American Society of Hematology

Figure 4.

$Figure 4. Expression and activity of scFv28.3-HaaT. (A) Prokaryotic expression: 10 μg protein extract from the periplasmic, soluble (S), and insoluble (I) fractions of E coli JM83 expressing scFv28.3-HaaT were resolved under reducing conditions on 10% polyacrylamide electrophoresis gels and blotted onto nylon membranes. Recombinant proteins were revealed by incubation of the membranes with HRP-conjugated anti–c-myc antibody and by enhanced chemiluminescence (ECL). (B) Fifty nanograms scFv28.3-HaaT produced in Cos cells, under native (N) or reducing (R) conditions, was analyzed by Western blotting as described in panel A. Molecular size markers (kDa) are shown on the left. (C) CD28+ Jurkat T cells were incubated with supernatant from transfected Cos cells containing 2 μg/mL scFv28.3-HaaT (black histogram, first row) or with supernatant from control Cos cells (white histogram, second row), revealed with anti-HaaT antibody plus FITC-conjugated donkey antirabbit Ig antibody (DAR) and analyzed by cytofluorometry. CD28- U937 cells were analyzed in parallel with supernatant containing scFv28.3-HaaT (dark gray histogram, third row) or with control supernatant (light gray histogram, last row). (D) The reactivity of Cos-produced scFv28.3-HaaT was examined by ELISA. CD28-Fc molecules were coated onto microtiter plates. Purified scFv28.3-HaaT at different dilutions in PBS-Tween was added and incubated for 1 hour at 37°C. After washing, bound scFv28.3-HaaT molecules were revealed with a rabbit anti-HaaT antibody followed by a peroxidase-conjugated DAR as a third-step reagent. Colorimetric analysis was performed after reaction of ABTS with peroxidase. X indicates signal without sc28.3-HaaT; ♦ and ⋄, signals obtained with incubations of HaaT alone and with an irrelevant scFv-M13 fusion antibody, revealed with a peroxidase-labeled anti-M13 antibody. Data are presented as means of triplicates.$

Expression and activity of scFv28.3-HaaT. (A) Prokaryotic expression: 10 μg protein extract from the periplasmic, soluble (S), and insoluble (I) fractions of E coli JM83 expressing scFv28.3-HaaT were resolved under reducing conditions on 10% polyacrylamide electrophoresis gels and blotted onto nylon membranes. Recombinant proteins were revealed by incubation of the membranes with HRP-conjugated anti–c-myc antibody and by enhanced chemiluminescence (ECL). (B) Fifty nanograms scFv28.3-HaaT produced in Cos cells, under native (N) or reducing (R) conditions, was analyzed by Western blotting as described in panel A. Molecular size markers (kDa) are shown on the left. (C) CD28⁺ Jurkat T cells were incubated with supernatant from transfected Cos cells containing 2 μg/mL scFv28.3-HaaT (black histogram, first row) or with supernatant from control Cos cells (white histogram, second row), revealed with anti-HaaT antibody plus FITC-conjugated donkey antirabbit Ig antibody (DAR) and analyzed by cytofluorometry. CD28^- U937 cells were analyzed in parallel with supernatant containing scFv28.3-HaaT (dark gray histogram, third row) or with control supernatant (light gray histogram, last row). (D) The reactivity of Cos-produced scFv28.3-HaaT was examined by ELISA. CD28-Fc molecules were coated onto microtiter plates. Purified scFv28.3-HaaT at different dilutions in PBS-Tween was added and incubated for 1 hour at 37°C. After washing, bound scFv28.3-HaaT molecules were revealed with a rabbit anti-HaaT antibody followed by a peroxidase-conjugated DAR as a third-step reagent. Colorimetric analysis was performed after reaction of ABTS with peroxidase. X indicates signal without sc28.3-HaaT; ♦ and ⋄, signals obtained with incubations of HaaT alone and with an irrelevant scFv-M13 fusion antibody, revealed with a peroxidase-labeled anti-M13 antibody. Data are presented as means of triplicates.

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