gh ha1  (Sino Biological)


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    Structured Review

    Sino Biological gh ha1
    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into <t>HA1</t> and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Gh Ha1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin"

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199683

    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Figure Legend Snippet: Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Techniques Used: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.
    Figure Legend Snippet: Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Techniques Used: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.
    Figure Legend Snippet: CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.

    Techniques Used: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption, HI Assay, Drug Susceptibility Assay

    2) Product Images from "Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin"

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0199683

    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Figure Legend Snippet: Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Techniques Used: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.
    Figure Legend Snippet: Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Techniques Used: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.
    Figure Legend Snippet: CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.

    Techniques Used: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption, HI Assay, Drug Susceptibility Assay

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: The pH sensitive mouse anti-HA mAbs: anti-H2 HA (H2N2) 1/87 and anti-H3 HA (H3N2) (HC31 and HC67) were used to determine binding activities to various pH-treated H2 and H3 rHAs, respectively. .. Confirmation of H3 rHA folding by ELISA using mouse monoclonal antibodiesEctodomain (in-house made) and GH HA1 (Sino Biological, Inc. China) of rHAs from A/Hong Kong/1/68 (H3N2) were coated on nickel-coated plates and an ELISA was performed with rabbit anti-H3N2 (A/Aichi/2/68) sera and a panel of mouse anti-H3 HA (A/Aichi/2/68) conformation specific mAbs HC3, HC31, HC67, HC100, and HC263, as described previously [ , ]. .. Determination of the optimal TPCK-treated trypsin concentration for cleavage of rHA bound to nickel-coated platesTo determine the N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (trypsin, Sigma, MO) concentration to cleave rHA, rHA was bound to nickel-coated plates and subjected to digestion with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only conditions at 37°C for 15 minutes.

    other:

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: As shown in , most conformational epitope specific mAbs reacted with the ectodomain of rHA and GH HA1 at similar levels, with the exception of HC31 and HC67, which was expected because these mAbs bind to the trimeric HA head domain at neutral pH conformation.

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: As shown in , the major epitopes of monomeric GH HA1 from A/Hong Kong/1/68 fold correctly; furthermore, HI antibodies were adsorbed completely , as expected GH HA1 did not bind to HC31 and HC67 antibodies which recognize trimeric epitopes ( ).

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: We also used another GH HA1 from A/Perth/16/2009 that was not evaluated for proper folding due to the lack of well-characterized mAbs.

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: Initially, the conformation of GH HA1 was confirmed using an anti-H3 HA mAb panel used in previous studies [ , ].

    Adsorption:

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: Additional steps were performed as described previously, except that rabbit antisera against A/Aichi/2/68 (H3N2) virus was used in the Western blot to reflect appropriate antisera for the H3 rHA. .. Serum adsorption with GH HA1 from A/Hong Kong/1/68 (H3N2) only or followed by adsorption with GH HA1 from A/Perth/16/2009 (H3N2)The convenient human serum pool (Pool) and the highest positive sample #115 in HCCIA were further analyzed by HI and traditional microneutralization (TMN) assays to explore correlations between HCCIA and existing assays. .. Many serum samples showed HI titers against the A/Aichi/2/68 (H3N2) and A/Perth/16/2009 (H3N2) viruses, indicating the presence of antibodies that bound to the HA head domain and showed hemagglutination inhibition activity.

    Drug Susceptibility Assay:

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
    Article Snippet: As shown in , sample #115 showed greater inhibition of low pH induced HA conformational changes compared to the human serum pool at a 1:400 dilution, and the ability of the mock or GH HA1 adsorbed samples to inhib it low pH induced conformational changes of the rHA was indistinguishable ( ). .. This observation was further confirmed using the protease susceptibility assay where low pH treated HAs were protected equally for both mock or GH HA1 adsorbed #115 serum samples ( ). .. The neutralizing activities of these serum samples after mock or GH HA1 adsorption were analyzed using A/Aichi/2/68 (H3N2) and A/Perth/16/2009 (H3N2) viruses.

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    Sino Biological gh ha1
    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into <t>HA1</t> and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.
    Gh Ha1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological mouse monoclonal anti ha
    Quercetin inhibited vRNP localization in the nucleus. MDCK cells were infected with virus in the presence of quercetin at the concentration of 100, 50 and 25 µg/mL. Viral NP protein was detected with NP-specific <t>monoclonal</t> antibody and Alexa 488-conjugated goat <t>anti-mouse</t> secondary antibody ( green ); the nuclei were counterstained with DAPI ( blue ). Original magnification, 40×.
    Mouse Monoclonal Anti Ha, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological mouse igg anti ha1 monoclonal antibodies
    Quercetin inhibited vRNP localization in the nucleus. MDCK cells were infected with virus in the presence of quercetin at the concentration of 100, 50 and 25 µg/mL. Viral NP protein was detected with NP-specific <t>monoclonal</t> antibody and Alexa 488-conjugated goat <t>anti-mouse</t> secondary antibody ( green ); the nuclei were counterstained with DAPI ( blue ). Original magnification, 40×.
    Mouse Igg Anti Ha1 Monoclonal Antibodies, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse igg anti ha1 monoclonal antibodies/product/Sino Biological
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    Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: Low pH induced conformational changes of H3 rHA were inhibited by the human serum samples. A. Inhibition of low pH induced H3 rHA conformation change by a convenient human serum pool (Pool) in HCCIA. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. rHA coated plates were incubated with diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour. The plate was washed and treated with a range of pH buffers followed by fixation with 0.05% glutaraldehyde/PBS. An ELISA was performed using a pH-specific mAb, HC31, and detected by measuring the OD at 450 nm. B. Inhibition of H3 rHA low pH induced conformation change by human serum pool in the proteinase susceptibility assay. The proteinase susceptibility assay was performed to confirm HA low pH conformational changes in Fig 4A . H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The rHA coated plate was incubated with either diluent only, 1:4000, 1:400, or 1:40 diluted Pool for 1 hour followed by treatment with pH 7.0 or pH 4.8 buffer. The rHAs were digested with 0, 0.1, 1, or 10 μg/ml trypsin, the samples including digestion mixture and rHA left on plate were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with rabbit anti A/Aichi/2/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody. C. Detection of the CCI against H3 rHA in normal human sera in HCCIA. In total, 150 normal human sera collected from US residents were tested at 1:400 dilution by HCCIA as described in the Fig 4A legend. The OD ratio of HC31 at pH 4.8 to pH 7.0 was plotted; the highest ratio positive sample, #115, highlighted as a filled circle.

    Article Snippet: Confirmation of H3 rHA folding by ELISA using mouse monoclonal antibodiesEctodomain (in-house made) and GH HA1 (Sino Biological, Inc. China) of rHAs from A/Hong Kong/1/68 (H3N2) were coated on nickel-coated plates and an ELISA was performed with rabbit anti-H3N2 (A/Aichi/2/68) sera and a panel of mouse anti-H3 HA (A/Aichi/2/68) conformation specific mAbs HC3, HC31, HC67, HC100, and HC263, as described previously [ , ].

    Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: Determination of low pH induced conformational change of H3 rHA on 96-well nickel-coated plate. Optimization of trypsin concentration for cleavage of rHA coated on nickel-coated plates. H3 rHA bound nickel-coated plates were digested with two-fold serially diluted trypsin starting from 16,000 ng/ml to 32 ng/ml in PBS and PBS only as control. A. rHAs were eluted by 1X reducing SLB supplemented with 0.5M Imidazole followed by Western blot using anti H3 rabbit sera. B. Trypsin treated rHAs were analyzed by ELISA using anti H3 monoclonal antibody HC3. C. Cleavage of HA0 into HA1 and HA2 of H3 rHA by trypsin was essential for low pH induced HA conformational changes. H3 rHAs bound to nickel-coated plates were treated with 100 μl of 200 ng/ml trypsin to cleave HA0 into HA1 and HA2. The plate was treated with a series of pH buffers followed by fixation with 0.05% glutaraldehyde in PBS. ELISA was performed by using pH-specific mAbs HC31 and HC67, and HC3 served as a control for H3 rHA. D. The proteinase susceptibility assay was performed to confirm the low pH induced HA conformational changes in Fig 2D . The H3 rHA bound nickel-coated plate was treated with pH 7.0 or pH 4.8 followed by 0, 0.1, 1, or 10 μg/ml trypsin digestion. Total sample which included the digestion mixture and rHA remaining bound to the plate were entirely eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with a rabbit anti A/Aichi/1/68 (H3N2) antisera. HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Article Snippet: Confirmation of H3 rHA folding by ELISA using mouse monoclonal antibodiesEctodomain (in-house made) and GH HA1 (Sino Biological, Inc. China) of rHAs from A/Hong Kong/1/68 (H3N2) were coated on nickel-coated plates and an ELISA was performed with rabbit anti-H3N2 (A/Aichi/2/68) sera and a panel of mouse anti-H3 HA (A/Aichi/2/68) conformation specific mAbs HC3, HC31, HC67, HC100, and HC263, as described previously [ , ].

    Techniques: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.

    Journal: PLoS ONE

    Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin

    doi: 10.1371/journal.pone.0199683

    Figure Lengend Snippet: CCI showed virus neutralizing activity in TMN after removal of head-binding antibodies. A. Correct folding of major epitopes for GH HA1 from A/Hong Kong/1/68 (H3N2) (Sino Biological, Inc. China) was confirmed by appropriate Ab binding profiles. GH HA1 from A/Hong Kong/1/68 (H3N2) and ectodomain H3 rHA from A/Aichi/1/68 (H3N2) were coated on a nickel-coated plate, an ELISA was performed by using rabbit antisera and a panel of conformation specific mAbs. B. Removal of HI antibodies by serum adsorption with GH HA1 rHAs. The convenient human serum pool (Pool) and #115, the highest positive sample in HCCIA, were adsorbed with GH HA1 from A/Hong Kong/1/68 (H3N2) or double adsorbed with GH HA1 proteins from A/Hong Kong/1/68 (H3N2) and A/Perth/16/2009 (H3N2). Hemagglutination inhibition assays were performed by using A/Aichi/2/68 and A/Perth/16/2009. CCI were consistently present after mock or serum adsorption with GH HA1 in HCCIA (C) and the proteinase susceptibility assay (D). E. CCI neutralized A/Aichi/2/68 (H3N2) and A/Perth/16/2009 after head-binding antibodies were removed by serum adsorption with GH HA1 rHAs. ND: not done.

    Article Snippet: Confirmation of H3 rHA folding by ELISA using mouse monoclonal antibodiesEctodomain (in-house made) and GH HA1 (Sino Biological, Inc. China) of rHAs from A/Hong Kong/1/68 (H3N2) were coated on nickel-coated plates and an ELISA was performed with rabbit anti-H3N2 (A/Aichi/2/68) sera and a panel of mouse anti-H3 HA (A/Aichi/2/68) conformation specific mAbs HC3, HC31, HC67, HC100, and HC263, as described previously [ , ].

    Techniques: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption, HI Assay, Drug Susceptibility Assay

    Quercetin inhibited vRNP localization in the nucleus. MDCK cells were infected with virus in the presence of quercetin at the concentration of 100, 50 and 25 µg/mL. Viral NP protein was detected with NP-specific monoclonal antibody and Alexa 488-conjugated goat anti-mouse secondary antibody ( green ); the nuclei were counterstained with DAPI ( blue ). Original magnification, 40×.

    Journal: Viruses

    Article Title: Quercetin as an Antiviral Agent Inhibits Influenza A Virus (IAV) Entry

    doi: 10.3390/v8010006

    Figure Lengend Snippet: Quercetin inhibited vRNP localization in the nucleus. MDCK cells were infected with virus in the presence of quercetin at the concentration of 100, 50 and 25 µg/mL. Viral NP protein was detected with NP-specific monoclonal antibody and Alexa 488-conjugated goat anti-mouse secondary antibody ( green ); the nuclei were counterstained with DAPI ( blue ). Original magnification, 40×.

    Article Snippet: Mouse monoclonal anti-HA (1:100 dilution; Sino Biological, Beijing, China) and rabbit monoclonal anti β-actin antibody (1:1000 dilution) were used as the primary antibodies.

    Techniques: Infection, Concentration Assay