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SouthernBiotech hrp conjugated secondary antibody
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% <t>glutaraldehyde/PBS.</t> 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 <t>HRP-conjugated</t> 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.
Hrp Conjugated Secondary Antibody, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 99/100, based on 33 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

Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.
Figure Legend Snippet: Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

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

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

Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.
Figure Legend Snippet: Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

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

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    SouthernBiotech horse radish peroxidase conjugated goat anti mouse igg
    In vitro <t>IgG</t> antibody secreting cell responses in splenocytes day 5 post challenge. The cells from the spleens were cultured for 1 day or 5 days and RSV specific IgG antibodies secreted into culture supernatants determined by ELISA. (A) IgG, (B) <t>IgG1,</t> (C) IgG2a and (D) Ratios of IgG2a/IgG1 isotypes levels were analyzed by ELISA using FI-RSV as a coating antigen. Mouse groups are the same as described in Figs 1 and 3 . Results are presented as mean (n = 5) ± SEM and were obtained from duplicate experiments. Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism; *** p
    Horse Radish Peroxidase Conjugated Goat Anti Mouse Igg, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech hrp conjugated secondary antibody
    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% <t>glutaraldehyde/PBS.</t> 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 <t>HRP-conjugated</t> 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.
    Hrp Conjugated Secondary Antibody, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    SouthernBiotech hrp conjugated anti rat igg
    Expression of XMRV Env, Gag and VLP. (A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and <t>HRP-conjugated</t> goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.
    Hrp Conjugated Anti Rat Igg, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SouthernBiotech hrp conjugated goat anti mouse secondary antibody
    Specific binding of PzE by monoclonal antibodies that recognize zE conformational epitopes. PzE was coated in microtitre plates and incubated with serial dilutions of ZV 1 or ZV 54 mA b. E16, a West Nile virus EDIIII ‐specific mA b was used as a negative control. The specific binding between various mA b and PzE was detected by an HRP ‐conjugated goat <t>anti‐mouse</t> IgG antibody. Mean ± SD of samples from three independent experiments is presented.
    Hrp Conjugated Goat Anti Mouse Secondary Antibody, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vitro IgG antibody secreting cell responses in splenocytes day 5 post challenge. The cells from the spleens were cultured for 1 day or 5 days and RSV specific IgG antibodies secreted into culture supernatants determined by ELISA. (A) IgG, (B) IgG1, (C) IgG2a and (D) Ratios of IgG2a/IgG1 isotypes levels were analyzed by ELISA using FI-RSV as a coating antigen. Mouse groups are the same as described in Figs 1 and 3 . Results are presented as mean (n = 5) ± SEM and were obtained from duplicate experiments. Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism; *** p

    Journal: PLoS ONE

    Article Title: Vaccination by microneedle patch with inactivated respiratory syncytial virus and monophosphoryl lipid A enhances the protective efficacy and diminishes inflammatory disease after challenge

    doi: 10.1371/journal.pone.0205071

    Figure Lengend Snippet: In vitro IgG antibody secreting cell responses in splenocytes day 5 post challenge. The cells from the spleens were cultured for 1 day or 5 days and RSV specific IgG antibodies secreted into culture supernatants determined by ELISA. (A) IgG, (B) IgG1, (C) IgG2a and (D) Ratios of IgG2a/IgG1 isotypes levels were analyzed by ELISA using FI-RSV as a coating antigen. Mouse groups are the same as described in Figs 1 and 3 . Results are presented as mean (n = 5) ± SEM and were obtained from duplicate experiments. Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism; *** p

    Article Snippet: The antibody responses were detected using the secondary antibodies of horse radish peroxidase-conjugated goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology).

    Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

    RSV specific IgG and isotype IgG antibody responses after prime immunization with FI-RSV-via IM or MN patch. RSV specific IgG, isotype IgG1 and Ig2a serum antibodies were determined at 2 weeks after prime immunization (n = 5) by ELISA using FI-RSV as a coating antigen. (A) IgG, (B) IgG1 and (C) IgG2a levels. IM: intramuscular (IM) immunization with FI-RSV (2.2 μg), IM+MPL: IM immunization with FI-RSV (2.2 μg) + MPL (1 μg), MN: MN (microneedle) patch immunization in skin with FI-RSV (2.2 μg), MN+MPL: MN patch immunization with FI-RSV (2.2 μg) + MPL (1 μg) injected intradermally. RSV specific IgG concentrations (μg/ml) are presented as mean ± SEM (n = 5). The experiments were performed in duplicates. PBS: Unimmunized (no vaccine in PBS) control. Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism; *** p

    Journal: PLoS ONE

    Article Title: Vaccination by microneedle patch with inactivated respiratory syncytial virus and monophosphoryl lipid A enhances the protective efficacy and diminishes inflammatory disease after challenge

    doi: 10.1371/journal.pone.0205071

    Figure Lengend Snippet: RSV specific IgG and isotype IgG antibody responses after prime immunization with FI-RSV-via IM or MN patch. RSV specific IgG, isotype IgG1 and Ig2a serum antibodies were determined at 2 weeks after prime immunization (n = 5) by ELISA using FI-RSV as a coating antigen. (A) IgG, (B) IgG1 and (C) IgG2a levels. IM: intramuscular (IM) immunization with FI-RSV (2.2 μg), IM+MPL: IM immunization with FI-RSV (2.2 μg) + MPL (1 μg), MN: MN (microneedle) patch immunization in skin with FI-RSV (2.2 μg), MN+MPL: MN patch immunization with FI-RSV (2.2 μg) + MPL (1 μg) injected intradermally. RSV specific IgG concentrations (μg/ml) are presented as mean ± SEM (n = 5). The experiments were performed in duplicates. PBS: Unimmunized (no vaccine in PBS) control. Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism; *** p

    Article Snippet: The antibody responses were detected using the secondary antibodies of horse radish peroxidase-conjugated goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology).

    Techniques: Enzyme-linked Immunosorbent Assay, Injection

    IgG antibody responses specific for F and G protein antigens, and RSV neutralizing titers in prime immune sera. RSV F and G protein specific IgG antibodies were determined by ELISA in sera collected at 3 weeks after prime immunization of mice (n = 5). (A) F protein specific IgG antibodies (μg/ml). (B) IgG antibodies (μg/ml) specific for G 130-230 fragment. (C) RSV neutralizing antibody titers. RSV neutralizing antibody titers were determined by 50% plaque forming reduction (IC50%) assay. The linear line represents a lower limit of detection in RSV neutralizing titers. Results are presented as mean ± SEM (n = 5) and representative of duplicate experiments. Live RSV: the group of mice that were intranasally inoculated with RSV A2 (1x10 4 PFU/50 μl) as a live RSV control. Other groups are the same as described in the legend Fig 1 . Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism or student T-test; ***p

    Journal: PLoS ONE

    Article Title: Vaccination by microneedle patch with inactivated respiratory syncytial virus and monophosphoryl lipid A enhances the protective efficacy and diminishes inflammatory disease after challenge

    doi: 10.1371/journal.pone.0205071

    Figure Lengend Snippet: IgG antibody responses specific for F and G protein antigens, and RSV neutralizing titers in prime immune sera. RSV F and G protein specific IgG antibodies were determined by ELISA in sera collected at 3 weeks after prime immunization of mice (n = 5). (A) F protein specific IgG antibodies (μg/ml). (B) IgG antibodies (μg/ml) specific for G 130-230 fragment. (C) RSV neutralizing antibody titers. RSV neutralizing antibody titers were determined by 50% plaque forming reduction (IC50%) assay. The linear line represents a lower limit of detection in RSV neutralizing titers. Results are presented as mean ± SEM (n = 5) and representative of duplicate experiments. Live RSV: the group of mice that were intranasally inoculated with RSV A2 (1x10 4 PFU/50 μl) as a live RSV control. Other groups are the same as described in the legend Fig 1 . Statistical significances were performed by one-way ANOVA and Tukey’s multiple-comparison tests in GraphPad Prism or student T-test; ***p

    Article Snippet: The antibody responses were detected using the secondary antibodies of horse radish peroxidase-conjugated goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology).

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay

    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: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

    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: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

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

    Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). 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: Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the 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 an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.

    Article Snippet: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.

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

    Expression of XMRV Env, Gag and VLP. (A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.

    Journal: PLoS ONE

    Article Title: Antibody Responses against Xenotropic Murine Leukemia Virus-Related Virus Envelope in a Murine Model

    doi: 10.1371/journal.pone.0018272

    Figure Lengend Snippet: Expression of XMRV Env, Gag and VLP. (A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.

    Article Snippet: After incubation with secondary antibody, HRP-conjugated anti-rat IgG (Southern Biotech, Birmingham, AL), protein bands were visualized with enhanced chemiluminescence detection reagents (Amersham Pharmacia).

    Techniques: Expressing, Western Blot, Infection, Cell Culture, Centrifugation, SDS Page, Molecular Weight, Flow Cytometry, Cytometry, Staining, Produced, Purification, Electron Microscopy

    Specific binding of PzE by monoclonal antibodies that recognize zE conformational epitopes. PzE was coated in microtitre plates and incubated with serial dilutions of ZV 1 or ZV 54 mA b. E16, a West Nile virus EDIIII ‐specific mA b was used as a negative control. The specific binding between various mA b and PzE was detected by an HRP ‐conjugated goat anti‐mouse IgG antibody. Mean ± SD of samples from three independent experiments is presented.

    Journal: Plant Biotechnology Journal

    Article Title: Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

    doi: 10.1111/pbi.12796

    Figure Lengend Snippet: Specific binding of PzE by monoclonal antibodies that recognize zE conformational epitopes. PzE was coated in microtitre plates and incubated with serial dilutions of ZV 1 or ZV 54 mA b. E16, a West Nile virus EDIIII ‐specific mA b was used as a negative control. The specific binding between various mA b and PzE was detected by an HRP ‐conjugated goat anti‐mouse IgG antibody. Mean ± SD of samples from three independent experiments is presented.

    Article Snippet: The plates were then incubated with a HRP‐conjugated goat anti‐mouse secondary antibody (Southern Biotech, Bermingham, AL), developed with tetramethylbenzidine (TMB) substrate, and read at 450 nm (KPL Inc, MA).

    Techniques: Binding Assay, Incubation, Negative Control