Quantifying Polyreactive IgM in Transplanted Patients

Nunc MaxiSorp ELISA plates (Thermo Fisher Scientific 44-2404-21) were coated overnight with goat anti–human IgM (4 μg/mL; SouthernBiotech 2020-01, RRID:AB_2795599). After blocking, the plates were incubated with serum from transplanted patients. Bound IgM was detected by adding HRP-conjugated goat anti–human IgM (4 μg/mL; SouthernBiotech 2020-05, RRID:AB_2795603). Alternatively, polyreactive natural IgM was detected by analyzing the amount of immunoglobulin bound to LPS adapted from the protocol Singh et al. described (59 (link)). Briefly, Nunc MaxiSorp ELISA plates were coated overnight at room temperature with LPS (10 μg/mL; Alpha Diagnostic International LPS12-1). After blocking, human sera were incubated for 2 hours at 37°C. Bound IgM was detected by adding goat anti–human IgM–HRP (4 μg/mL; SouthernBiotech 2020-05, RRID:AB_2795603). The reactions were visualized by subsequent addition of 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate (SouthernBiotech 0202-01). All readings were recorded at 405 nm.