Rabbit anti human c1q polyclonal antibody

AEC was applied for development. Primary antibodies included rabbit anti-human IgA polyclonal antibody (DAKO), rabbit anti-human IgM polyclonal antibody (DAKO), rabbit anti-human IgG polyclonal antibody (DAKO), mouse anti-human IgG4 monoclonal antibody (Invitrogen), rabbit anti-human C1q polyclonal antibody (DAKO), rabbit anti-human C3d polyclonal antibody (Abcam), and rabbit anti-human C4d polyclonal antibody (Biomedica). Horseradish peroxidase-labelled secondary antibodies were purchased from LSBio (Maixing Company, China). For plasma cells expressing IgG and IgG4 assessment, five random fields were checked and photographed for each slide, whereby positive cells were counted and averaged. Primary isotopes were used as negative controls.

Patient sera (1:50) and milk (1:2) were incubated in microplate wells coated with SARS-CoV-2 S recombinant protein (S1/S2), as described above. In order to assess the capability of anti-SARS-CoV-2 antibodies to activate the C system, wells were then incubated with AB Rh+ pooled sera (1:100 in PBS + 2% w/v BSA + 0.7mM Ca⁺⁺Mg⁺⁺) for 30 min at 37 °C. After washing with PBS-T, the binding of C1q was evaluated using rabbit anti-human C1q polyclonal antibody (1:2000, Dako, Santa Clara, CA, USA) for 1 h at 37 °C and anti-rabbit IgG AP-conjugated (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) as a secondary antibody. Simultaneously, the deposition of C3 was detected using goat anti-human polyclonal antibodies (1:5000, Quidel, San Diego, CA, USA) for 1 h at 37 °C and anti-goat IgG AP-conjugated (1:30,000, Sigma-Aldrich). The formation of the terminal C complex C5b-9 was assessed using anti-human C5b-9 (1:50, clone: aE11, Dako) for 1 h at 37 °C and anti-mouse polyvalent Igs (G,A,M)-AP (1:30,000, Sigma Merck). The binding was revealed with pNPP and the absorbance was read at 405 nm using a PowerWave X Microplate Reader (Bio-Tek Instruments).