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.

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