105 Chinese hamster ovary cells per well into a 96-well plate for incubation at 37 degrees Celsius in a 6% carbon dioxide atmosphere for 18 to 24 hours.
When the cells reach 80% to 90% confluence, dilute the pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with one microliter of Lipofectamine 2000 and 49 microliters of Opti-MEM.
After 20 minutes of incubation, add 100 microliters of the mixture to each well of a 96-well plate containing CHO cells. At four to six hours post-transfection, replace the medium with DMEM/F12 supplemented with 10% FBS.
After 48 hours of incubation, add 400 micrograms per microliter G418 to each well to select the stably transfected cells. After 10 days of selection using DMEM/F12 medium supplemented with 10% FBS and 400 micrograms per microliter G418, sort the cells with fluorescence-activated cell sorting. Serially dilute the harvested positive cells. Seed them at an average of 0.5 to two cells per well in a 96-well plate, and culture in 37 degrees Celsius 6% carbon dioxide incubator.
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