Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Characterization of hPD1(25-167)-His and hPD1(25-167)-3S-IG

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–c Characterization of the assay reference standard-recombinant hPD1(25-167)-His protein. a SDS-PAGE analysis of the purified hPD1(25-167)-His protein expressed in HEK293 cells. Lane 1: molecular weight marker, lane 2: blank, lane 3: 1 μg of the purified hPD1(25-167)-His protein. The gel was stained with Simply Blue SafeStain. b Concentration series of hPDL-1 (0.125–2 μM) binding to captured hPD1(25-167)-His protein. c Equilibrium analysis of hPDL-1 binding to hPD1(25-167)-His yields a K D of 2.8 μM (average value over multiple surfaces)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, SDS Page, Purification, Molecular Weight, Marker, Staining, Concentration Assay, Binding Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: Binding Affinity of Anti-PD-1 Antibody MIH4 for Recombinant Human sPD-1 Proteins

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Binding Assay, Recombinant

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Detection of endogenous sPD-1 protein and spike recovery in sera from normal and cancer individuals. a Levels of sPD-1 in serum samples from normal and cancer individuals: normal, n = 15; melanoma, n = 15; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. b Spike and recovery study: 500 pg/mL hPD1(25-167)-His protein was spiked into serum samples from normal and cancer individuals: normal, n = 10; melanoma n = 8; renal cell carcinoma, n = 11; squamous lung cancer, n = 7; non-squamous lung cancer, n = 8; multiple myeloma, n = 10; Hodgkin’s lymphoma, n = 7. % Spike recovery = 100*(sample value post-spike − sample value pre-spike)/spiked value. Results are shown with box and whiskers graphs. The box included data within 5 to 95 percentiles with the median line in the middle and extended values in the whiskers. The dotted lines depict the nominal spiked value ± 25%

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Dilution linearity studies in melanoma and spiked normal sera. a Three melanoma serum samples (M1, M2, M3) were each diluted 1.5–8-fold with assay buffer; recombinant hPD1(25-167)-His protein standard in a SeraSub solution was diluted in same concentration range. b Three normal human serum samples (N1, N2, N3) were spiked with 5000 pg/mL of human hPD1(25-167)-His and were each diluted 2–256-fold with assay buffer. The back-calculated sPD-1 concentrations (observed concentrations times the dilution factor) were plotted against dilution factors. The dotted lines depict 75–125% range of the nominal spiked value

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques: Recombinant, Concentration Assay

Journal: The AAPS Journal

Article Title: Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

doi: 10.1208/s12248-015-9762-4

Figure Lengend Snippet: a–b Effect of PDL-1, PDL-2, and nivolumab on QC sample performance. a sPD-1 in a melanoma serum sample measured in the absence or presence of 20 nM (1 μg/mL) of human PDL-1 or PDL-2 proteins. b QC performance from analytical runs in the absence or presence of 685 nM (100 μg/mL) nivolumab. Results are shown as mean and SD of each group (n = 2–4)

Article Snippet: Binding of Human PDL-1 to hPD1(25-167)-His Surface plasmon resonance (SPR) experiments to characterize the binding of hPDL-1 to hPD1(25-167)-His were performed using a ProteOn XPR36 instrument (BioRad, Hercules, CA, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Tyrosine phosphorylation switching of a G protein

doi: 10.1074/jbc.RA117.000163

Figure Lengend Snippet: AtGPA1 Y166E changes its binding specificity with AtRGS1. A, direct interaction between GST-tagged AtRGS1 (RGS + Ct) and His-tagged AtGPA1 wildtype or Y166E mutant in the presence of GDP, GTPγS, or GDP-AlF4− was detected by an in vitro pulldown assay. Protein complexes were purified by glutathione-Sepharose, separated by SDS-PAGE, and detected by anti-GST or anti-His antibodies. B, the intensities of His-AtGPA1 wildtype and Y166E mutant were quantitated with ImageJ and normalized as relative values to each interaction in the presence of GDP-AlF4−. The quantitative results were expressed as the means ± S.D. (error bars) of four experiments. Statistical significance was determined by ANOVA. *, differences with p values of <0.05. **, differences with p values of <0.01. The binding affinity constants of AtRGS1 and AtGPA1 wildtype or Y166E mutant were measured by surface plasmon resonance with a ProteOn XPR36 instrument. The GST-tagged AtRGS1 (RGS + Ct) was immobilized on the surface of a GLC sensor chip as ligand, and the His-tagged AtGPA1 wildtype or Y166E in the GDP-bound, GTPγS-bound, or GDP-AlF4−-bound state were diluted into dosage concentrations as analyte. The affinity constants were calculated by kinetic analysis (C–H) or equilibrium analysis (I-K) and are shown in Table 2. IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: Surface plasmon resonance Binding affinity constants were measured by the ProteOn XPR36 protein interaction array system and a ProteOn TM GLC Sensor Chip (Bio-Rad, catalog no. 1765011).

Techniques: Binding Assay, Mutagenesis, In Vitro, Purification, SDS Page, SPR Assay, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tyrosine phosphorylation switching of a G protein

doi: 10.1074/jbc.RA117.000163

Figure Lengend Snippet: Summary of affinity constants between AtRGS1 and Gα subunits Binding affinity constants were measured using a ProteOn XPR36 surface plasmon resonance instrument. For the kinetic analysis, the original curves were fit with a 1:1 Langmuir binding model. k a , association rate constant; k d , dissociation rate constant; K D , calculated by k a / k d ; R max , maximum response; χ 2 , the average of squared residuals. For the equilibrium analysis, K D was calculated by response units at steady state. RU, response units; NA, no specific binding was detected with the GDP-bound state of wildtype AtGPA1.

Article Snippet: Surface plasmon resonance Binding affinity constants were measured by the ProteOn XPR36 protein interaction array system and a ProteOn TM GLC Sensor Chip (Bio-Rad, catalog no. 1765011).

Techniques: Binding Assay, SPR Assay

Journal: Journal of Virology

Article Title: H5N1 Virus-Like Particle Vaccine Elicits Cross-Reactive Neutralizing Antibodies That Preferentially Bind to the Oligomeric Form of Influenza Virus Hemagglutinin in Humans

doi: 10.1128/jvi.05406-11

Figure Lengend Snippet: FIG. 2. Characterization of bacterially expressed and purified H5N1 HA proteins. (A and B) Characterization of purified H5N1 HA proteins from E. coli by Superdex S-200 gel filtration chromatography. Purified H5N1 HA1 proteins with an intact N terminus (positions 1 to 320) (A) and HA1 with an N-terminal deletion (28 to 320) (B) were subjected to gel filtration. The panels show superimposed elution profiles of purified HA1 proteins (red lines) overlaid with calibration standards (gray lines). The elution volumes of protein species are shown in parentheses. (C and D) Steady-state binding equilibrium analysis of conformation-dependent human H5N1 neutralizing MAbs FLD21.40 (C) and FLA3.14 (D) at 10 g/ml to purified bacterially expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group and on a blank flow cell, free of peptide. Binding was recorded using a ProteOn system surface plasmon resonance biosensor instrument (Bio-Rad Labs, Hercules, CA). (E) Agglutination of human RBCs by properly folded oligomeric H5N1 rHA1(1-320) protein and its monomeric H5N1 rHA1(28-320) counterpart along with rgH5N1 virus. Serial dilutions of purified HA1 proteins were mixed with washed RBCs, and hemagglutination was read after 30 min at room temperature. Reassorted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1) was used as a positive control.

Article Snippet: Binding kinetics for the selected human vaccine sera and data analyses were calculated using Bio-Rad ProteOn manager software (version 2.0.1).

Techniques: Chromatography, Binding Assay, SPR Assay, Agglutination, Virus, Positive Control