Journal: Scientific reports

Article Title: Biparatopic anti-PCSK9 antibody enhances the LDL-uptake in HepG2 cells.

doi: 10.1038/s41598-024-66290-9

Figure Lengend Snippet: Figure 2. The epitope binning assay- comparison between VHH-H2/D8/B11 and Repatha by SPR. The epitope binning assay consists of four steps: chip capturing; binding PCSK9 to saturation; adding secondary antibody; Protein A chip regeneration. (A) It shows the binding-epitope differences between VHH-H2/D8/B11 and Repatha by SPR. (B) It shows the binding-epitope differences between VHH-H2/D8 and VHH-B11 by SPR. The horizontal axis represents the timeline. The vertical axis represents the relative response unit.

Article Snippet: Firstly, evolocumab (also known as the Repatha, CAS# 1256937-27-5, Amgen, USA), diluted to 2 μg/mL, was captured on the flow cell 2 (FC2) of the Protein A chip (Cat# 29127555, GE Healthcare, USA) at approximately 300 RU level, while FC1 served as the blank control.

Techniques: Comparison, Binding Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet:

Article Snippet: Sensor Chip Protein A , GE Healthcare , Cat# 29127556.

Techniques: Recombinant, Software

Journal: Journal of cellular and molecular medicine

Article Title: Aryl Hydrocarbon Receptor Alleviates Hepatic Fibrosis by Inducing Hepatic Stellate Cell Ferroptosis.

doi: 10.1111/jcmm.70278

Figure Lengend Snippet: FIGURE 1 | AHR directly regulates Mrp1 transcription in mHSCs. (A–C) The expression of Ahr, Mrp1 or Cyp1a1 was detected by QPCR. (D) There are two potential AHR exogenous response elements on the Mrp1 promoter sequence. (E) Promoter sequences containing XREL1 and XREL2 were cloned onto PGL3 plasmids, and mutant plasmids were constructed. (F) Double luciferase assay to detect luciferase activity. (G) EMSA detects AHR binding to specific elements XREL1 and XREL2. (H) CHIP detects AHR binding to specific elements XREL1 and XREL2. Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test or one-way ANOVA.

Article Snippet: ChIP assays were performed using the ChIP Assay kit (Beyotime, P2078). mHSCs were treated with YH439 (5 μM) for 24 h. Subsequently, mHSCs were sonicated and then immunoprecipitated with the antibody against AHR (1:100 dilution BOSTER Cat# A00225- 4, RRID: AB_3095576) with IgG (1:100 dilution Proteintech Cat# 30000- 0- AP RRID AB_2819035) as a negative control.

Techniques: Expressing, Sequencing, Clone Assay, Mutagenesis, Construct, Luciferase, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Reverse engineering of a pathogenic antibody reveals the molecular mechanism of vaccine-induced immune thrombotic thrombocytopenia

doi: 10.1101/2023.06.30.547300

Figure Lengend Snippet: Surface-plasmon resonance sensograms obtained for the binding of the PF4 to the RVT1-captured Protein A chip. The concentration of the PF4 was titrated from 100nM till 2uM concentrations, and the signal was normalized to the sensograms with the blank solution whose composition matches the solution of PF4.

Article Snippet: The Protein A conjugated chip was acquired from Xantec Ltd (Düsseldorf, Germany).

Techniques: SPR Assay, Binding Assay, Concentration Assay

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