α hbz  (R&D Systems)

Journal: Retrovirology

Article Title: Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status

doi: 10.1186/s12977-016-0263-z

Figure Lengend Snippet: Generation and characterization of anti-HTLV-1 bZIP factor (HBZ) monoclonal antibodies (mAbs). a HBZ protein levels in HBZ-transfected 293T cells were analyzed by indirect immunofluorescence using anti-HBZ mAbs P6-A7 (raised against peptide #1, mouse IgG2b), #20-H12 (raised against peptide #2, mouse IgG1), #91-1 (raised against peptide #3, rat IgG1), and #7-1 (raised against recombinant HBZ, mouse IgG2b). b HBZ expression vector-transfected 293T cells (HBZ) and mock-transfected 293T cells (Mock) were analyzed by western blotting using the novel anti-HBZ mAbs. c HBZ expression vector-transfected 293T cells ( red line ) and mock-transfected 293T cells ( blue line ) were analyzed by flow cytometry using the novel anti-HBZ mAbs. Three independent experiments were performed, and representative data are shown in each figure

Article Snippet: PVDF membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) and probed with anti-HBZ mAbs or anti-Histone H3 mAb (Cell Signaling Technology, Danvers, MA).

Techniques: Bioprocessing, Transfection, Immunofluorescence, Recombinant, Expressing, Plasmid Preparation, Western Blot, Flow Cytometry

Journal: Retrovirology

Article Title: Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status

doi: 10.1186/s12977-016-0263-z

Figure Lengend Snippet: HBZ protein expression in HTLV-1-infected cell lines. a HBZ protein expression in HTLV-1-infected cell lines was analyzed by western blotting using anti-HBZ mAb #91-1 (raised against peptide #3, rat IgG1), which gave highest sensitivity among four mAbs (i.e. P6-A7, #20-H12, #91-1, #7-1). Histone H3 was used as a loading control for the nuclear fraction. b HBZ protein expression in HTLV-1-infected cell lines (MT-1 and SLB1) was analyzed by immunofluorescence microscopy using anti-HBZ mAb #7-1 (raised against recombinant HBZ, mouse IgG2b), which gave highest sensitivity among four mAbs (i.e. P6-A7, #20-H12, #91-1, #7-1). Three independent experiments were performed, and representative data are shown in each figure . c Immunoprecipitation assay using four different anti-HBZ mAbs. P6-A7, #20-H12, and #7-1 were available for immunoprecipitation

Article Snippet: PVDF membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) and probed with anti-HBZ mAbs or anti-Histone H3 mAb (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Infection, Western Blot, Control, Immunofluorescence, Microscopy, Recombinant, Immunoprecipitation

Journal: Retrovirology

Article Title: Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status

doi: 10.1186/s12977-016-0263-z

Figure Lengend Snippet: Quantification of HBZ protein expression levels in HTLV-1-infected T-cell lines. The absolute intracellular concentration of an endogenous HBZ protein in HTLV-1-infected T-cell lines was analyzed by a novel HBZ sandwich ELISA. a Western blot image of twofold serially diluted recombinant HBZ protein probed with anti-HBZ mAb (clone #91-1). b A representative standard curve for the ELISA comprised of twofold serial dilutions of recombinant HBZ protein (from 4000 to 3.91 ng/mL). c Absolute quantification of HBZ protein in HTLV-1-infected T-cell lines. Results are presented as mean ± SD of three independent experiments with duplicate wells

Article Snippet: PVDF membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) and probed with anti-HBZ mAbs or anti-Histone H3 mAb (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Infection, Concentration Assay, Sandwich ELISA, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics

Journal: Retrovirology

Article Title: Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status

doi: 10.1186/s12977-016-0263-z

Figure Lengend Snippet: Quantification of HBZ protein expression levels and comparison to HBZ mRNA load, anti-HBZ antibody levels, and PVL in clinical samples from HTLV-1-infected individuals with different clinical status. a HBZ protein expression levels in naturally infected PBMCs of HTLV-1-infected individuals with different clinical status, evaluated by an in-house sandwich ELISA using mAbs against HBZ. HBZ protein was detected in three out of five acute ATL patients examined, but not in HAM/TSP patients (zero out of ten) or ACs (zero out of four). The dotted line represents the cutoff, i.e. the mean OD plus twice the standard deviation of four HTLV-1 uninfected cells (CEM, Jurkat, and two normal uninfected PBMCs). b Anti-HBZ antibody levels in plasma of HTLV-1-infected individuals with different clinical status determined by ELISA using a recombinant HBZ protein. c HBZ mRNA levels in PBMCs of HTLV-1-infected individuals with different clinical status determined by real-time PCR. HBZ mRNA expression were normalized to the expression of the hypoxanthine phosphoribosyltransferase gene (Human HPRT1 Endogenous Control 4333768; Applied Biosystems). d HTLV-1 proviral load (PVL) in PBMCs of HTLV-1-infected individuals with different clinical status. The HTLV-1 PVL was determined using the following formula: HTLV-1 tax copy number per 1 × 10 4 PBMCs = [( tax copy number)/(β-actin copy number/2)] × 10 4 . All samples were analyzed in triplicate

Article Snippet: PVDF membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) and probed with anti-HBZ mAbs or anti-Histone H3 mAb (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Comparison, Infection, Sandwich ELISA, Standard Deviation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Control