human c3a c3a desarg (Hycult Biotech)


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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

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

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

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

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

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

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