Journal:

Article Title: HLA-DM mediates peptide exchange by interacting transiently and repeatedly with HLA-DR1

doi: 10.1016/j.molimm.2009.07.001

Figure Lengend Snippet: (a) DM mediated peptide binding is blocked by SEA. 2.4 μM DR1-HAY308A complexes were produced, isolated, and incubated with or without 1μM DM or 7μM SEA for 20 minutes, and injected over either HA306–318 or HAAnchorless peptide-bound CM5 chip surface in a BIAcore experiment. Samples in Citrate Phosphate (CP) pH 6.0 were injected at 4μL/minute followed by washout (CP pH 6.0 + 0.01% Tween). Raw real-time binding data of DR1 to the peptide chip is shown; in the presence of DM, blue; in the presence of SEA, red; in the presence of DM + SEA, black; in the presence of DM + SEA + EDTA, green. (b) Same as (a), except that SEB or TSST were used instead of SEA. Real-time binding data of DR1 to peptide in the presence of DM, red; SEB + DM, blue; or TSST + DM, black. (c) DM mediated peptide dissociation is blocked by SEA and SEH. 2.4 μM DR1-FITC-HAAnchorless complexes were allowed to dissociate for 30 minutes in CP pH 5.5, in the presence or absence of 1μM DM, either without SAg (black), or in the presence of 7μM SEA (grey) or 7μM SEH (white). The fluorescence of the complexes is expressed as a fraction of complexes dissociated at 0 minutes, which was assigned an arbitrary value of 1.0. (d) Same as (c), except that the experiment was performed over 20 minutes, and the SAgs used were SEA (grey), SEB (stripes), or a mutant SEA that does not bind β81His (dotted).

Article Snippet: 2.4 μM DR1-HA Y308A complexes were produced, isolated, and incubated with or without 1μM DM or 7μM SEA for 20 minutes, and injected over either HA 306–318 or HA Anchorless peptide-bound CM5 chip surface in a BIAcore experiment.

Techniques: Binding Assay, Produced, Isolation, Incubation, Injection, Fluorescence, Mutagenesis

Journal: Analytical Chemistry

Article Title: Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis

doi: 10.1021/acs.analchem.3c05607

Figure Lengend Snippet: Variability in the percent activity of distinct sLAG3 reagent lots may account for perceived discrepancies in kinetic fits of the respective binding parameters. (A) A pie chart depicting the active and inactive species that may proportionally change between reagent lots, producing lot-to-lot discrepancies in assay responses, using domains of PDB: 7TZG . (B) The molar active concentrations reported for different epitopes from Lot1 of sLAG3 calibrator (Bradford: n = 2, Capture-ProteinG: n = 7, Capture-ProteinA: n = 2, Capture-CM5: n = 2, Detection(u)-CM5: n = 2, Detection(s)-CM5: n = 2). The Capture-CM5 and Detection(s)-CM5 chips were calibrated with a ProteinG-CM5 chip from the same chip lot, while the Detection(u)-CM5 chip was calibrated with a ProteinA-CM5 chip. Capture mAb was biotinylated, Detection(u) mAb was unconjugated, and Detection(s) mAb was sulfo-tagged. (C) An expansion of the data shown in panel (B) including all three sLAG3 lots, comparing each active concentration value to the respective lot’s Bradford total protein concentration to measure the percent activity of each epitope in each lot. (D) Biolayer interferometry was performed to measure the binding kinetic on-rates of the sLAG3 reagent lots to the capture or detection mAbs. Apparent lot-to-lot discrepancies measured using Bradford-defined concentrations were much less prominent when sLAG3 lots were instead defined by mAb-specific active concentrations ( n = 2). (E) The respective dissociation constants from the fits in panel (D) similarly demonstrate that using the active concentrations of each sLAG3 reagent leads to increased lot-to-lot agreement compared with using the total concentration.

Article Snippet: Additionally, the active concentrations for the capture mAb epitope measured using a CM5, ProteinA, or ProteinG chip were similar for each lot of sLAG3, emphasizing the utility of independently calibrating each flow cell/sensor ( Figure B,C).

Techniques: Activity Assay, Binding Assay, Concentration Assay, Protein Concentration

Journal: Analytical Chemistry

Article Title: Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis

doi: 10.1021/acs.analchem.3c05607

Figure Lengend Snippet: Defining sLAG3 calibrator lots by the capture or detection epitope active concentration moderately unifies immunoassay responses over total protein concentration. (A) Venn diagram representing the overlap of protein species with active capture mAb and/or detection mAb epitopes. (B) MSD titration of three sLAG3 lots, defining each lot by their respective Bradford concentration, capture mAb concentration (Capture-ProteinG), or detection mAb concentration (Detection(s)-CM5). Pairwise %CV comparisons were conducted using data from a prior sLAG3 bridging study, containing responses from 50 patient serum samples. These signals were reinterpolated using the standard curves shown above to have %CVs spanning the clinically relevant range of MSD signals. The overall %CVs for the three lots (100* stdev of all three/mean of all three) were 42.4% for Bradford, 19.0% for capture mAb, and 18.5% for detection mAb.

Article Snippet: Additionally, the active concentrations for the capture mAb epitope measured using a CM5, ProteinA, or ProteinG chip were similar for each lot of sLAG3, emphasizing the utility of independently calibrating each flow cell/sensor ( Figure B,C).

Techniques: Concentration Assay, Protein Concentration, Titration