isotype matched human igg4  (Novus Biologicals)

Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Treatment of keratinocytes with hAK23 and 4B3 antibodies results in Dsg3 depletion, and 4B3 abrogates FcRn protein level. ( a ) hTert cells were plated on 6-well plates, grown in KGM2 medium with 0.05 mM CaCl 2 until confluent, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. Thereafter, the cells were treated for 24 h with the recombinant antibodies hAK23 (12.5 µg/mL) or 4B3 (50 µg/mL). As controls, mock incubation (untreated), an isotype-matched hIgG (12.5 µg/mL IgG4, or 50 µg/mL hIgG1), and mAK23 (75 µg/mL) were used. The cells were lysed, and the level of Dsg3, Dsg1 and FcRn was analyzed by Western blot. GAPDH was included as a loading control. A representative experiment is shown. ( b – d ) Western blot signals were quantified using ImageJ software, normalized against GAPDH, and expressed as relative values compared to the untreated control. The error bars represent the SD of values obtained from four independent experiments. Statistical analysis was done using one-way analysis of variance (ANOVA) with Dunnett´s post-test. Statistically significant differences are indicated by ** = p ≤ 0.01; *** = p ≤ 0.001.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Recombinant, Incubation, Western Blot, Control, Software

Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Efgartigimod treatment protects keratinocyte monolayers against dissociation induced by PV IgG. hTert keratinocytes were plated on 24-well plates, grown in KGM2 medium with 0.05 mM CaCl 2 until confluent, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. The cells were left untreated, or efgartigimod (25 µg/mL) was applied for 30 min prior to the 24 h treatment with hAK23 (12.5 µg/mL), control IgG4 (12.5 µg/mL), PV IgG (150 µg/mL) or human control IgG (150 µg/mL). Monolayer dissociation assay was performed in triplicates, and the number of fragments was quantified using ImageJ software. A representative experiment is shown. The error bars show the SD of the mean values obtained from five independent experiments. Statistical analysis was done using two-way ANOVA with Bonferroni’s post-test. Statistically significant differences are indicated by *** = p ≤ 0.001.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Control, Software

Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Efgartigimod does not rescue the aberrant Dsg3 staining pattern induced by hAK23 and 4B3. hTert keratinocytes were cultured on coverslips in KGM2 medium with 0.05 mM CaCl 2 for at least three days, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. The cells were either mock-treated, or efgartigimod (25 µg/mL) was applied for 30 min, after which a 24 h treatment with the recombinant antibodies hAK23 (12.5 µg/mL), 4B3 (50 µg/mL), an isotype-matched human IgG4 (12.5 µg/mL) or IgG1 control (50 µg/mL) coupled to Alexa Fluor 488 (green) was initiated. After methanol fixation, the control IgG-treated cells were stained with the 5H10 anti-Dsg3 antibody, detected with a fluorochrome coupled secondary antibody (anti-mouse Alexa Fluor 546, red). Representative images from one out of at least three independent experiments are shown. Scale bar: 20 µm.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Staining, Cell Culture, Recombinant, Control