Figure 2.

Quantitative staining of IgG rich lupus kidney. (A) The human IgG1 mAb heavy chain vector was genetically modified to express the FLAG sequence (DYKDDDDK) at the C-terminus of each heavy chain, enabling detection with an anti-FLAG secondary antibody. Top = Protein schematic of respective domains of IgG1 with inserted tag in green. Bottom = C-terminal amino acid and 3’ nucleotide sequences of heavy chain construct. (B) SDS PAGE and subsequent Coomassie staining or western blotting with anti-human IgG (hum.IgG) or anti-FLAG IgG on the selected and reverted PB5 TII mAb in the absence or incorporation of the FLAG tag. (C) Reactivity of FLAG-tagged PB4 (top) and PB5 (bottom) variants with Vim^hi and nuclear zones of inflamed lupus kidney as determined by multi-color confocal microscopy. (i) Each dot represents the MPI of the TII mAb channel for the indicated subcellular region for a respective FOV (ii) Co-variances of MPIs between the AVA and V9 or Hoechst channel. Dots represent correlation coefficients between the TII mAb channel and V9 or Hoechst channel for respective FOVs. Wilcoxon matched-pairs tests were performed between MPIs of Vim^hi and nuc subcellular areas. Mann-Whitney tests were performed between somatically hypermutated (mut) and germline reversion values. na = not applicable; ns = not statistically significant. *p < 0.05, **p < 0.001, ***p < 0.001, ****p < 0.0001. (D) Representative examples of co-staining of lupus renal tissue with FLAG-tagged PB4 and PB5 AVA variants. Black scale bar = 25 microns.