Ab97205

The IgM fraction was purified using a home-made IgM resin. Briefly, the IgM antibody (SICGEN ANTIBODIES, AB0405-500) was covalently bonded to glyoxal agarose beads resin (ABT, 6BCL-GM3) according to the manufacturer’s instructions. IgA and IgG from plasma samples were purified through Peptide M/Agarose (Invivogen, 6457-43-01) or Protein G (Thermo Fisher Scientific, 20398), respectively, according to the manufacturers’ instructions. Briefly, 100 μL of plasma was incubated with 200 μL of Peptide M/Agarose, Protein G, or IgM resin for 20 minutes. The resins/beads were washed 5 times with wash buffer (10 mM sodium phosphate, 150 mM sodium chloride; pH 7.2) and eluted in 100 μL fractions with 0.1 M glycine pH 2.76. The pH of the collected fractions was adjusted to 7 with 1 M Tris (pH 8.83). All steps were carried out at 4°C. Western blotting was performed according to standard procedures. Secondary antibodies used were from Abcam, diluted 1:5000: goat anti–human IgA alpha chain HRP (ab97215), goat anti–human IgG Fc HRP (ab97225), and goat anti–human IgM mu chain HRP (ab97205). Protein bands were imaged using ECL on a GE Amersham Imager 680.

The SARS-CoV-2 S trimer, RBD, S2 and nucleocapsid protein (N) (Acro Biosystems SPN-C52H9, SPD-C52H3, S2N-C52H5, and NUN-C5227, respectively) were coated on 96-well plates at 0.5 μg/ml in PBS overnight at 4 °C. After the plates were blocked with buffer (1× PBS with 3% BSA (Solarbio, A8020) and 0.05% Tween-20 (Sigma, P9416)) for 1 h at 37 °C, plasma samples were added and incubated for 1 h at 37 °C. The plasma samples were assayed at a 1:100 starting dilution and 7 additional threefold serial dilutions in blocking buffer (1× PBS with 1% BSA (Solarbio, A8020) and 0.05% Tween-20). The plates were washed 5 times with washing buffer and then incubated with anti-human IgG (Abcam, ab97225) or IgM (Abcam, ab97205) secondary antibody conjugated to horseradish peroxidase (HRP) in blocking buffer at a 1:50,000 dilution (and IgG) or 1:20,000 dilution (IgM) for 0.5 h at 37 °C. After the plates were washed 5 times, the HRP substrate TMB (Solarbio, PR1200) was added for 10 min, followed by the addition of 50 μl of 1 M H₂SO₄ (Solarbio, C1058) to stop the reaction. The absorbance was measured at 450 nm with an ELISA microplate reader (TECAN Infinite 200 PRO).