N-glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin 
that metabolically incorporates into diverse native glycoconjugates in humans. 
Anti-Neu5Gc antibodies are detected in all human sera, though with variable 
levels and epitope-recognition profiles. These antibodies likely play a role in 
several inflammation-mediated pathologies including cardiovascular diseases and 
cancer. In cancer, they have dualistic and opposing roles, either stimulating or 
repressing disease, as a function of their dose, and some of these antibodies 
serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of 
inflammation-mediated diseases, and changes in their levels could potentially be 
used to monitor disease progression and/or response to therapy. Currently, it is 
difficult to determine levels of anti-Neu5Gc antibodies in individual human 
samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we 
describe a simple and specific method for detection and overall estimation of 
human anti-Neu5Gc antibodies. We exploit the difference between two mouse models 
that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah(-/-) 
knockout). We characterize mouse serum from both strains by HPLC, lectin and 
mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use 
Cmah(-/-) knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding 
to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We 
applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in 
sera of patients with Kawasaki disease (KD) at various stages compared to 
controls. KD is an acute childhood febrile disease characterized by inflammation 
of coronary arteries that untreated may lead to coronary artery aneurysms with 
risk of thrombosis and myocardial infarction. This estimated response is 
comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a 
sialoglycan microarray. Both assays revealed an elevated response in acute KD 
patients with normal coronaries compared to patients with aneurysm or dilated 
coronaries. Implications of these findings are discussed.