Transient plant production of Salmonella Typhimurium diagnostic antibodies

Kopertekh, Lilya; Meyer, Torsten; Freyer, Cornelia; Hust, Michael

Antigen binding of TM43-E10 antibody formats was determined by ELISA in 96-well microtitre plates (Greiner Bio-One) coated overnight at 4 C with OmpD recombinant protein or plant material infected with S. Typhimurium. After coating the wells were washed three times with phosphate buffer (PBST) and blocked through 2 h incubation in PBST supplemented with 2% skimmed milk (PBST-M). Blocked microtitre plates were washed three times with PBST-M and incubated with purified antibody fragments for 2 h at room temperature. After further washing bound scFv and scFv-Fc were detected with the anti-c-Myc mouse and anti-human lgG (Fc) mouse antibody, respectively, followed by the addition of anti-mouse goat antibody. The visualization was carried out with p-nitrophenyl phosphate as substrate and the reaction was stopped by adding 2 M NaOH. Absorbance at 450 nm was measured by using a SUNRISE TM microtitre plate reader (Tecan). For antigen titration ELISA different dilutions of OmpD recombinant protein ranging from 0.1 ng/ml to 1000 ng/ml were coated in duplicates to wells overnight at 4 C. The detection was carried out with a concentration of antibody at half maximum saturation (determined by antibody titration ELISA) as described above. 56 nM of scFv-TM43-E10 and 25 nM of scFv-Fc-TM43-E10 antibody were used in the experiments. The antibody detection limit was defined as 3 SDs above the background. Antigen and antibody titration ELISA tests have been repeated twice.