Carbodiimide nhs ester coupling

Antibody-dependent complement deposition was conducted as previously described (38 (link)). Briefly, SARS-CoV-2 antigens were coupled to magnetic Luminex beads (Luminex Corp.) by carbodiimide-NHS ester-coupling (Thermo Fisher). Coupled beads were incubated for 2 h at 37°C with serum samples (1:10 dilution) to form immune complexes and then washed to remove unbound immunoglobulins. Lyophilized guinea pig complement (Cedarlane) was diluted in gelatin veronal buffer with calcium and magnesium (GBV++; Boston BioProducts) and added to immune complexes to measure antibody-dependent deposition of C3. C3 was then detected with an anti-C3 fluorescein-conjugated goat IgG fraction detection antibody (MP Bio). Flow cytometry was performed using iQue (IntelliCyt) and an S-Lab robot (PAA). ADCD was reported as the median fluorescence of C3 deposition.

ADCD was conducted using a 384-well based customized multiplexed assay. Protein antigens were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide-NHS ester-coupling (Thermo Fisher). OPS and LPS antigens were modified by 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium and conjugated to Luminex Magplex carboxylated beads Schlottmann et al., 2006 (link)). To form immune complexes, a mix of four antigen-coupled beads was incubated for 2 h at 37°C with diluted samples (1:10) and then washed to remove unbound immunoglobulins. Lyophilized guinea pig complement (Cedarlane) was resuspended according to manufacturer’s instructions and diluted in gelatin veronal buffer with calcium and magnesium (Boston BioProducts). Resuspend guinea pig complement was added to immune complexes and incubated for 20 min at 37°C. Post incubation, C3 was detected with Fluorescein-Conjugated Goat IgG Fraction to Guinea Pig Complement C3 (Mpbio).

SARS-CoV-2 specific antibody subclass and isotypes, and FcγR binding was analyzed using a custom Luminex multiplexed assay^{36 (link)}. SARS2-CoV2-RBD, SARS2-CoV2-N, and SARS2-CoV2-S were coupled to magnetic Luminex beads (Luminex Corp, TX, USA) by carbodiimide-NHS ester-coupling (Thermo Fisher). Dilution curves were performed on pooled samples from the cohort to determine dilutions in the linear range for each detection reagent. Coupled beads were then incubated with different plasma dilutions (between 1:100 and 1:1000 depending on the secondary reagent) for 2 h at room temperature in 384-well plates (Greiner Bio-One, Germany). Unbound antibodies were washed away and PE-conjugated antibody (Southern Biotech, AL, USA, see Life Science Reporting Summary) at a 1:100 dilution used to detect IgG1 (#9054-09), IgG3 (#9210-09), IgM (#9020-09) or IgA1 (#9130-09), respectively. For the FcγR3b binding, a PE-Streptavidin (Agilent Technologies, CA, USA) coupled recombinant biotinylated human FcγR3b protein (Duke Protein Production Facility) was used as a secondary probe. After 1h incubation, the excessive secondary reagent was washed away and the relative antibody concentration per antigen determined on an IQue analyzer (IntelliCyt, NM, USA). Samples with mean signals plus five times the standard deviation of the PBS-control wells were considered as positive.