Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid...

We have adapted a commercially-available kit for the enzymatic determination of sialic acid so that the reaction can be carried out in microtitre wells and the coloured end product quantitated on an ELISA plate reader. The small volume of serum used allows sialic acid to be measured in capillary blood samples. The assay was based on release of sialic acid from glycoconjugates by neuraminidase, cleavage of sialic acid by N-acetyl neuraminic acid aldolase to pyruvate, and then oxidation of pyruvate to hydrogen peroxide by pyruvate oxidase. Hydrogen peroxide was determined by the red product formed in the presence of peroxidase, 4-aminoantipyrine and N-ethyl-N-2-hydroxyethyl-3-toluidine. The assay was linear to at least 10 mmol/l and unaffected by haemolysis, and by the addition of glucose, 3-hydroxybutyrate, bilirubin and pyruvate. Capillary serum sialic acid concentrations were not significantly different from simultaneously measured venous serum sialic acid levels. Self-collected capillary blood samples were obtained from healthy subjects over 6 h during the day. No variations in serum sialic acid concentrations were found in response to a meal. We conclude that this micro-adaptation of a specific sialic acid assay will be suitable for epidemiological surveys of serum sialic acid collected by patients and normal subjects.