Structured Review

Rockland Immunochemicals hrp conjugated secondary antibody
Hrp Conjugated Secondary Antibody, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hrp conjugated secondary antibody/product/Rockland Immunochemicals
Average 90 stars, based on 8 article reviews
Price from $9.99 to $1999.99
hrp conjugated secondary antibody - by Bioz Stars, 2020-07
90/100 stars

Images

Related Articles

Incubation:

Article Title: Alzheimer's-Related Peptide Amyloid-? Plays a Conserved Role in Angiogenesis
Article Snippet: .. Blots were washed 3×10 min with TBS/0.05% Tween-20 and incubated with HRP-conjugated secondary antibodies for an hour at room temperature in a TBS/0.1% tween-20 buffer solution; secondary antibodies used were Anti-Goat HRP - 1∶25,000 (Rockland), Anti-Rabbit HRP - 1∶5000, Anti-Mouse HRP - 1∶5000 (GE Healthcare). .. After secondary antibody incubations, blots were washed 3×10 min in TBS/0.05% Tween-20, and then developed with ECL Detection reagent (GE Healthcare).

Article Title: Decreased Surface Expression of the δ Subunit of the GABAA Receptor Contributes to Reduced Tonic Inhibition in Dentate Granule Cells in a Mouse Model of Fragile X Syndrome
Article Snippet: .. Blots were incubated overnight at 4°C with primary polyclonal rabbit antibodies against the following: δ (aa1-44) ( ); α4 (aa379-421) ( ); (both at 1 µg/ml, gift of W. Sieghart); α4 (1:1000; Millipore, AB5457); or mouse monoclonal β-actin (1: 1000; Sigma Aldrich, A2228), followed by HRP-conjugated secondary antibodies (1:5000; Rockland) for 2 h at room temperature. .. Bands were detected using ECL detection kit (GE Healthcare) and exposed to X-ray films (MidSci).

Article Title: Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling
Article Snippet: .. Following several washes with 4% milk in PBS-T membranes were incubated with HRP-conjugated secondary antibodies (Rockland) for 1 h at room temperature. .. Membranes were developed using ECL-Plus reagent (GE Healthcare Bio-Science) and acquired in a chemiluminescence imager coupled to a CCD camera (ImageQuant LAS 4000 GE Healthcare).

Article Title: Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling
Article Snippet: .. Then, membrane was incubated with anti-TRAF6 antibody, and followed with HRP-conjugated secondary antibody (TrueBlot; Rockland). .. Signal development was performed by chemiluminescent kit (Millipore).

Article Title: 5,7-Dimethoxycoumarin prevents chronic mild stress induced depression in rats through increase in the expression of heat shock protein-70 and inhibition of monoamine oxidase-A levels
Article Snippet: .. The membrane washing was performed with PBS before 1 h incubation with HRP-conjugated secondary antibodies (Rockland, Gilbertsville, PA, USA). .. The protein bands were analyzed using the system (Odyssey; LI-COR, Inc., Lincoln, NE, USA).

other:

Article Title: Kaposi's Sarcoma-Associated Herpesvirus K-Cyclin Interacts with Cdk9 and Stimulates Cdk9-Mediated Phosphorylation of p53 Tumor Suppressor ▿
Article Snippet: HRP-conjugated secondary antibodies were purchased from Rockland Immunochemicals (Gilbertsville, PA).

Article Title: Increased Cell-Matrix Adhesion upon Constitutive Activation of Rho Proteins by Cytotoxic Necrotizing Factors from E. Coli and Y. Pseudotuberculosis
Article Snippet: The following reagents were obtained from commercial sources: RhoA (mAb-26C4), Rac1 (mAb-23A8) (Santa Cruz); β -actin (mAb AC-40) (Sigma); Cdc42 (mAb-44) (BD Transduction Laboratories); pS144/141-PAK1/2 (mAb EP656Y) (Abcam); vinculin (mAb hVIN-1) (Abcam); horseradish peroxidise-conjugated secondary antibodies (Rockland); anti-rabbit IgG Alexa Fluor 488 goat secondary antibody (Invitrogen).

Article Title: Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors
Article Snippet: HRP-conjugated secondary antibodies were anti-rabbit and anti-mouse antibodies from Zymax and anti-goat antibody from Rockland.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Rockland Immunochemicals rabbit anti mouse peroxidase conjugated igg1 abs
    Inhibition of <t>IgG1</t> antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.
    Rabbit Anti Mouse Peroxidase Conjugated Igg1 Abs, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mouse peroxidase conjugated igg1 abs/product/Rockland Immunochemicals
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mouse peroxidase conjugated igg1 abs - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    93
    Rockland Immunochemicals hrp conjugated mouse trueblot ultra secondary antibody
    Validation of candidate DG-binding proteins. (A) Coimmunoprecipitation (co-IP) of DG-associated scaffold proteins. Monolayers of A549 and SAEC were lysed in cold Triton X-100 containing buffer, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) using MAb anti-HA matrix (HA) as a control. Immunocomplexes were probed in Western blots for functional α-DG (MAb IIH6), β-DG (MAb 8D9), utrophin (MAb 20C5), α1-syntrophin (goat pAb 5941), β1-syntrophin (rabbit pAb 98977), β2-syntrophin (MAb 1351), and β-dystrobrevin (rabbit pAb 152133). Primary rabbit and goat antibodies as well as MAb IIH6 (mouse IgM) were detected as described in the legend to Fig. 1B and C . For detection of mouse MAb IgG, <t>HRP-conjugated</t> mouse <t>TrueBlot</t> <t>ULTRA</t> secondary antibody was used as detailed in Materials and Methods. For a positive control (+), total lysates of differentiated human myotubes were included. One representative example out of three independent experiments is shown. (B) Detection of sarcoglycans at the surfaces of A549 cells. Intact monolayers of A549 cells were subjected to cell surface biotinylation using the membrane-impermeable reagent sulfo-NHS-X-biotin (+) or reaction buffer only (−). After reaction quenching, cells were lysed, and biotinylated proteins were precipitated with streptavidin agarose beads. Proteins were eluted and probed in Western blots for β-DG (MAb 8D9), α-sarcoglycan (SG) (rabbit pAb R98), β-sarcoglycan (MAb 5B1), γ-sarcoglycan (MAb 21B5), δ-sarcoglycan (rabbit pAb R214), ε-sarcoglycan (rabbit pAb 155651), and sarcospan (SSN) (rabbit MAb 186730), followed by detection as described above for panel A. The positive control (+) was differentiated human myotube lysate. One representative example of two independent experiments is shown. (C) Flow chart for the live cell surface cross-linking approach. For details, please see text. (D) Chemical cross-linking of DG with sarcoglycans at the cell surface. Live intact A549 monolayers were treated with the membrane-impermeable thiol-cleavable cross-linking reagent DTSSP (+) or reaction buffer only (−). After quenching, cells were lysed, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or anti-HA matrix (HA) as described above for panel A. Eluted proteins were treated with DTT and analyzed in Western blot probing for functional α-DG, β-DG, the indicated sacroglycans, and sarcospan as described above for panel B. Please note that the blots for α- and β-DG (top) correspond each to 1% of the material, and the blots for the sacroglycans and sarcospan each correspond to 15% of the sample. One representative example of two independent experiments is shown. (E) Schema of a working model of the DG complex in A549 cells (this study) compared to the DG complex in skeletal muscle (based on published data [ 40 , 41 ]). The α-DG-linked matriglycan sugar polymers and β-DG are indicated, as well as α-, β-, γ-, δ-, and ε-sarcoglycans (SG), sarcospan (SPN), α- and β-dystrobrevin (DTN), α1, β1, and β2-syntrophin (SNT), and nitric oxide synthase (nNOS).
    Hrp Conjugated Mouse Trueblot Ultra Secondary Antibody, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated mouse trueblot ultra secondary antibody/product/Rockland Immunochemicals
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated mouse trueblot ultra secondary antibody - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    94
    Rockland Immunochemicals goat anti rabbit igg hrp conjugated
    Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human <t>IgG</t> (H+L) and goat anti-rabbit IgG <t>HRP</t> conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P
    Goat Anti Rabbit Igg Hrp Conjugated, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit igg hrp conjugated/product/Rockland Immunochemicals
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg hrp conjugated - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    88
    Rockland Immunochemicals goat anti rat igg
    Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins. ( A ) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. ( B ) Binding of anti-CD36 mAb and control mouse <t>IgA</t> to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. ( C ) Binding of rCD36 to plate-adsorbed proteins. ( D ) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. ( E ) Binding of polyclonal anti-mouse CD36 Ab and control goat <t>IgG</t> to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. ( F ) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. ( G ) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. ( H ) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p
    Goat Anti Rat Igg, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rat igg/product/Rockland Immunochemicals
    Average 88 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    goat anti rat igg - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    Image Search Results


    Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.

    Journal: Frontiers in Immunology

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    doi: 10.3389/fimmu.2017.00659

    Figure Lengend Snippet: Inhibition of IgG1 antibodies (Abs) in mice immune sera with OS ligands and CP. Enzyme-linked immunosorbent assay inhibition assays were performed using streptavidin-coated plates with tetra-biotin, hexa-biotin, and octa-biotin adsorbed on their surfaces. (A–C) Inhibition of IgG1 Abs in the pooled sera of BALB/c mice ( n = 6) that were immunized twice intraperitoneally with glycoconjugates at 10 µg/dose. Sera were obtained 14 days after the second immunization. Serum samples for all glycoconjugates were diluted 1:4,000. The tetra-, hexa-, and octasaccharide ligands, synCP, and bacCP were used as inhibitors and applied in amounts ranging from 0 to 10 µg/well. The horizontal line indicates the IC 50 at the point of intersection of the inhibition curves. (A) Tetra-bovine serum albumin (BSA) conjugate antiserum was tested against tetra-biotin capture material. (B) Hexa-BSA conjugate antiserum was tested against hexa-biotin capture material (C) . Octa-BSA conjugate antiserum was tested against octa-biotin capture material. (D) Inhibition of IgG1 Abs was measured in the pooled sera of BALB/c mice immunized twice intraperitoneally over 2 weeks with the conjugated pneumococcal vaccine, Prevenar-13, at 1.1 µg of Streptococcus pneumoniae type 14 CP per single dose. The dilutions of sera tested against the tetra-biotin and octa-biotin coating antigens were 1:500; hexa-biotin was diluted 1:300. (E) Inhibition of IgG Abs was measured in serum harvested from rabbits that were immunized multiple times with inactivated S. pneumonia type 14 bacteria. The dilution of rabbit sera tested against tetra-biotin coating antigens was 1:300, against hexa-biotin and octa-biotin coating antigens was 1:3,000; n = 3 per data point.

    Article Snippet: Rabbit anti-mouse peroxidase-conjugated IgG1 Abs (gamma 1 chain; Rockland Immunochemicals, Inc., USA) were used as secondary Abs.

    Techniques: Inhibition, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P

    Journal: Frontiers in Immunology

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    doi: 10.3389/fimmu.2017.00659

    Figure Lengend Snippet: Anti-CP IgG1 antibody (Ab) titers in mice immunized with the glycoconjugates. BALB/c mice were immunized intraperitoneally with tetra-bovine serum albumin (BSA) (A) , hexa-BSA (B) , and octa-BSA (C) conjugates adsorbed on aluminum hydroxide twice over 14 days at 1.25–10 µg/dose (the hexa-BSA conjugate was injected at 2.5–10 µg/dose). Anti-CP IgG1 Ab titers in murine blood sera were determined by enzyme-linked immunosorbent assay 2 weeks after the second immunization. Streptococcus pneumoniae type 14 bacterial CP was used as the coating antigens. The data from two experiments were summarized. For each glycoconjugate, blood was taken from 6 to 12 mice. The data represent individual anti-CP IgG1 Ab titers, bars indicates median ± SD. Mann–Whitney Rank Sum tests were used to evaluate significance. Differences in the anti-CP IgG1 Ab titers between tetra-BSA and octa-BSA conjugates at the immunizing dose of 10 µg/mouse, * P

    Article Snippet: Rabbit anti-mouse peroxidase-conjugated IgG1 Abs (gamma 1 chain; Rockland Immunochemicals, Inc., USA) were used as secondary Abs.

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P

    Journal: Frontiers in Immunology

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    doi: 10.3389/fimmu.2017.00659

    Figure Lengend Snippet: IgG1 antibody (Ab) titer in mice immunized with Streptococcus pneumoniae type 14 CP. BALB/c mice received two intraperitoneal immunizations with the conjugated pneumococcal vaccine Prevenar-13 with aluminum phosphate as adjuvant at 1.1 µg (content of S. pneumoniae type 14 CP) per dose. The tetra-bovine serum albumin (BSA), hexa-BSA, and octa-BSA conjugates, and synthetic (synCP) and bacterial CP (bacCP) were capture antigens, coating the enzyme-linked immunosorbent assay plates. The data are represented by individual titers of IgG1 Abs, bars indicate median ± SD. Mann–Whitney Rank Sum tests were used to determine significance. For differences in the level of Abs detected against tetra-BSA and octa-BSA conjugates, * P

    Article Snippet: Rabbit anti-mouse peroxidase-conjugated IgG1 Abs (gamma 1 chain; Rockland Immunochemicals, Inc., USA) were used as secondary Abs.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P

    Journal: Frontiers in Immunology

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    doi: 10.3389/fimmu.2017.00659

    Figure Lengend Snippet: Anti-OS IgG1 antibody (Ab) titer in mice immunized with the OS-conjugates, CP, or bacteria. The biotinylated oligosaccharides tetra-biotin, hexa-biotin, and octa-biotin were applied as coating antigens. (A) Fourteen days after the second immunization, IgG1 Ab titers were determined in the pooled sera of BALB/c mice ( n = 6) intraperitoneally vaccinated with a single dose (10 µg) of each conjugate adsorbed on aluminum hydroxide. (B) Fourteen days after the second immunization, the IgG1 Ab titers were measured in the pooled sera of two groups of BALB/c mice ( n = 6) who were intraperitoneally vaccinated with single doses of either 1.1 or 2.2 µg (CP content) of Streptococcus pneumoniae type 14 CP-CPM 197 adsorbed on aluminum phosphate. (C) The level of IgG Abs in rabbits ( n = 2) immunized with inactive S. pneumoniae type 14 bacterial cells. Ab titers were transformed to log 10 . n = 6 assays per antiserum. The data are displayed as a mean value ± SD. Mann–Whitney Rank Sum tests were used to determine significance, * P

    Article Snippet: Rabbit anti-mouse peroxidase-conjugated IgG1 Abs (gamma 1 chain; Rockland Immunochemicals, Inc., USA) were used as secondary Abs.

    Techniques: Mouse Assay, Transformation Assay, MANN-WHITNEY

    Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.

    Journal: Frontiers in Immunology

    Article Title: Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates

    doi: 10.3389/fimmu.2017.00659

    Figure Lengend Snippet: Inhibition of IgG antibodies (Abs) recognizing SynCP as the coating antigen with OS ligands and CP. Pooled immune sera were obtained after double intraperitoneal immunization of BALB/c mice ( n = 6 for each glycoconjugate) with the OS-bovine serum albumin (BSA) conjugates adsorbed on aluminum hydroxide (10 µg of carbohydrate/single dose). The tetra-, hexa-, and octasaccharide ligands, as well as synCP and bacCP, were used as inhibitory materials at concentrations of 0–10 µg/well. The horizontal line indicates the IC 50 level at the point of intersection of the inhibition curves. (A) Inhibition of IgG Abs was measured in tetra-BSA conjugate sera at a dilution of 1:400. (B) Inhibition of IgG Abs was measured in hexa-BSA conjugate sera at a dilution of 1:400. (C) Inhibition of IgG Abs was measured in octa-BSA conjugate sera at a dilution of 1:1,600. (D) Inhibition of IgG Abs was measured in Streptococcus pneumoniae type 14 CP-CRM 197 at a dilution of 1:200. Immune sera were obtained after immunization of mice ( n = 6) with Prevenar-13 adsorbed on aluminum phosphate at 2.2 µg (content of S. pneumoniae type 14 CP) per single dose.

    Article Snippet: Rabbit anti-mouse peroxidase-conjugated IgG1 Abs (gamma 1 chain; Rockland Immunochemicals, Inc., USA) were used as secondary Abs.

    Techniques: Inhibition, Mouse Assay

    Validation of candidate DG-binding proteins. (A) Coimmunoprecipitation (co-IP) of DG-associated scaffold proteins. Monolayers of A549 and SAEC were lysed in cold Triton X-100 containing buffer, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) using MAb anti-HA matrix (HA) as a control. Immunocomplexes were probed in Western blots for functional α-DG (MAb IIH6), β-DG (MAb 8D9), utrophin (MAb 20C5), α1-syntrophin (goat pAb 5941), β1-syntrophin (rabbit pAb 98977), β2-syntrophin (MAb 1351), and β-dystrobrevin (rabbit pAb 152133). Primary rabbit and goat antibodies as well as MAb IIH6 (mouse IgM) were detected as described in the legend to Fig. 1B and C . For detection of mouse MAb IgG, HRP-conjugated mouse TrueBlot ULTRA secondary antibody was used as detailed in Materials and Methods. For a positive control (+), total lysates of differentiated human myotubes were included. One representative example out of three independent experiments is shown. (B) Detection of sarcoglycans at the surfaces of A549 cells. Intact monolayers of A549 cells were subjected to cell surface biotinylation using the membrane-impermeable reagent sulfo-NHS-X-biotin (+) or reaction buffer only (−). After reaction quenching, cells were lysed, and biotinylated proteins were precipitated with streptavidin agarose beads. Proteins were eluted and probed in Western blots for β-DG (MAb 8D9), α-sarcoglycan (SG) (rabbit pAb R98), β-sarcoglycan (MAb 5B1), γ-sarcoglycan (MAb 21B5), δ-sarcoglycan (rabbit pAb R214), ε-sarcoglycan (rabbit pAb 155651), and sarcospan (SSN) (rabbit MAb 186730), followed by detection as described above for panel A. The positive control (+) was differentiated human myotube lysate. One representative example of two independent experiments is shown. (C) Flow chart for the live cell surface cross-linking approach. For details, please see text. (D) Chemical cross-linking of DG with sarcoglycans at the cell surface. Live intact A549 monolayers were treated with the membrane-impermeable thiol-cleavable cross-linking reagent DTSSP (+) or reaction buffer only (−). After quenching, cells were lysed, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or anti-HA matrix (HA) as described above for panel A. Eluted proteins were treated with DTT and analyzed in Western blot probing for functional α-DG, β-DG, the indicated sacroglycans, and sarcospan as described above for panel B. Please note that the blots for α- and β-DG (top) correspond each to 1% of the material, and the blots for the sacroglycans and sarcospan each correspond to 15% of the sample. One representative example of two independent experiments is shown. (E) Schema of a working model of the DG complex in A549 cells (this study) compared to the DG complex in skeletal muscle (based on published data [ 40 , 41 ]). The α-DG-linked matriglycan sugar polymers and β-DG are indicated, as well as α-, β-, γ-, δ-, and ε-sarcoglycans (SG), sarcospan (SPN), α- and β-dystrobrevin (DTN), α1, β1, and β2-syntrophin (SNT), and nitric oxide synthase (nNOS).

    Journal: mBio

    Article Title: Dynamic Dystroglycan Complexes Mediate Cell Entry of Lassa Virus

    doi: 10.1128/mBio.02869-18

    Figure Lengend Snippet: Validation of candidate DG-binding proteins. (A) Coimmunoprecipitation (co-IP) of DG-associated scaffold proteins. Monolayers of A549 and SAEC were lysed in cold Triton X-100 containing buffer, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) using MAb anti-HA matrix (HA) as a control. Immunocomplexes were probed in Western blots for functional α-DG (MAb IIH6), β-DG (MAb 8D9), utrophin (MAb 20C5), α1-syntrophin (goat pAb 5941), β1-syntrophin (rabbit pAb 98977), β2-syntrophin (MAb 1351), and β-dystrobrevin (rabbit pAb 152133). Primary rabbit and goat antibodies as well as MAb IIH6 (mouse IgM) were detected as described in the legend to Fig. 1B and C . For detection of mouse MAb IgG, HRP-conjugated mouse TrueBlot ULTRA secondary antibody was used as detailed in Materials and Methods. For a positive control (+), total lysates of differentiated human myotubes were included. One representative example out of three independent experiments is shown. (B) Detection of sarcoglycans at the surfaces of A549 cells. Intact monolayers of A549 cells were subjected to cell surface biotinylation using the membrane-impermeable reagent sulfo-NHS-X-biotin (+) or reaction buffer only (−). After reaction quenching, cells were lysed, and biotinylated proteins were precipitated with streptavidin agarose beads. Proteins were eluted and probed in Western blots for β-DG (MAb 8D9), α-sarcoglycan (SG) (rabbit pAb R98), β-sarcoglycan (MAb 5B1), γ-sarcoglycan (MAb 21B5), δ-sarcoglycan (rabbit pAb R214), ε-sarcoglycan (rabbit pAb 155651), and sarcospan (SSN) (rabbit MAb 186730), followed by detection as described above for panel A. The positive control (+) was differentiated human myotube lysate. One representative example of two independent experiments is shown. (C) Flow chart for the live cell surface cross-linking approach. For details, please see text. (D) Chemical cross-linking of DG with sarcoglycans at the cell surface. Live intact A549 monolayers were treated with the membrane-impermeable thiol-cleavable cross-linking reagent DTSSP (+) or reaction buffer only (−). After quenching, cells were lysed, and cleared lysates were subjected to IP with MAb VIA4 matrix (α-DG) or anti-HA matrix (HA) as described above for panel A. Eluted proteins were treated with DTT and analyzed in Western blot probing for functional α-DG, β-DG, the indicated sacroglycans, and sarcospan as described above for panel B. Please note that the blots for α- and β-DG (top) correspond each to 1% of the material, and the blots for the sacroglycans and sarcospan each correspond to 15% of the sample. One representative example of two independent experiments is shown. (E) Schema of a working model of the DG complex in A549 cells (this study) compared to the DG complex in skeletal muscle (based on published data [ 40 , 41 ]). The α-DG-linked matriglycan sugar polymers and β-DG are indicated, as well as α-, β-, γ-, δ-, and ε-sarcoglycans (SG), sarcospan (SPN), α- and β-dystrobrevin (DTN), α1, β1, and β2-syntrophin (SNT), and nitric oxide synthase (nNOS).

    Article Snippet: For detection of mouse MAb IgG in IPs using mouse IgG, HRP-conjugated mouse TrueBlot ULTRA secondary antibody was used (Rockland Inc.).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Functional Assay, Positive Control, Flow Cytometry

    Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG (H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P

    Journal: Journal of Neuroinflammation

    Article Title: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages

    doi: 10.1186/s12974-014-0195-2

    Figure Lengend Snippet: Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG (H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P

    Article Snippet: Rabbit-anti-human IgG(H+L) (1:1,000 dilution) (Rockland) and goat anti-rabbit IgG HRP-conjugated (1:3,000 dilution) (Rockland) were used before the exposure to a metal enhanced DAB substrate (PIERCE).

    Techniques: Binding Assay, Incubation, Cell Culture, Positive Control, CTL Assay, Negative Control, Functional Assay, MTT Assay

    Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins. ( A ) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. ( B ) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. ( C ) Binding of rCD36 to plate-adsorbed proteins. ( D ) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. ( E ) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. ( F ) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. ( G ) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. ( H ) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p

    Journal: PLoS ONE

    Article Title: Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

    doi: 10.1371/journal.pone.0123293

    Figure Lengend Snippet: Roles of SR CD36 (A-E) and SR-A (G-I) as receptors for HOCl-modified proteins. ( A ) Uptake of fluorescently-labelled proteins by CHO cells transfected with human CD36 as compared to non-transfected cells. ( B ) Binding of anti-CD36 mAb and control mouse IgA to CD36-transfected and non-transfected CHO cells, determined by cellular ELISA. ( C ) Binding of rCD36 to plate-adsorbed proteins. ( D ) Effects of 10 or 100 μg/ml of indicated, soluble ligands on rCD36 binding to plate-adsorbed OVA-Cl. ( E ) Binding of polyclonal anti-mouse CD36 Ab and control goat IgG to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is displayed on the left graph. ( F ) Binding of anti-mouse SR-A mAb and control rat IgG2b to BM-DC, splenic DC and PEM. A representative histogram of Ab binding to BM-DC is shown on the left graph. ( G ) Binding of SR-A present in lysates of PEM to plate-adsorbed proteins. ( H ) Effects of anti-SR-A 2F8 mAb and AcLDL on the uptake of fluorescently-labelled proteins by BM-DC. Shown are results of single experiments, each representative of at least 3 similar experiments performed (A-D, G, H) or averages +SEM from 4–6 independent experiments (E, F). The data were analysed with the unpaired (A-C, G) or one-sample (H) Student’s t-test or with ANOVA, followed by the Tukey-Kramer post-test (D). *, p

    Article Snippet: Other reagents Rat anti-mouse scavenger receptor A (SR-A) mAb (clone 2F8) was obtained from AbD Serotec; mouse anti-mouse CD36 mAb (CRF D-2712) from Hycult Biotech; mouse IgA isotype control mAb (M18-254), rat IgG2b isotype control mAb (A95-1), rat anti-mouse CD11b mAb (M1/70) and phycoerythrin (PE)-streptavidin conjugate from BD Biosciences; rat IgG2a isotype control mAb (54447), normal goat IgG, polyclonal goat anti-mouse CD36, anti-mouse LOX-1 (lectin-type oxidised LDL receptor-1), anti-human SREC-I (scavenger receptor expressed by endothelial cells-I) Ab and PE-conjugated rat anti-mouse LOX-1 mAb (214012) from R & D Systems; polyclonal goat anti-mouse SREC-I, anti-mouse RAGE (receptor for advanced glycation end products), anti-mouse stabilin-1 and rabbit anti-mouse-stabilin-1 Ab from Santa Cruz Biotechnology; PE-conjugated donkey anti-goat IgG Ab from SouthernBiotech; rat anti-mouse CD206/mannose receptor mAb (MR5D3) and PE-conjugated goat anti-rat IgG Ab from BioLegend; horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgA, F(ab’)2 fragments of goat anti-rat IgG and donkey anti-goat IgG Ab from Rockland.

    Techniques: Modification, Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay, End-sequence Profiling