Determination of α2,3-Sialic Acid PSA

The determination of % α2,3-sialic acid of PSA was performed using a previously published method [9 (link)]. Briefly, ethanolamine 5 M was added to 0.75 mL of each serum sample to a final concentration of 1 M to release the PSA complexed to α1-antichymotrypsin. Total PSA was immunopurified using the Access Hybritech PSA assay Kit (Beckman Coulter, Brea, CA, USA). Amicon Ultra-0.5 3K Centrifugal Filter Devices (Millipore, Cork, Ireland) were used for desalting and concentrating the immunopurified tPSA samples up to a final volume of 40 μL. Samples were then applied to a lectin chromatography using Sambucus nigra (SNA)-agarose lectin (Vector Laboratories, Inc., Burlingame, CA, USA). Eluted unbound and bound chromatographic fractions were collected by centrifugation and quantification of free PSA of these fractions was performed using the Roche ELECSYS platform and used to determine the percentages of fPSA in the unbound fraction, corresponding to α2,3-sialic acid PSA, and in the bound fractions, which correspond to α2,6-sialic acid PSA.