Fig 1: Anti-ACE2 IgM antibodies are found in patients with COVID-19.A: Antibodies were assayed by ELISA in the combined COVID cohort (N = 118 patients). Left panel: the number of patients with and without anti-ACE2 IgM antibodies is shown grouped by disease severity. 27.2% of severe patients were anti-ACE2 positive compared to 3.8% with moderate COVID (p = 0.0009; Fisher’s exact test). In the center and right panels, data from anti-ACE2 IgM and IgG ELISA assays, respectively, is presented as corrected OD 450 absorbance units. This data was obtained on all the COVID patients presented in the left panel, as well as from 30 healthy controls. Red dots in the IgG panel denote IgG-positive samples that also have anti-ACE2 IgM antibodies. The horizontal line on each plot represents the cutoff for assigning a positive antibody status. B: Longitudinal analysis of anti-ACE2 IgM antibodies. For all those anti-ACE2 IgM-positive patients with multiple banked sera available (16/18), anti-ACE2 IgM and IgG antibodies were quantitated over time. Red and blue lines on each plot denote anti-ACE2 IgM and IgG antibodies, respectively. Solid black bars represent steroid treatment periods. Additional examples are shown in Suppl Fig 3.
Fig 2: Properties of anti-ACE2 IgM antibodies. (A–C): Kinetic analysis.A: Kinetic traces of the binding interactions between immobilized human ACE2 and purified IgM, as determined by biolayer interferometry. Percentages represent twofold dilutions of IgM from patient CV-1 and Control B. B: Equilibrium binding titrations. Normalized responses at the indicated concentrations of purified IgM from the donors shown in (A) are plotted. C: Quantitation of the data obtained in A&B, and a separate patient and control shown in Supp. Fig 5A&B. D: Anti-ACE2 IgM antibodies do not inhibit ACE2 activity. ACE2 activity, in the presence or absence of IgM from patient CV-1 or Control B, was measured using a fluorescent substrate in a time course assay. The positive control was ACE2 alone, and the negative control was ACE2 plus ACE2 inhibitor (see Suppl Fig.5D for data obtained from another patient and control). E: Complement activation induced by IgM antibodies to ACE2. Dynabeads containing immune complexes of ACE2 and purified IgM from controls or anti-ACE2-positive COVID-19 (CV) patients were incubated with human complement. Deposition of C1q and C3 was visualized by immunoblotting. ACE2 is shown as a loading control. Markedly enhanced C1q binding in CV-1 observed in 3 separate experiments.
Fig 3: IgM isolated from SARS-CoV-2–infected patients, but not healthy donors, binds to ACE2.Equilibrium binding titrations (A–C) and kinetic traces (D–K) of immobilized ACE2 and IgM purified from human serum, as measured by biolayer interferometry. (A–C) Normalized responses at the indicated concentrations of purified IgM from 3 healthy donors and 5 SARS-CoV-2–infected patients are plotted (data from 3 independent experiments). Equilibrium dissociation constants (KD) calculated by fitting to a 4-parameter logistic regression model are provided. (D–K) The indicated percentages (color-coded on the right side of K) represent 2-fold dilutions of the 8 purified IgM preparations shown in A–C. Binding constants were determined via global fitting for each IgM sample using Octet Data Analysis software (assuming a 1:1 binding model). Quantitation of the data shown in A–K is provided in Supplemental Table 2.
Fig 4: Flow diagram for identification of anti-ACE2 IgM antibodies.Anti-ACE2 IgM antibodies were assayed by ELISA in serum from 66 patients with COVID-19, 52 COVID-19 patients with multiple bleeds available, 133 disease controls, and 30 healthy controls. The functional consequences of these antibodies were investigated with 3 different assays using IgM purified from anti-ACE2 IgM–positive sera. MDA5 DM, melanoma differentiation-associated 5 dermatomyositis.
Fig 5: ACE2 and GlycACE2 expression in human cardiomyocytes. A Representative images of Western blotting analysis of ACE2 and GlycACE2 in explanted heart tissue, basal and follow-up biopsies from nondiabetic and diabetic patients with poor (HbA1c > 7%) or good (HbA1c < 7%) glycemic control. ACE2 and Glyc ACE2 levels were normalized using a-Tubulin. Glyc ACE2 levels in B nondiabetic and diabetic patients and C diabetic patients with HbA1c < 7% and diabetic patients with HbA1c > 7%. Analysis was performed by ImageJ 1.52n software; values were expressed as percentage and data were shown as box and whisker plots using GraphPad Prism 9.1.2. software. *P < 0.05 vs nondiabetic patients; †P < 0.05 vs diabetic patients in good glycemic control (< 7%), §P < 0.05 vs basal values
Supplier Page from Abcam for Recombinant Human ACE2 protein