Hi All,
I'm trying to express a rAb in CHO or HEK293 cells and I am having an issue getting functional antibody. WB shows bands for HC and LC under reducing conditions and non-reduced shows a single band at the expected MW for antibody dimer.
Right now we are using adherent cells (both CHO and HEK293) in DMEM/P+S/10% FBS and transfections are being performed with 20 uL of Effectene and anywhere from 400ng-2ug of DNA (many transfections) in 6-well plates.
We are testing the functionality of the antibody through an ELISA and even when we have good bands on the WB we see no activity on the ELISA. We are using two single gene vectors (pcDNA3.1) and have been transfecting in different ratios of LC to HC (all the way from 5:1 to 1:5) and still have no success.
Not sure what else we can try here aside from sequencing our mAb and starting over from scratch.
Does your rAb has a glycosylation site (N-X-S/T) in the CDRs? glycosylation can depend on cells and culture medium
Firstly, as long as you're using FBS, you also have certain amount of bovine Ig's in your media, be cautious with sds-page bands interpretation. WB detection antibody must be specific of your target antibody, check for cross reactivity loading a control lane with culture media.
Second, check your controls in ELISA, you need a positive for your antigen. ELISA can be tricky sometimes, include as many controls as you can (Blocking agents, blocking by FBS, secondary Ab control, TMB, etc...)
Third, is a synthetic antibody?Or is a Chinese/Humanized antibody based on an hybridoma one? If the second is true and the original one is working fine, it could be an issue with humanisation process....
Check this points first and if they're fine, we can discuss others possibilities...
Thanks for your answers all, I took another look at our sequence data and it doesn't match up with what we would expect, therefore we are going back to cloning the LC and will be doing transfections over again. Ramon Roman Roldan I'll be sure to keep this in mind when we start doing ELISAs again, thanks!
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