Journal: bioRxiv

Article Title: Tracing production instability in a clonally-derived CHO cell line using single cell transcriptomics

doi: 10.1101/2020.11.04.368480

Figure Lengend Snippet: (a) The expression construct for the anti-IL8 mAb was designed to express the heavy and light chains of the antibody under the control of two separate SV40 promoters. Upon confirming that complete anti-IL8 monoclonal antibody could not be detected by either western blot or by mass spectrometry, we analysed the heavy and light chain of the mAb in the cell lysate and the supernatant at Day 3 and Day 10 of cell culture. Under reducing conditions, (b) the light chain was detected at both day 3 and day 10 in the lysate, however the heavy chain could not be detected. The heavy chain was detected (c) only in the supernatant harvested after 10 days of culture (Day 10). When non-reducing conditions (d) were used for western blotting a light chain dimer was present in the supernatant harvested after 3 (Day 3) and 10 days (Day 10) of culture. (0.125 µg per lane of a recombinant human IgG1 kappa (Biorad, HCA192) was used as a control antibody). Mass spectrometry analysis following SEC separation of the supernatant at Day 10 was used to (e) confirm the presence of light chain dimer containing one inter molecular disulphide bond.

Article Snippet: The heavy chain was detected by the anti-human IgG Fcγ fragment specific primary antibody (1:1,000; Jackson, cat.no.109-005-008), the light chain by the anti-human kappa light chain antibody (1:2,500; Biorad, cat.no.

Techniques: Expressing, Construct, Control, Western Blot, Mass Spectrometry, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: Tracing production instability in a clonally-derived CHO cell line using single cell transcriptomics

doi: 10.1101/2020.11.04.368480

Figure Lengend Snippet: Western blot analysis of intracellular and extracellular anti-IL8 antibody under denaturing non-reducing conditions failed to detect the anti-IL8 antibody. Cells and supernatant were harvested after 3 and 10 days of culture and a recombinant human IgG1 kappa antibody (0.125 µg per lane -Biorad, HCA192) was used as a positive control.

Article Snippet: The heavy chain was detected by the anti-human IgG Fcγ fragment specific primary antibody (1:1,000; Jackson, cat.no.109-005-008), the light chain by the anti-human kappa light chain antibody (1:2,500; Biorad, cat.no.

Techniques: Western Blot, Recombinant, Positive Control

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: ( a ) Three-dimensional representation of the crystal structures of a human IgG1 (PDB 1HZH) and the hinge deleted Mcg Antibody (PDB 1MCO). ( b ) Sequence alignment of the IgG1 and IgG4 hinge (IgG1 HC, IgG4 HC) and resulting sequence after deletion (IgG1 Δhinge and IgG4 Δhinge).

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Sequencing

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Biochemical and biophysical characterization of the hinge deleted IgG4 and its corresponding full-length control mAb by ( a ) SEC-UPLC coupled to UV (A280) and Multi Angle Laser Light Scattering (MALS). The UV elution profiles of the hinge deleted IgG4 molecule (bottom trace) and the corresponding full-length control (top trace) are shown. MALS data show measured molecular masses of 132 (hinge-deleted) and 149 kDa (full-length mAb). ( b ) Nonreduced CE-SDS coupled to UV (A280). Electropherograms of the buffer control (bottom trace), hinge deleted IgG4 molecule (middle trace) and the corresponding full-length control (top trace) are shown. ( c ) Differential Scanning Fluorimetry. An overlay of the melting profiles of the hinge deleted IgG4 molecule (orange) and the corresponding full-length control (teal) are shown. Melting temperatures/onset were 54.6/47.9 °C (hinge-deleted) and 62.7/56.6 °C (full-length mAb). ( d ) Intact reduced LC–MS. Total Ion Counts of the hinge deleted IgG4 molecule heavy chain (bottom trace) and the corresponding heavy chain of the full-length control (top trace) are shown. The peak corresponding to the dominant Fc N-Glycan G0F species is shown.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Biochemical and biophysical characterization of the hinge deleted IgG1 and its corresponding full-length control mAb by ( a ) SEC-UPLC coupled to UV (A280) and MALS. The UV elution profiles of the hinge deleted IgG1 molecule (bottom trace) and the corresponding full-length control (top trace) are shown. Molecular masses of 140 and 150 kDa were measured by MALS for the hinge deleted molecule and its full-length counterpart. ( b ) Nonreduced CE-SDS coupled to UV (A280). Electropherograms of the buffer control (bottom trace), hinge deleted IgG1 molecule (middle trace) and the corresponding full-length control (top trace) are shown. ( c ) Differential Scanning Fluorimetry. An overlay of the melting profiles of the hinge deleted IgG1 molecule (orange) and the corresponding full-length control (teal) are shown. Melting temperatures/onset were 67.8/56.5 °C for the hinge deleted molecule and 71.1/63.2 °C for the full-length control. ( d ) Intact reduced LC–MS. Total Ion Counts of the hinge deleted IgG1 molecule heavy chain (top trace) and the corresponding heavy chain of the full-length control (bottom trace) are shown. The peak corresponding to the dominant Fc N-Glycan G0F and G1F species are shown.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Analysis of the hinge deleted IgG4 and its corresponding full-length control mAb by LC–MS. ( a ) Deconvoluted spectrum of a nonreduced analysis. Under denaturating conditions, the hinge deleted IgG4 molecule has an apparent mass of 72,161.0 Da consistent with a Heavy-Light (HL) molecule with G0F N-Glycans. The full length mAb control has the expected mass of 146,829.0 Da consistent with the H2L2 structure (with G0F N-Glycans). Under reducing conditions, the heavy chain ( d) and Light chains ( b ) can be separated. The hinge deleted IgG4 and the full-length control molecule have the same light chain (23,399.5 Da).

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Control, Liquid Chromatography with Mass Spectroscopy

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Binding of hinge deleted IgG4 (blue dilution curves) and its corresponding full-length control mAb (green dilution curves) to ( a ) FcγRI by ELISA, ( b ) FcRn by ELISA. ( c ) Enzymatic inhibition of the MMP9 target antigen.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Binding of hinge deleted IgG1 (orange dilution curves) and its corresponding full-length control mAb (blue dilution curves) to ( a ) FcγRI by competitive AlphaScreen ® assay, ( b ) FcγRIIIa V158 by binding ELISA, ( c ) FcRn by binding ELISA. Incorporation of point mutations in the Fc of the hinge deleted IgG1 (green dilution series) almost rescues binding to the level of the full-length control. ( d ) Engagement of the gp120 target antigen by a HEK290 cell-based ELISA reflective of the mAb mode of action.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Binding Assay, Control, Amplified Luminescent Proximity Homogenous Assay, Enzyme-linked Immunosorbent Assay, In-Cell ELISA

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Blocking Fab arm exchange of hinge deleted mAbs by CH3 stabilization monitored by Förster resonance energy transfer analysis (FAE) which was performed under 1 mM glutathione reducing conditions ( a ) or in the absence of reducing agent as the negative control ( b ). Förster resonance energy transfer (FRET) signal (ex 490 nm/Em 515 nm) was recorded for the positive control (Natalizumab x Natalizumab), the hinge deleted IgG4 (Natalizumab x hinge deleted IgG4), the hinge deleted IgG4 with R409K point mutation in the CH3 domain (Natalizumab x R409K hinge deleted IgG4), for the negative control (S228P full length IgG4 x Natalizumab) and for the blank background control. ( c ) Bar graph summary representing the propensity of each of the test molecules to engage in FAE with Natalizumab.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Blocking Assay, Förster Resonance Energy Transfer, Negative Control, Positive Control, Mutagenesis, Control

Journal: Antibodies

Article Title: Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

doi: 10.3390/antib9040050

Figure Lengend Snippet: Blocking Fab arm exchange of hinge deleted mAbs by CH3 stabilization monitored by chromatographic separation. Propensity of IgG4 hinge deleted (T1), R409K CH3 stabilized IgG4 hinge deleted (T2), and S228P hinge stabilized full length IgG4 (WT) to engage in Fab arm exchange with Tysabri ® in the presence ( a ) or the absence ( c ) of 1 mM GSH reducing agent was analyzed by chromatographic separation. An FAE positive control reaction was conducted by monitoring the formation of the bispecific antibody by chromatographic separation between Tysabri ® and a nonhinge stabilized aGFP IgG4 antibody (IgG4 Kappa Antibody) in the presence ( b ) or the absence ( d ) of 1 mM GSH reducing agent.

Article Snippet: The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).

Techniques: Blocking Assay, Positive Control

Journal: Frontiers in Immunology

Article Title: Identification of a Novel Mutation in TNFAIP3 in a Family With Poly-Autoimmunity

doi: 10.3389/fimmu.2022.804401

Figure Lengend Snippet: Reduced A20 function in vivo . (A) Western Blot and densitometric analysis of A20 protein levels in PBMCs isolated from patients (Pt1-4) and 2 healthy donors (HD). GAPDH was used as loading control. Densitometric quantification of A20 was reported (right graph). (B) PBMCs isolated from Pt1, Pt4 and one healthy subject (HD) were starved for 2 h in media with 0.5% of FBS and lysed or stimulated for 30 minutes with 0.01 μg/ml LPS and then lysed. Phosphorylated (S536) p65 NF-kB (P-p65) and total p65 NF-kB protein levels were assessed by Western blot analyses. GAPDH was used as loading control. The phosphorylated-p65 NF-kB/total p65 NF-kB densitometry comparative ratio was also reported (right graph). Similar results were obtained in two independent experiments.

Article Snippet: 30 μg protein extracts were resolved by TGX precast mini Gels (4568084 BioRAD), transferred to nitrocellulose membranes (Amersham Life Sciences) and probed with antibody to A20 (D13H3), pS536- NF-kB (3033S), NF-kB (8242) and GAPDH (5174s), all from Cell Signaling Technology.

Techniques: In Vivo, Western Blot, Isolation, Control

Journal: Theranostics

Article Title: Immunization with the Gly 1127 -Cys 1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies

doi: 10.7150/thno.37305

Figure Lengend Snippet: P3 immunization reduces aortic pro-inflammatory mediators in the aorta . Representative Western blot analysis of LRP1, TNFR1, pNF-kB (p65), total NF-kB (p65), and β-actin bands ( A ) and bar graphs showing band quantification of LRP1 and TNFR1 normalized by a-actin ( B,C ) and pNF-kB (p65)/total NF-kB (p65) ratio ( E ). Results are shown as mean ± SD in chow IrP (N=6), chow P3 (N=6), HFD Ctr (N=3), HFD IrP (N=6) and HFD P3 (N=9). *P<0.005 versus chow diet; #P<0.005 versus control or IrP-rabbits. Representative confocal microscopy images of TNFR1 ( D ) and NF-kB levels ( F ) in aortic samples from the different groups. Scale bar = 50 µm.

Article Snippet: Blots were incubated with antibodies against mouse LRP1 (β - chain, clone 5A6 RDI-PRO61066), SREBP-2 (Santa Cruz Biotechnology; sc-13552), phospho-NF-kB p65 (Cell Signaling; #3039), NF-kB p65 (C22B4) (Cell Signalling; #4764) and TNFR1 (MyBiosource MBS840939) Protein extracts (10 μg) were loaded, resolved on 12% SDS-PAGE and transferred to nitrocellulose membranes (BioRad).

Techniques: Western Blot, Control, Confocal Microscopy

Journal: Theranostics

Article Title: Immunization with the Gly 1127 -Cys 1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies

doi: 10.7150/thno.37305

Figure Lengend Snippet: HFD serum from control and IrP serum, but not P3 serum, dramatically increased intracellular CE and pro-inflammatory mediators in human macrophages . Quiescent human macrophages (hMΦ) were exposed to serum from the different groups (0.5%, 2 hours). Cells were then exhaustively washed and collected in NaoH 0.1N for lipid extraction or in lysis buffer for Western blot analysis. A ) Representative TLC and bar graphs showing the cholesteryl ester (CE) /free cholesterol ratio. Representative Western blot analysis of LRP1, SREBP-2, TNFR1, pNF-kB (p65), total NF-kB (p65), and a-actin bands ( B ) and bar graphs showing band quantification of LRP1, SREBP-2 and TNFR1 normalized by a-actin ( C,D,E ) and pNF-kB (p65)/total NF-kB (p65) ratio ( F ). Results are shown as mean ± SD of three experiments performed in duplicate. *P<0.005 versus chow serum; #P<0.005 versus control or IrP-serums.

Article Snippet: Blots were incubated with antibodies against mouse LRP1 (β - chain, clone 5A6 RDI-PRO61066), SREBP-2 (Santa Cruz Biotechnology; sc-13552), phospho-NF-kB p65 (Cell Signaling; #3039), NF-kB p65 (C22B4) (Cell Signalling; #4764) and TNFR1 (MyBiosource MBS840939) Protein extracts (10 μg) were loaded, resolved on 12% SDS-PAGE and transferred to nitrocellulose membranes (BioRad).

Techniques: Control, Extraction, Lysis, Western Blot

Journal: Theranostics

Article Title: Immunization with the Gly 1127 -Cys 1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies

doi: 10.7150/thno.37305

Figure Lengend Snippet: HFD serum from control and IrP serum, but not P3 serum, dramatically increased intracellular CE and pro-inflammatory mediators in human coronary vascular smooth muscle cells . Quiescent human coronary vascular smooth muscle cells (hcVSMC) were exposed to serum from the different groups (0.5%, 2 hours). Cells were then exhaustively washed and collected in NaoH 0.1N for lipid extraction or in lysis buffer for Western blot analysis. A ) Representative TLC and bar graphs showing the cholesteryl ester (CE) /free cholesterol ratio. Representative Western blot analysis of LRP1, SREBP-2, TNFR1, pNF-kB (p65), total NF-kB (p65), and a-actin bands ( B ) and bar graphs showing band quantification of LRP1, SREBP-2 and TNFR1 normalized by a-actin ( C,D,E ) and pNF-kB (p65)/total NF-kB (p65) ratio ( F ). Results are shown as mean ± SD of three experiments performed in duplicate. *P<0.005 versus chow serum; #P<0.005 versus control or IrP-serums.

Article Snippet: Blots were incubated with antibodies against mouse LRP1 (β - chain, clone 5A6 RDI-PRO61066), SREBP-2 (Santa Cruz Biotechnology; sc-13552), phospho-NF-kB p65 (Cell Signaling; #3039), NF-kB p65 (C22B4) (Cell Signalling; #4764) and TNFR1 (MyBiosource MBS840939) Protein extracts (10 μg) were loaded, resolved on 12% SDS-PAGE and transferred to nitrocellulose membranes (BioRad).

Techniques: Control, Extraction, Lysis, Western Blot