Figure 4. Immunoglobulin expression in transfected 293F cells.

(A) RT-PCR analysis for neo (560 bp), L (720 bp) and H (1420 bp) chains in cells transfected with plasmid pN4 at passage 4 (p4) to show that the transgenes were being transcribed at equivalent levels. (B) ELISA result shows AGR2 binding by IgG1 from transfected 293F cells (in triplicate). The color intensity indicates similar binding affinity between the IgG1 constructs p7-2 and p13-1 vs. P3A5. Background consists of untransfected 293F and p6-2, which is a non-productive construct. Detection is by either HRP anti-human IgG for the transfected cell media or HRP anti-mouse IgG2a for P3A5. (C) ELISA shows IgG isolation by spin filtration. Background level was detected in the 100 μL flow-through well (4^th) vs. the three wells (1^st – 3^rd) containing different amounts of the filtrate/retentate. (D) Histogram representation of ELISA data shows comparable binding between 293F-synthesized human IgG and P3A5 in tissue digestion media of LuCaP lines (147, 35CR, 86.2, 105) with different levels of AGR2 expression. p12-1 is a defective construct, and served as negative control. Absorbance units are indicated on the y-axis. (E) RT-PCR analysis of 293F/p40-1 P1G4 chimeric detected transcripts for bsr (420 bp), V_L, V_LC_κ, V_H, and V_HC_γ1. (F) The histogram bars show AGR2 binding activities detected in media supernatant of p40, p50, and p40 clones A1-A6 (absorbance units on the y-axis). The positive control is mouse P1G4 and the negative control 293F cells. “Early” and “later” indicate media harvested during early and late passages, respectively, of cultures after drug selection.