Antibody-dependent complement deposition (ADCD) was quantified using a 96-well-based customized multiplexed Luminex assay, as described previously [13 (link)]. Bulk IgG was purified and separated from other serum proteins using Melon Gel resin according to the manufacturer's instructions (Thermo Scientific #45208). Purified IgG at a 1:75 dilution was incubated with antigen-coupled beads for 2 hours at 37°C on a 700-rpm orbital shaker. After incubation, each sample was incubated with human complement serum (Sigma #S1764) at a concentration of 1:50 at 37°C for 30 minutes. Samples were then incubated at RT for 30 minutes with biotinylated monoclonal antihuman C3d (Quidel #207) at a 1-µg/mL final concentration. Finally, 1 µg/mL of Streptavidin-RPE (Prozyme #PJ31S) was added to each well and incubated at 37°C in the dark for 1 hour. Complement deposition was determined on a BioPlex-200 (Bio-Rad) and measured as median fluorescence intensity (MFI). Assays performed without IgG and with heat-inactivated human complement serum were used as negative controls.
Biotinylated monoclonal antihuman c3d
Manufactured by Quidel
Biotinylated monoclonal antihuman C3d is a laboratory reagent used for the detection and quantification of the C3d complement component in biological samples. It is a monoclonal antibody that has been labeled with biotin, a small molecule that can be used to enable signal amplification and detection.
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Lab products found in correlation
2 protocols using biotinylated monoclonal antihuman c3d
1
Quantifying Antibody-Mediated Complement Activation
2
Multiplexed Luminex ADCD Assay
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Antibody-Dependent Complement Deposition (ADCD) was quantified using 96-well based customized multiplexed Luminex assay. Bulk IgG was purified and separated from other serum proteins using Melon Gel resin according to the manufacturer’s instructions (Thermo Scientific #45208). Purified IgG at 1:75 dilution for IgG anti-RBD and at 1:150 dilution for IgG anti-S1, -S2, -S, -NP and -TT, was incubated with antigen-coupled beads for 2 h at 37°C on 700 rpm orbital shaker. After incubation, each sample was incubated with human complement serum (Sigma #S1764) at a concentration of 1:50 at 37°C for 30 min. Samples were then incubated at RT for 30 min with biotinylated monoclonal anti-human C3d (Quidel #207) at 1 µg/ml final concentration. Finally, 1 µg/ml of Streptavidin-RPE (Prozyme #PJ31S) was added to each well and incubated at 37°C in the dark for 1 h. Complement deposition was determined on a BioPlex-200 equipment (Bio-Rad) and measured as MFI. Assays performed without IgG and with heat-inactivated human complement serum were used as negative controls.
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