Journal: Current Protocols

Article Title: Efficient On‐Column Removal of Endotoxin from Immunoglobulins Such as AK23

doi: 10.1002/cpz1.70238

Figure Lengend Snippet: Overview of the IgG sanitization procedure. Created in BioRender (Müller, E.J., 2025; https://BioRender.com/f0fv9ee ).

Article Snippet: Primary human epidermal keratinocyte progenitor (HPEK) cells (CELLnTEC, cat. no. HPEKs, lot no. ES1503029, Müller et al., ; human or mouse keratinocytes, including HaCat cells, are also suitable) Materials for cell culture (see Müller et al., , Basic Protocol 4) CnT‐NX‐EX culture medium (CELLnTEC, cat. no. CnT‐NX‐EX) 226 mM CaCl stock solution (see Müller et al., , Basic Protocol 4) PBS+ (Müller et al., ), ice cold 1% (v/v) bovine serum albumin (BSA) in PBS+ AK23 IgG (Basic Protocol ; IgG1 isotype; Tsunoda et al., ), both before and after sanitization Isotype control mouse IgG1 (mIgG1; BioXCell, cat. no. BE0083, custom‐made, ≤0.05 EU/mg) Methanol‐free 16% (w/v) formaldehyde (Pierce, cat. no. 28906), diluted to 4% (w/v) formaldehyde with PBS+ Goat anti‐mouse IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen, cat. no. A‐11005) or equivalent 1 mg/ml Hoechst 33342 (AAT BioQuest, cat. no. 17530, or equivalent) Fluorescence mounting medium (Dako, cat. no. S302380‐2 or equivalent) 8‐well removable chambered glass slides (ibidi, cat. no. 80841, or equivalent) 24 × 60‐mm rectangular glass cover slips, no. 1 (Corning, cat. no. CLS2975246, or equivalent) 37°C, 5% CO incubator, set to 90% humidity Orbital shaker (Rotamax 120, P/N 544‐41200‐00, Heidolph, or equivalent) Fluorescence microscope (Nikon, cat. no. Eclipse Ti‐E, or equivalent) NOTE : It is important to perform direct immunofluorescence staining with AK23 IgG under high‐calcium conditions without fixation and at 4°C, as AK23 does not bind to fixed cells or under low‐calcium conditions, and the low temperature prevents its internalization.

Techniques:

Journal: Current Protocols

Article Title: Efficient On‐Column Removal of Endotoxin from Immunoglobulins Such as AK23

doi: 10.1002/cpz1.70238

Figure Lengend Snippet: AK23 IgG purification: Flowthrough analysis for the MabSelect Protein A HiTrap column after IgG loading, depicting washing, sanitization, and elution steps. The UV monitor measures protein concentrations based on A 260 / A 280 , and the conductivity monitor measures ion concentrations (Fig. ).

Article Snippet: Primary human epidermal keratinocyte progenitor (HPEK) cells (CELLnTEC, cat. no. HPEKs, lot no. ES1503029, Müller et al., ; human or mouse keratinocytes, including HaCat cells, are also suitable) Materials for cell culture (see Müller et al., , Basic Protocol 4) CnT‐NX‐EX culture medium (CELLnTEC, cat. no. CnT‐NX‐EX) 226 mM CaCl stock solution (see Müller et al., , Basic Protocol 4) PBS+ (Müller et al., ), ice cold 1% (v/v) bovine serum albumin (BSA) in PBS+ AK23 IgG (Basic Protocol ; IgG1 isotype; Tsunoda et al., ), both before and after sanitization Isotype control mouse IgG1 (mIgG1; BioXCell, cat. no. BE0083, custom‐made, ≤0.05 EU/mg) Methanol‐free 16% (w/v) formaldehyde (Pierce, cat. no. 28906), diluted to 4% (w/v) formaldehyde with PBS+ Goat anti‐mouse IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen, cat. no. A‐11005) or equivalent 1 mg/ml Hoechst 33342 (AAT BioQuest, cat. no. 17530, or equivalent) Fluorescence mounting medium (Dako, cat. no. S302380‐2 or equivalent) 8‐well removable chambered glass slides (ibidi, cat. no. 80841, or equivalent) 24 × 60‐mm rectangular glass cover slips, no. 1 (Corning, cat. no. CLS2975246, or equivalent) 37°C, 5% CO incubator, set to 90% humidity Orbital shaker (Rotamax 120, P/N 544‐41200‐00, Heidolph, or equivalent) Fluorescence microscope (Nikon, cat. no. Eclipse Ti‐E, or equivalent) NOTE : It is important to perform direct immunofluorescence staining with AK23 IgG under high‐calcium conditions without fixation and at 4°C, as AK23 does not bind to fixed cells or under low‐calcium conditions, and the low temperature prevents its internalization.

Techniques: Purification

Journal: Current Protocols

Article Title: Efficient On‐Column Removal of Endotoxin from Immunoglobulins Such as AK23

doi: 10.1002/cpz1.70238

Figure Lengend Snippet: Functional AK23 testing on primary human keratinocytes before and after AK23 IgG sanitization. ( A ) Mechanical disruption of confluent keratinocyte sheet by a KDA. Note that the functional activity of AK23 before and after sanitization is similar. 20 µg/ml AK23 and mIgG1 (BioXCell, BE0083, custom‐made ≤0.05 EU/mg) were used. ns, non‐significant; **** p <.0001; n = 3. ( B ) Direct immunofluorescence.

Article Snippet: Primary human epidermal keratinocyte progenitor (HPEK) cells (CELLnTEC, cat. no. HPEKs, lot no. ES1503029, Müller et al., ; human or mouse keratinocytes, including HaCat cells, are also suitable) Materials for cell culture (see Müller et al., , Basic Protocol 4) CnT‐NX‐EX culture medium (CELLnTEC, cat. no. CnT‐NX‐EX) 226 mM CaCl stock solution (see Müller et al., , Basic Protocol 4) PBS+ (Müller et al., ), ice cold 1% (v/v) bovine serum albumin (BSA) in PBS+ AK23 IgG (Basic Protocol ; IgG1 isotype; Tsunoda et al., ), both before and after sanitization Isotype control mouse IgG1 (mIgG1; BioXCell, cat. no. BE0083, custom‐made, ≤0.05 EU/mg) Methanol‐free 16% (w/v) formaldehyde (Pierce, cat. no. 28906), diluted to 4% (w/v) formaldehyde with PBS+ Goat anti‐mouse IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen, cat. no. A‐11005) or equivalent 1 mg/ml Hoechst 33342 (AAT BioQuest, cat. no. 17530, or equivalent) Fluorescence mounting medium (Dako, cat. no. S302380‐2 or equivalent) 8‐well removable chambered glass slides (ibidi, cat. no. 80841, or equivalent) 24 × 60‐mm rectangular glass cover slips, no. 1 (Corning, cat. no. CLS2975246, or equivalent) 37°C, 5% CO incubator, set to 90% humidity Orbital shaker (Rotamax 120, P/N 544‐41200‐00, Heidolph, or equivalent) Fluorescence microscope (Nikon, cat. no. Eclipse Ti‐E, or equivalent) NOTE : It is important to perform direct immunofluorescence staining with AK23 IgG under high‐calcium conditions without fixation and at 4°C, as AK23 does not bind to fixed cells or under low‐calcium conditions, and the low temperature prevents its internalization.

Techniques: Functional Assay, Disruption, Activity Assay, Immunofluorescence