Journal: Proteomics

Article Title: Resurrection of a Clinical Antibody: Template ProteoGenomic de novo Proteomic Sequencing and Reverse Engineering of an Anti-Lymphotoxin Alpha Antibody

doi: 10.1002/pmic.201000487

Figure Lengend Snippet: LT-3F12.ascites specifically binds LT-α3. (a) LT-3F12.ascites blocked binding of LT-α3 to microtiter plates coated with TNFRII-Fc with an IC-50 similar to TNFRII-Fc in solution. Human LT-α3-biotin was preincubated with titrations of 3F12.ascites or TNFRII-Fc, and then added to microtiter plates coated with TNFRII-Fc. LTα3-biotin bound to coated TNFRII-Fc was detected with streptavadin-HRP followed by TMB substrate. (b). FACS of 293-LTαβ cells stained with anti-LT-3F12.ascites (blue histogram) or isotype control antibody (grey histogram) and detected using APC-conjugated anti-IgG.

Article Snippet: 100 μL of 80 ng/mL horseradish peroxidase conjugated goat anti-mouse IgG (all subtypes) (Jackson ImmunoResearch Laboratories Inc., PA, USA), diluted in AD was then added to each well and incubated for 1 hour.

Techniques: Binding Assay, Staining

Journal: Proteomics

Article Title: Resurrection of a Clinical Antibody: Template ProteoGenomic de novo Proteomic Sequencing and Reverse Engineering of an Anti-Lymphotoxin Alpha Antibody

doi: 10.1002/pmic.201000487

Figure Lengend Snippet: Equivalent potency of LT-3F12 ascites vs. rescued chimera in binding and blocking LT-α3. (a). Binding of anti-LT-3F12.chim, anti-LT-3F12.ascites, TNFRII-Fc and LTβR-Fc to plates coated with LT-α3. EC50 values for each molecule are shown. (b) FACS of 293-LTαβ cells stained with anti-LT-3F12.ascites (blue histogram), anti-LT-3F12.chim (green histogram), LTβR-Fc (black line) or isotype control antibody (grey histogram) and detected using APC-conjugated anti-IgG. (c) HUVEC grown in microtiter plates were stimulated with LT-α3 pre-incubated with titrations of anti-LT-3F12.ascites, anti-LT-3F12.chim, TNFRII-Fc, or isotype control mouse IgG2a antibody. Blocking of LT-α3 induction of ICAM expression on HUVEC and IC50 values are shown. (D) Surface plasmon resonance (BIACORE) analyses of the LT-3F12 chimeric and ascites IgG binding kinetics to LT-α3 demonstrate sub-nanomolar binding (KD 0.3 Nm. IgG was captured by the Fc region on a CM5 sensor chip covalently coupled with goat anti-species Fc Fab’2 pAb. Traces are shown illustrating the on-rate and off-rate kinetic profile for a titration of LT-α3 from 1.25 to 10 nM flowed over LT-3F12 chimeric IgG.

Article Snippet: 100 μL of 80 ng/mL horseradish peroxidase conjugated goat anti-mouse IgG (all subtypes) (Jackson ImmunoResearch Laboratories Inc., PA, USA), diluted in AD was then added to each well and incubated for 1 hour.

Techniques: Binding Assay, Blocking Assay, Staining, Incubation, Expressing, SPR Assay, Titration