Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: A next generation peripherally restricted Cavα2δ-1 ligand with inhibitory action on Cav2.2 channels and utility in neuropathic pain.

doi: 10.1016/j.biopha.2024.116472

Figure Lengend Snippet: Fig. 3. Specific SPR binding of the recombinant Cavα2δ-1 fragment comprising the VGGC_α2 domain to NVA1309, covalently coupled to the surface of a Biacore CM5 optical sensor chip (surface density 20 RU) (a). Binding sensorgram obtained with CUSABIO fragment; specificity is confirmed by lack of binding of non related proteins (human IgG and rec. human leukemia inhibiting factor (LIF), respectively). The binding sensorgram was analysed by mathematical curve fitting applying a Langmuir 1:1 interaction algorithm (BiaEvaluation 4.1 software). The fitted curve is shown in (b). Binding of human recombinant full length wild type and mutated α2δ-1 to NVA1309, covalently immobilised on the surface of a Biacore CM5 optical sensor chip: Each Cavα2δ-1 recombinant protein was run as analyte at three concentrations across the NVA1309 sensor chip surface. (NVA1309 surface density = 204RU). Kinetic binding constants and affinities (ka, kd; KD) were determined from double referenced sensorgrams by mathematical sensorgram fitting, with fitted curves shown in c-f.

Article Snippet: Synthetic Cavα2δ-1-derived peptides containing a carboxy-terminal GGGC tag were covalently coupled via their carboxy-terminal cysteine to the surface of Biacore CM5 optical sensor chips at high surface density (> 10000 RU) using the Biacore Thiol Coupling Kit following the Biacore thiol coupling procedure.

Techniques: Binding Assay, Recombinant, Software

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