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human c3a c3a desarg  (Hycult Biotech)


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    Hycult Biotech human c3a c3a desarg
    Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine <t>C3a</t> desArg formation. B , principle of HTRF assay to detect C3 depletion.
    Human C3a C3a Desarg, supplied by Hycult Biotech, 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 "Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P"

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.101136

    Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine C3a desArg formation. B , principle of HTRF assay to detect C3 depletion.
    Figure Legend Snippet: Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine C3a desArg formation. B , principle of HTRF assay to detect C3 depletion.

    Techniques Used: Activation Assay, HTRF Assay

    MSD method for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , specificity of the coating antibody. Purified C3 (■), C3a (●), C3a desArg (○) and iC3b (□) were diluted to various concentrations up to ∼22.2 nM and captured in an MSD plate coated with neo-epitope of human C3a/C3a-desArg monoclonal antibody. In B – D , pooled human serum from Complement Technology was used and the final serum volume was 70% in the complement mixture. B , impact of dilution factor on quantitation of C3a desArg formation yielded from complement activation. Dezamizumab and SAP antigen was mixed at a 4:1 ratio (μg/ml) in the complement activation mixture. The final Dezamizumab concentration was 500 μg/ml (○), 250 μg/ml (●), and serum-only control (□). The reaction was terminated after 40 min at ambient temperature. The complement activation mixture and serum-only control were diluted with HBSP buffer at a range from 5 to 10,935-fold in threefold serial dilution before testing in the MSD assay. C , time course of complement activation at 37 °C. D , time course of complement activation at ambient temperature. In both C and D , Dezamizumab:SAP antigen ratio was 4:1 in μg/ml in the complement mixture. Complement activation was terminated with the stop solution at the indicated time. The complement activation mixtures were diluted 10,000-fold before testing in the MSD method. The MSD signal was back calculated by dilution factors and normalized by subtracting T = 0 signal. The final Dezamizumab concentration was 1000 μg/ml (○), 500 μg/ml (●), 250 μg/ml (□), 125 μg/ml (■), 62.5 μg/ml (Δ), and serum-only control (▲). Error bars for all panels represent the mean ± SD from n = 2 determination.
    Figure Legend Snippet: MSD method for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , specificity of the coating antibody. Purified C3 (■), C3a (●), C3a desArg (○) and iC3b (□) were diluted to various concentrations up to ∼22.2 nM and captured in an MSD plate coated with neo-epitope of human C3a/C3a-desArg monoclonal antibody. In B – D , pooled human serum from Complement Technology was used and the final serum volume was 70% in the complement mixture. B , impact of dilution factor on quantitation of C3a desArg formation yielded from complement activation. Dezamizumab and SAP antigen was mixed at a 4:1 ratio (μg/ml) in the complement activation mixture. The final Dezamizumab concentration was 500 μg/ml (○), 250 μg/ml (●), and serum-only control (□). The reaction was terminated after 40 min at ambient temperature. The complement activation mixture and serum-only control were diluted with HBSP buffer at a range from 5 to 10,935-fold in threefold serial dilution before testing in the MSD assay. C , time course of complement activation at 37 °C. D , time course of complement activation at ambient temperature. In both C and D , Dezamizumab:SAP antigen ratio was 4:1 in μg/ml in the complement mixture. Complement activation was terminated with the stop solution at the indicated time. The complement activation mixtures were diluted 10,000-fold before testing in the MSD method. The MSD signal was back calculated by dilution factors and normalized by subtracting T = 0 signal. The final Dezamizumab concentration was 1000 μg/ml (○), 500 μg/ml (●), 250 μg/ml (□), 125 μg/ml (■), 62.5 μg/ml (Δ), and serum-only control (▲). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Techniques Used: Activation Assay, Purification, Quantitation Assay, Concentration Assay, Serial Dilution

    Effect of Dezamizumab:SAP ratio and concentration on complement activation. MSD method was used. Pooled human serum from Complement Technology was used and the final serum volume was 70%. After 40 min at the room temperature, the complement activation was terminated and diluted 10,000-fold to detect for C3a desArg formation. The Ratio of C3a desArg formation was calculated by sample signal divided by serum-only signal. A , two-fold serial dilutions were performed for Dezamizumab and SAP respectively. The diluted Dezamizumab and SAP were added to the assay plate followed by the addition of human serum with a final volume of 70%. After 40 min at ambient temperature, the complement activation was terminated with the stop buffer. The final Dezamizumab concentration in the complement activation ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM) and SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C3a desArg formation was detected using the MSD method. The signal ratio of complement activation versus an average of serum-only background from eight determinations was plotted against the indicated Dezamizumab concentration at the indicated ratio between Dezamizumab and SAP in molar. Dezamizumab:SAP molar ratios were 84.4 (○), 42.2 (●), 21.1 (□), 10.6 (■), 5.3 (Δ), 2.6 (▲), and 1.3 (▽). B , SAP concentrations without the addition of Dezamizumab. SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C , Dezamizumab concentrations without the addition of SAP. Dezamizumab ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM). D , Dezamizumab was mixed with SAP at the indicated molar ratio ranging from 3.6 to 9.9. The final Dezamizumab concentration was 500 μg/ml (○) or 250 μg/ml (●). Error bars for all panels represent the mean ± SD from n = 2 determination.
    Figure Legend Snippet: Effect of Dezamizumab:SAP ratio and concentration on complement activation. MSD method was used. Pooled human serum from Complement Technology was used and the final serum volume was 70%. After 40 min at the room temperature, the complement activation was terminated and diluted 10,000-fold to detect for C3a desArg formation. The Ratio of C3a desArg formation was calculated by sample signal divided by serum-only signal. A , two-fold serial dilutions were performed for Dezamizumab and SAP respectively. The diluted Dezamizumab and SAP were added to the assay plate followed by the addition of human serum with a final volume of 70%. After 40 min at ambient temperature, the complement activation was terminated with the stop buffer. The final Dezamizumab concentration in the complement activation ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM) and SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C3a desArg formation was detected using the MSD method. The signal ratio of complement activation versus an average of serum-only background from eight determinations was plotted against the indicated Dezamizumab concentration at the indicated ratio between Dezamizumab and SAP in molar. Dezamizumab:SAP molar ratios were 84.4 (○), 42.2 (●), 21.1 (□), 10.6 (■), 5.3 (Δ), 2.6 (▲), and 1.3 (▽). B , SAP concentrations without the addition of Dezamizumab. SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C , Dezamizumab concentrations without the addition of SAP. Dezamizumab ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM). D , Dezamizumab was mixed with SAP at the indicated molar ratio ranging from 3.6 to 9.9. The final Dezamizumab concentration was 500 μg/ml (○) or 250 μg/ml (●). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Techniques Used: Concentration Assay, Activation Assay

    Minimal human serum component needed for complement activation. Dezamizumab: SAP antigen ratio was 4:1 in μg/ml (molar ratio 6.3:1) in the complement activation mixture. The final Dezamizumab concentration was 100 μg/ml (○) and 250 μg/ml (●). Pooled human serum from Innovative Research was used. The final serum volume in the complement activation mixture ranged from 10% to 70%. After 45 min at ambient temperature, complement activation was terminated and diluted 3645 and 10,935-fold respectively to detect C3a desArg formation in the MSD method. The ratio of C3a desArg formation was calculated by sample signal divided by corresponding serum-only signal. The ratio of C3a desArg formation from the 3645 and 10,935-fold dilution was plotted against percentage of serum volume in complement activation mixture. Error bars represent the mean ± SD from n = 2 determination.
    Figure Legend Snippet: Minimal human serum component needed for complement activation. Dezamizumab: SAP antigen ratio was 4:1 in μg/ml (molar ratio 6.3:1) in the complement activation mixture. The final Dezamizumab concentration was 100 μg/ml (○) and 250 μg/ml (●). Pooled human serum from Innovative Research was used. The final serum volume in the complement activation mixture ranged from 10% to 70%. After 45 min at ambient temperature, complement activation was terminated and diluted 3645 and 10,935-fold respectively to detect C3a desArg formation in the MSD method. The ratio of C3a desArg formation was calculated by sample signal divided by corresponding serum-only signal. The ratio of C3a desArg formation from the 3645 and 10,935-fold dilution was plotted against percentage of serum volume in complement activation mixture. Error bars represent the mean ± SD from n = 2 determination.

    Techniques Used: Activation Assay, Concentration Assay

    Relative potency determination between Dezamizumab reference standard and the thermal-stressed samples. Dose–response determination was performed with Dezamizumab reference standard (○), and Dezamizumab stored in a formulation buffer at pH 7.0 for 3 months at 2 to 8 °C (●), 25 °C (□), and 40 °C (■), or reference standard stored at −70 °C (○). Human serum in the complement activation mixture was either prepared from the blood of a single donor ( A and B ) or the pooled serum purchased from Complement Technology ( C and D ). C3a desArg formation by MSD method ( A and C ) and C3 depletion by HTRF method ( B and D ) were determined. The twofold serial dilution was performed with a fixed Dezamizumab:SAP molar ratio of 6.3:1. The final Dezamizumab concentration in the complement activation mixture ranged from 0.007 to 3.36 μM. The final serum volume in the complement activation mixture was 40%. The complement activation time was 40 min for A , C , and D , and 10 min for B at the ambient temperature. The complement mixture was diluted 10,000-fold for A and C in the MSD method, and 100-fold for B and 40-fold for D in the HTRF method. The ratio of C3a desArg formation in Dezamizumab samples to serum-only control was plotted against Dezamizumab concentration in the MSD method. Similarly, the %C3 remained in Dezamizumab samples versus the serum-only control was plotted against Dezamizumab concentration in the HTRF method. The average of n = 2 data with ± SD was fit using the full four-parameter EC50 model as described in . Error bars represent the mean ± SD.
    Figure Legend Snippet: Relative potency determination between Dezamizumab reference standard and the thermal-stressed samples. Dose–response determination was performed with Dezamizumab reference standard (○), and Dezamizumab stored in a formulation buffer at pH 7.0 for 3 months at 2 to 8 °C (●), 25 °C (□), and 40 °C (■), or reference standard stored at −70 °C (○). Human serum in the complement activation mixture was either prepared from the blood of a single donor ( A and B ) or the pooled serum purchased from Complement Technology ( C and D ). C3a desArg formation by MSD method ( A and C ) and C3 depletion by HTRF method ( B and D ) were determined. The twofold serial dilution was performed with a fixed Dezamizumab:SAP molar ratio of 6.3:1. The final Dezamizumab concentration in the complement activation mixture ranged from 0.007 to 3.36 μM. The final serum volume in the complement activation mixture was 40%. The complement activation time was 40 min for A , C , and D , and 10 min for B at the ambient temperature. The complement mixture was diluted 10,000-fold for A and C in the MSD method, and 100-fold for B and 40-fold for D in the HTRF method. The ratio of C3a desArg formation in Dezamizumab samples to serum-only control was plotted against Dezamizumab concentration in the MSD method. Similarly, the %C3 remained in Dezamizumab samples versus the serum-only control was plotted against Dezamizumab concentration in the HTRF method. The average of n = 2 data with ± SD was fit using the full four-parameter EC50 model as described in . Error bars represent the mean ± SD.

    Techniques Used: Activation Assay, Serial Dilution, Concentration Assay

    Schematic overview of the complement cascade illustrating the three activation pathways (classical, lectin, and alternative). The classical pathway is activated when C1q binds to antibody complexed with antigen. The interaction activates C1r and C1s, which cleave C4 and C2. The lectin pathway is activated when mannose-binding lectin (MBL) binds to conserved pathogenic carbohydrate motifs. The interaction activates the MBL-associated serine proteases (MASPs) and cleaves C4 and C2. The products from C4 and C2 cleavage form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. C3b associates with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated by spontaneous hydrolysis of C3. In the presence of Factors B and D, the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b) are eventually formed. C5 convertase from all three pathways cleaves C5 to form C5a and C5b. C5b together with C6-C9 forms the membrane attack complex (MAC) that lyses targeted pathogens or damaged self-cells.
    Figure Legend Snippet: Schematic overview of the complement cascade illustrating the three activation pathways (classical, lectin, and alternative). The classical pathway is activated when C1q binds to antibody complexed with antigen. The interaction activates C1r and C1s, which cleave C4 and C2. The lectin pathway is activated when mannose-binding lectin (MBL) binds to conserved pathogenic carbohydrate motifs. The interaction activates the MBL-associated serine proteases (MASPs) and cleaves C4 and C2. The products from C4 and C2 cleavage form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. C3b associates with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated by spontaneous hydrolysis of C3. In the presence of Factors B and D, the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b) are eventually formed. C5 convertase from all three pathways cleaves C5 to form C5a and C5b. C5b together with C6-C9 forms the membrane attack complex (MAC) that lyses targeted pathogens or damaged self-cells.

    Techniques Used: Activation Assay, Binding Assay



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    Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine <t>C3a</t> desArg formation. B , principle of HTRF assay to detect C3 depletion.
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    Bacteria were incubated in serum in increasing amounts of FH6-7/hFc (A) or FH18-20/Fc (B) and analyzed for binding of serum FH. (C) Flow cytometry was used to measure surface bound C9, the final step of complement activation. FH18-20 did not increase C9 on AP1. (D) Complement C9 deposition was not affected by FH6-7/hFc or FH18-20/hFc on BM27.6 that does not bind either molecule. FH6-7/Fc immobilized on microtiter wells activates the classical and lectin pathways as measured by C3b (E) and C4b (F) deposition. (G) Plasma was incubated with FH6-7/hFc, PBS or zymosan and analyzed for <t>C3a</t> generation as a sign of complement activation. Mean (±SD) from at least 3 independent experiments are shown. Statistical significance of differences was calculated using a 2-way ANOVA with Dunnett’s post-test (overall p value in C p> 0.0001); *, p<0.05; **, p<0.01 and ****, p<0.0001.
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    Bacteria were incubated in serum in increasing amounts of FH6-7/hFc (A) or FH18-20/Fc (B) and analyzed for binding of serum FH. (C) Flow cytometry was used to measure surface bound C9, the final step of complement activation. FH18-20 did not increase C9 on AP1. (D) Complement C9 deposition was not affected by FH6-7/hFc or FH18-20/hFc on BM27.6 that does not bind either molecule. FH6-7/Fc immobilized on microtiter wells activates the classical and lectin pathways as measured by C3b (E) and C4b (F) deposition. (G) Plasma was incubated with FH6-7/hFc, PBS or zymosan and analyzed for <t>C3a</t> generation as a sign of complement activation. Mean (±SD) from at least 3 independent experiments are shown. Statistical significance of differences was calculated using a 2-way ANOVA with Dunnett’s post-test (overall p value in C p> 0.0001); *, p<0.05; **, p<0.01 and ****, p<0.0001.
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    Hycult Biotech human c3a desarg
    ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to <t>C3a</t> and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the <t>complement</t> <t>activation</t> products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.
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    ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to <t>C3a</t> and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the <t>complement</t> <t>activation</t> products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.
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    ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to <t>C3a</t> and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the <t>complement</t> <t>activation</t> products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.
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    Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine C3a desArg formation. B , principle of HTRF assay to detect C3 depletion.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine C3a desArg formation. B , principle of HTRF assay to detect C3 depletion.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Activation Assay, HTRF Assay

    MSD method for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , specificity of the coating antibody. Purified C3 (■), C3a (●), C3a desArg (○) and iC3b (□) were diluted to various concentrations up to ∼22.2 nM and captured in an MSD plate coated with neo-epitope of human C3a/C3a-desArg monoclonal antibody. In B – D , pooled human serum from Complement Technology was used and the final serum volume was 70% in the complement mixture. B , impact of dilution factor on quantitation of C3a desArg formation yielded from complement activation. Dezamizumab and SAP antigen was mixed at a 4:1 ratio (μg/ml) in the complement activation mixture. The final Dezamizumab concentration was 500 μg/ml (○), 250 μg/ml (●), and serum-only control (□). The reaction was terminated after 40 min at ambient temperature. The complement activation mixture and serum-only control were diluted with HBSP buffer at a range from 5 to 10,935-fold in threefold serial dilution before testing in the MSD assay. C , time course of complement activation at 37 °C. D , time course of complement activation at ambient temperature. In both C and D , Dezamizumab:SAP antigen ratio was 4:1 in μg/ml in the complement mixture. Complement activation was terminated with the stop solution at the indicated time. The complement activation mixtures were diluted 10,000-fold before testing in the MSD method. The MSD signal was back calculated by dilution factors and normalized by subtracting T = 0 signal. The final Dezamizumab concentration was 1000 μg/ml (○), 500 μg/ml (●), 250 μg/ml (□), 125 μg/ml (■), 62.5 μg/ml (Δ), and serum-only control (▲). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: MSD method for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , specificity of the coating antibody. Purified C3 (■), C3a (●), C3a desArg (○) and iC3b (□) were diluted to various concentrations up to ∼22.2 nM and captured in an MSD plate coated with neo-epitope of human C3a/C3a-desArg monoclonal antibody. In B – D , pooled human serum from Complement Technology was used and the final serum volume was 70% in the complement mixture. B , impact of dilution factor on quantitation of C3a desArg formation yielded from complement activation. Dezamizumab and SAP antigen was mixed at a 4:1 ratio (μg/ml) in the complement activation mixture. The final Dezamizumab concentration was 500 μg/ml (○), 250 μg/ml (●), and serum-only control (□). The reaction was terminated after 40 min at ambient temperature. The complement activation mixture and serum-only control were diluted with HBSP buffer at a range from 5 to 10,935-fold in threefold serial dilution before testing in the MSD assay. C , time course of complement activation at 37 °C. D , time course of complement activation at ambient temperature. In both C and D , Dezamizumab:SAP antigen ratio was 4:1 in μg/ml in the complement mixture. Complement activation was terminated with the stop solution at the indicated time. The complement activation mixtures were diluted 10,000-fold before testing in the MSD method. The MSD signal was back calculated by dilution factors and normalized by subtracting T = 0 signal. The final Dezamizumab concentration was 1000 μg/ml (○), 500 μg/ml (●), 250 μg/ml (□), 125 μg/ml (■), 62.5 μg/ml (Δ), and serum-only control (▲). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Activation Assay, Purification, Quantitation Assay, Concentration Assay, Serial Dilution

    Effect of Dezamizumab:SAP ratio and concentration on complement activation. MSD method was used. Pooled human serum from Complement Technology was used and the final serum volume was 70%. After 40 min at the room temperature, the complement activation was terminated and diluted 10,000-fold to detect for C3a desArg formation. The Ratio of C3a desArg formation was calculated by sample signal divided by serum-only signal. A , two-fold serial dilutions were performed for Dezamizumab and SAP respectively. The diluted Dezamizumab and SAP were added to the assay plate followed by the addition of human serum with a final volume of 70%. After 40 min at ambient temperature, the complement activation was terminated with the stop buffer. The final Dezamizumab concentration in the complement activation ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM) and SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C3a desArg formation was detected using the MSD method. The signal ratio of complement activation versus an average of serum-only background from eight determinations was plotted against the indicated Dezamizumab concentration at the indicated ratio between Dezamizumab and SAP in molar. Dezamizumab:SAP molar ratios were 84.4 (○), 42.2 (●), 21.1 (□), 10.6 (■), 5.3 (Δ), 2.6 (▲), and 1.3 (▽). B , SAP concentrations without the addition of Dezamizumab. SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C , Dezamizumab concentrations without the addition of SAP. Dezamizumab ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM). D , Dezamizumab was mixed with SAP at the indicated molar ratio ranging from 3.6 to 9.9. The final Dezamizumab concentration was 500 μg/ml (○) or 250 μg/ml (●). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: Effect of Dezamizumab:SAP ratio and concentration on complement activation. MSD method was used. Pooled human serum from Complement Technology was used and the final serum volume was 70%. After 40 min at the room temperature, the complement activation was terminated and diluted 10,000-fold to detect for C3a desArg formation. The Ratio of C3a desArg formation was calculated by sample signal divided by serum-only signal. A , two-fold serial dilutions were performed for Dezamizumab and SAP respectively. The diluted Dezamizumab and SAP were added to the assay plate followed by the addition of human serum with a final volume of 70%. After 40 min at ambient temperature, the complement activation was terminated with the stop buffer. The final Dezamizumab concentration in the complement activation ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM) and SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C3a desArg formation was detected using the MSD method. The signal ratio of complement activation versus an average of serum-only background from eight determinations was plotted against the indicated Dezamizumab concentration at the indicated ratio between Dezamizumab and SAP in molar. Dezamizumab:SAP molar ratios were 84.4 (○), 42.2 (●), 21.1 (□), 10.6 (■), 5.3 (Δ), 2.6 (▲), and 1.3 (▽). B , SAP concentrations without the addition of Dezamizumab. SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C , Dezamizumab concentrations without the addition of SAP. Dezamizumab ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM). D , Dezamizumab was mixed with SAP at the indicated molar ratio ranging from 3.6 to 9.9. The final Dezamizumab concentration was 500 μg/ml (○) or 250 μg/ml (●). Error bars for all panels represent the mean ± SD from n = 2 determination.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Concentration Assay, Activation Assay

    Minimal human serum component needed for complement activation. Dezamizumab: SAP antigen ratio was 4:1 in μg/ml (molar ratio 6.3:1) in the complement activation mixture. The final Dezamizumab concentration was 100 μg/ml (○) and 250 μg/ml (●). Pooled human serum from Innovative Research was used. The final serum volume in the complement activation mixture ranged from 10% to 70%. After 45 min at ambient temperature, complement activation was terminated and diluted 3645 and 10,935-fold respectively to detect C3a desArg formation in the MSD method. The ratio of C3a desArg formation was calculated by sample signal divided by corresponding serum-only signal. The ratio of C3a desArg formation from the 3645 and 10,935-fold dilution was plotted against percentage of serum volume in complement activation mixture. Error bars represent the mean ± SD from n = 2 determination.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: Minimal human serum component needed for complement activation. Dezamizumab: SAP antigen ratio was 4:1 in μg/ml (molar ratio 6.3:1) in the complement activation mixture. The final Dezamizumab concentration was 100 μg/ml (○) and 250 μg/ml (●). Pooled human serum from Innovative Research was used. The final serum volume in the complement activation mixture ranged from 10% to 70%. After 45 min at ambient temperature, complement activation was terminated and diluted 3645 and 10,935-fold respectively to detect C3a desArg formation in the MSD method. The ratio of C3a desArg formation was calculated by sample signal divided by corresponding serum-only signal. The ratio of C3a desArg formation from the 3645 and 10,935-fold dilution was plotted against percentage of serum volume in complement activation mixture. Error bars represent the mean ± SD from n = 2 determination.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Activation Assay, Concentration Assay

    Relative potency determination between Dezamizumab reference standard and the thermal-stressed samples. Dose–response determination was performed with Dezamizumab reference standard (○), and Dezamizumab stored in a formulation buffer at pH 7.0 for 3 months at 2 to 8 °C (●), 25 °C (□), and 40 °C (■), or reference standard stored at −70 °C (○). Human serum in the complement activation mixture was either prepared from the blood of a single donor ( A and B ) or the pooled serum purchased from Complement Technology ( C and D ). C3a desArg formation by MSD method ( A and C ) and C3 depletion by HTRF method ( B and D ) were determined. The twofold serial dilution was performed with a fixed Dezamizumab:SAP molar ratio of 6.3:1. The final Dezamizumab concentration in the complement activation mixture ranged from 0.007 to 3.36 μM. The final serum volume in the complement activation mixture was 40%. The complement activation time was 40 min for A , C , and D , and 10 min for B at the ambient temperature. The complement mixture was diluted 10,000-fold for A and C in the MSD method, and 100-fold for B and 40-fold for D in the HTRF method. The ratio of C3a desArg formation in Dezamizumab samples to serum-only control was plotted against Dezamizumab concentration in the MSD method. Similarly, the %C3 remained in Dezamizumab samples versus the serum-only control was plotted against Dezamizumab concentration in the HTRF method. The average of n = 2 data with ± SD was fit using the full four-parameter EC50 model as described in . Error bars represent the mean ± SD.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: Relative potency determination between Dezamizumab reference standard and the thermal-stressed samples. Dose–response determination was performed with Dezamizumab reference standard (○), and Dezamizumab stored in a formulation buffer at pH 7.0 for 3 months at 2 to 8 °C (●), 25 °C (□), and 40 °C (■), or reference standard stored at −70 °C (○). Human serum in the complement activation mixture was either prepared from the blood of a single donor ( A and B ) or the pooled serum purchased from Complement Technology ( C and D ). C3a desArg formation by MSD method ( A and C ) and C3 depletion by HTRF method ( B and D ) were determined. The twofold serial dilution was performed with a fixed Dezamizumab:SAP molar ratio of 6.3:1. The final Dezamizumab concentration in the complement activation mixture ranged from 0.007 to 3.36 μM. The final serum volume in the complement activation mixture was 40%. The complement activation time was 40 min for A , C , and D , and 10 min for B at the ambient temperature. The complement mixture was diluted 10,000-fold for A and C in the MSD method, and 100-fold for B and 40-fold for D in the HTRF method. The ratio of C3a desArg formation in Dezamizumab samples to serum-only control was plotted against Dezamizumab concentration in the MSD method. Similarly, the %C3 remained in Dezamizumab samples versus the serum-only control was plotted against Dezamizumab concentration in the HTRF method. The average of n = 2 data with ± SD was fit using the full four-parameter EC50 model as described in . Error bars represent the mean ± SD.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Activation Assay, Serial Dilution, Concentration Assay

    Schematic overview of the complement cascade illustrating the three activation pathways (classical, lectin, and alternative). The classical pathway is activated when C1q binds to antibody complexed with antigen. The interaction activates C1r and C1s, which cleave C4 and C2. The lectin pathway is activated when mannose-binding lectin (MBL) binds to conserved pathogenic carbohydrate motifs. The interaction activates the MBL-associated serine proteases (MASPs) and cleaves C4 and C2. The products from C4 and C2 cleavage form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. C3b associates with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated by spontaneous hydrolysis of C3. In the presence of Factors B and D, the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b) are eventually formed. C5 convertase from all three pathways cleaves C5 to form C5a and C5b. C5b together with C6-C9 forms the membrane attack complex (MAC) that lyses targeted pathogens or damaged self-cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

    doi: 10.1016/j.jbc.2021.101136

    Figure Lengend Snippet: Schematic overview of the complement cascade illustrating the three activation pathways (classical, lectin, and alternative). The classical pathway is activated when C1q binds to antibody complexed with antigen. The interaction activates C1r and C1s, which cleave C4 and C2. The lectin pathway is activated when mannose-binding lectin (MBL) binds to conserved pathogenic carbohydrate motifs. The interaction activates the MBL-associated serine proteases (MASPs) and cleaves C4 and C2. The products from C4 and C2 cleavage form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. C3b associates with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated by spontaneous hydrolysis of C3. In the presence of Factors B and D, the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b) are eventually formed. C5 convertase from all three pathways cleaves C5 to form C5a and C5b. C5b together with C6-C9 forms the membrane attack complex (MAC) that lyses targeted pathogens or damaged self-cells.

    Article Snippet: Monoclonal antibody 2991 recognizing the neo-epitope of human C3a/C3a-desArg used in the MSD method was from Hycult Biotech.

    Techniques: Activation Assay, Binding Assay

    Bacteria were incubated in serum in increasing amounts of FH6-7/hFc (A) or FH18-20/Fc (B) and analyzed for binding of serum FH. (C) Flow cytometry was used to measure surface bound C9, the final step of complement activation. FH18-20 did not increase C9 on AP1. (D) Complement C9 deposition was not affected by FH6-7/hFc or FH18-20/hFc on BM27.6 that does not bind either molecule. FH6-7/Fc immobilized on microtiter wells activates the classical and lectin pathways as measured by C3b (E) and C4b (F) deposition. (G) Plasma was incubated with FH6-7/hFc, PBS or zymosan and analyzed for C3a generation as a sign of complement activation. Mean (±SD) from at least 3 independent experiments are shown. Statistical significance of differences was calculated using a 2-way ANOVA with Dunnett’s post-test (overall p value in C p> 0.0001); *, p<0.05; **, p<0.01 and ****, p<0.0001.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Factor H – IgG chimeric proteins as a therapeutic approach against the Gram-positive bacterial pathogen Streptococcus pyogenes

    doi: 10.4049/jimmunol.1700426

    Figure Lengend Snippet: Bacteria were incubated in serum in increasing amounts of FH6-7/hFc (A) or FH18-20/Fc (B) and analyzed for binding of serum FH. (C) Flow cytometry was used to measure surface bound C9, the final step of complement activation. FH18-20 did not increase C9 on AP1. (D) Complement C9 deposition was not affected by FH6-7/hFc or FH18-20/hFc on BM27.6 that does not bind either molecule. FH6-7/Fc immobilized on microtiter wells activates the classical and lectin pathways as measured by C3b (E) and C4b (F) deposition. (G) Plasma was incubated with FH6-7/hFc, PBS or zymosan and analyzed for C3a generation as a sign of complement activation. Mean (±SD) from at least 3 independent experiments are shown. Statistical significance of differences was calculated using a 2-way ANOVA with Dunnett’s post-test (overall p value in C p> 0.0001); *, p<0.05; **, p<0.01 and ****, p<0.0001.

    Article Snippet: Antibodies for Western blot analysis: anti human C3a/C3a des-Arg (Hycult) and anti-mouse Ig/HRP (DAKO).

    Techniques: Incubation, Binding Assay, Flow Cytometry, Activation Assay

    ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to C3a and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the complement activation products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to C3a and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the complement activation products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.

    Article Snippet: Purified recombinant human C3a-desArg (1 µg, HC2127), C5a-desArg (1 µg, HC2102, Hycult Biotech, Beutelsbach, Germany), sC5b-9 (1 µg/0.1 µg, A127, Complement Technology, Tyler, TX, USA) or normal human serum (20 µg, NHS) were separated under reducing and non-reducing conditions in either 10% or 15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes.

    Techniques: Activation Assay, Multiplex Assay, Luminex, Magnetic Beads, In Vitro

    ( A ) Serial dilutions of native, human C3a-desArg (black circle), recombinant, human C5a-desArg (black square) and native human sC5b-C9 (black triangle) depicted different assay performances for the simultaneous analysis of complement activation markers. The target antigens were only detected by respective coupled magnetic beads and not by control beads (grey). Limit of detection (orange) and assay cutoffs (red square) for each analyte are shown. ( B ) The concentration range of complement activation markers determined with the novel multiplex assay from a human serum pool (black square with dot) corresponded to previously reported ranges for C3a, C5a and sC5b-C9 single detection either from plasma (grey), serum (black) or unknown sample matrix (white), respectively [ – ].

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A ) Serial dilutions of native, human C3a-desArg (black circle), recombinant, human C5a-desArg (black square) and native human sC5b-C9 (black triangle) depicted different assay performances for the simultaneous analysis of complement activation markers. The target antigens were only detected by respective coupled magnetic beads and not by control beads (grey). Limit of detection (orange) and assay cutoffs (red square) for each analyte are shown. ( B ) The concentration range of complement activation markers determined with the novel multiplex assay from a human serum pool (black square with dot) corresponded to previously reported ranges for C3a, C5a and sC5b-C9 single detection either from plasma (grey), serum (black) or unknown sample matrix (white), respectively [ – ].

    Article Snippet: Purified recombinant human C3a-desArg (1 µg, HC2127), C5a-desArg (1 µg, HC2102, Hycult Biotech, Beutelsbach, Germany), sC5b-9 (1 µg/0.1 µg, A127, Complement Technology, Tyler, TX, USA) or normal human serum (20 µg, NHS) were separated under reducing and non-reducing conditions in either 10% or 15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes.

    Techniques: Recombinant, Activation Assay, Magnetic Beads, Concentration Assay, Multiplex Assay

    ( A–C ) The histologic grade ( G ) describes the cell differentiation in the patients’ tumors/ neoplasms. Patients with G2 tumor grading had significantly elevated (A) C3a, as well as (B) C5a and (C) sC5b-9 serum levels compared to the control group. ( D–F ) The T-classification system categorizes the extension of the patients′ primary tumors. (D) There was no relation between C3a serum levels and T-classification. (E) All tumor extension classifications (T-classifications) showed a significant increase in systemic C5a levels compared to the control group. (F) Invasive growth (T4) is associated with increased sC5b-9 serum levels compared to the control, T2 and T3 group. ( G–J ) The N-classification describes the degree of spread of tumor cells to regional lymph nodes. (G) C3a concentration was significantly correlated with tumor spread to lymph nodes. C3a concentration was highest in N2 classified lymph node metastases. (H, I) There was no significant correlation between complement markers C5a, sC5b-C9 and lymph node metastases. (J) Patients, that presented a N2-tumor, were significantly younger than patients with a N0 or N1 tumor. ( K–M ) Age was not associated with T-classification or correlated with complement activation marker concentration in serum. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 two-tailed, unpaired t -test).

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A–C ) The histologic grade ( G ) describes the cell differentiation in the patients’ tumors/ neoplasms. Patients with G2 tumor grading had significantly elevated (A) C3a, as well as (B) C5a and (C) sC5b-9 serum levels compared to the control group. ( D–F ) The T-classification system categorizes the extension of the patients′ primary tumors. (D) There was no relation between C3a serum levels and T-classification. (E) All tumor extension classifications (T-classifications) showed a significant increase in systemic C5a levels compared to the control group. (F) Invasive growth (T4) is associated with increased sC5b-9 serum levels compared to the control, T2 and T3 group. ( G–J ) The N-classification describes the degree of spread of tumor cells to regional lymph nodes. (G) C3a concentration was significantly correlated with tumor spread to lymph nodes. C3a concentration was highest in N2 classified lymph node metastases. (H, I) There was no significant correlation between complement markers C5a, sC5b-C9 and lymph node metastases. (J) Patients, that presented a N2-tumor, were significantly younger than patients with a N0 or N1 tumor. ( K–M ) Age was not associated with T-classification or correlated with complement activation marker concentration in serum. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 two-tailed, unpaired t -test).

    Article Snippet: Purified recombinant human C3a-desArg (1 µg, HC2127), C5a-desArg (1 µg, HC2102, Hycult Biotech, Beutelsbach, Germany), sC5b-9 (1 µg/0.1 µg, A127, Complement Technology, Tyler, TX, USA) or normal human serum (20 µg, NHS) were separated under reducing and non-reducing conditions in either 10% or 15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes.

    Techniques: Cell Differentiation, Concentration Assay, Activation Assay, Marker, Two Tailed Test

    ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to C3a and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the complement activation products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A ) The complement system is a part of the innate immunity and activated by three different pathways: the classical, the lectin, and the alternative pathway. Activation results in enzymatic cleavage of C3 to C3a and C3b. The latter triggers the terminal complement pathway by cleavage of C5 and the formation of the terminal complement complex sC5b-C9. The formation of sC5b-C9 and the anaphylatoxins C3a and C5a determines complement activation. ( B , C ) The detection of the complement activation products was performed in multiplex suspension bead array based on the x-MAP technology (Luminex ® ). (B) Activation product specific antibodies were immobilized to distinct fluorescent, magnetic beads to capture either C3a, C5a or sC5b-C9 from solution, respectively. Antigen specific biotinylated antibodies and phycoerythrin conjugated to streptavidin detected the captured activation products simultaneously. (C) False positive signals by cross-reacting human anti-species antibodies and in vitro complement activation were excluded by utilizing an appropriate reaction buffer.

    Article Snippet: A mixture of serial 1:3 diluted human native C3a-desArg (197.5–0.001 ng/mL, cat. HC2127, Hycult Biotech, Beutelsbach, Germany), human recombinant C5a-desArg (28.1–0.004 ng/mL, Hycult Biotech, Beutelsbach, Germany) and human native sC5b-9 (66.6–0.0004 µg/mL, cat. A127, Complement Technology, Tyler, TX, USA) were used as standard curve.

    Techniques: Activation Assay, Multiplex Assay, Luminex, Magnetic Beads, In Vitro

    ( A ) Serial dilutions of native, human C3a-desArg (black circle), recombinant, human C5a-desArg (black square) and native human sC5b-C9 (black triangle) depicted different assay performances for the simultaneous analysis of complement activation markers. The target antigens were only detected by respective coupled magnetic beads and not by control beads (grey). Limit of detection (orange) and assay cutoffs (red square) for each analyte are shown. ( B ) The concentration range of complement activation markers determined with the novel multiplex assay from a human serum pool (black square with dot) corresponded to previously reported ranges for C3a, C5a and sC5b-C9 single detection either from plasma (grey), serum (black) or unknown sample matrix (white), respectively [ – ].

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A ) Serial dilutions of native, human C3a-desArg (black circle), recombinant, human C5a-desArg (black square) and native human sC5b-C9 (black triangle) depicted different assay performances for the simultaneous analysis of complement activation markers. The target antigens were only detected by respective coupled magnetic beads and not by control beads (grey). Limit of detection (orange) and assay cutoffs (red square) for each analyte are shown. ( B ) The concentration range of complement activation markers determined with the novel multiplex assay from a human serum pool (black square with dot) corresponded to previously reported ranges for C3a, C5a and sC5b-C9 single detection either from plasma (grey), serum (black) or unknown sample matrix (white), respectively [ – ].

    Article Snippet: A mixture of serial 1:3 diluted human native C3a-desArg (197.5–0.001 ng/mL, cat. HC2127, Hycult Biotech, Beutelsbach, Germany), human recombinant C5a-desArg (28.1–0.004 ng/mL, Hycult Biotech, Beutelsbach, Germany) and human native sC5b-9 (66.6–0.0004 µg/mL, cat. A127, Complement Technology, Tyler, TX, USA) were used as standard curve.

    Techniques: Recombinant, Activation Assay, Magnetic Beads, Concentration Assay, Multiplex Assay

    ( A–C ) The histologic grade ( G ) describes the cell differentiation in the patients’ tumors/ neoplasms. Patients with G2 tumor grading had significantly elevated (A) C3a, as well as (B) C5a and (C) sC5b-9 serum levels compared to the control group. ( D–F ) The T-classification system categorizes the extension of the patients′ primary tumors. (D) There was no relation between C3a serum levels and T-classification. (E) All tumor extension classifications (T-classifications) showed a significant increase in systemic C5a levels compared to the control group. (F) Invasive growth (T4) is associated with increased sC5b-9 serum levels compared to the control, T2 and T3 group. ( G–J ) The N-classification describes the degree of spread of tumor cells to regional lymph nodes. (G) C3a concentration was significantly correlated with tumor spread to lymph nodes. C3a concentration was highest in N2 classified lymph node metastases. (H, I) There was no significant correlation between complement markers C5a, sC5b-C9 and lymph node metastases. (J) Patients, that presented a N2-tumor, were significantly younger than patients with a N0 or N1 tumor. ( K–M ) Age was not associated with T-classification or correlated with complement activation marker concentration in serum. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 two-tailed, unpaired t -test).

    Journal: Oncotarget

    Article Title: A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma

    doi: 10.18632/oncotarget.22963

    Figure Lengend Snippet: ( A–C ) The histologic grade ( G ) describes the cell differentiation in the patients’ tumors/ neoplasms. Patients with G2 tumor grading had significantly elevated (A) C3a, as well as (B) C5a and (C) sC5b-9 serum levels compared to the control group. ( D–F ) The T-classification system categorizes the extension of the patients′ primary tumors. (D) There was no relation between C3a serum levels and T-classification. (E) All tumor extension classifications (T-classifications) showed a significant increase in systemic C5a levels compared to the control group. (F) Invasive growth (T4) is associated with increased sC5b-9 serum levels compared to the control, T2 and T3 group. ( G–J ) The N-classification describes the degree of spread of tumor cells to regional lymph nodes. (G) C3a concentration was significantly correlated with tumor spread to lymph nodes. C3a concentration was highest in N2 classified lymph node metastases. (H, I) There was no significant correlation between complement markers C5a, sC5b-C9 and lymph node metastases. (J) Patients, that presented a N2-tumor, were significantly younger than patients with a N0 or N1 tumor. ( K–M ) Age was not associated with T-classification or correlated with complement activation marker concentration in serum. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 two-tailed, unpaired t -test).

    Article Snippet: A mixture of serial 1:3 diluted human native C3a-desArg (197.5–0.001 ng/mL, cat. HC2127, Hycult Biotech, Beutelsbach, Germany), human recombinant C5a-desArg (28.1–0.004 ng/mL, Hycult Biotech, Beutelsbach, Germany) and human native sC5b-9 (66.6–0.0004 µg/mL, cat. A127, Complement Technology, Tyler, TX, USA) were used as standard curve.

    Techniques: Cell Differentiation, Concentration Assay, Activation Assay, Marker, Two Tailed Test