Development of a Fucoidan-Specific Antibody and Measurement of Fucoidan in Serum and Urine by Sandwich ELISA

Abstract

Fucoidan exhibits various biological properties. We raised a novel antibody against fucoidan extracted from Cladosiphon okamuranus and developed a sandwich ELISA method to measure fucoidan. The fucoidan antibody was specific and did not cross-react with other polysulfated polysaccharides. Fucoidan recovery from serum and urine by ELISA was 86-113%. Intra-and inter-assay CVs were 1.5-13.4%. Assay linearity was maintained after 3-fold dilution of each sample with phosphate-buffer saline (PBS). In the serum and urine of healthy volunteers (n ¼ 10), fucoidan was not detected before administration, and the levels markedly increased 6 and 9 h after oral administration. The molecular weight of the serum fucoidan determined by HPLC gel filtration remained unchanged, whereas that of urine fucoidan was significantly reduced. This is the first ELISA method of measuring serum and urine fucoidan levels after oral administration. The method is simple, reliable, and practical for the analysis of samples, especially urine samples.

A fluorometric assay for quantification of fucoidan, a sulfated polysaccharide from brown algae

“…The method that used methylene blue staining of fucoidan blotted on a membrane measured in excess of 1 µg/spot (Lee et al 2012), while Mizuno et al (2009) reported a standard curve at fucoidan concentrations above 5 ng µl −1 using the competitive ELISA method. On the other hand, the sandwich ELISA method described by Tokita et al (2010) showed higher sensitivity (LOD, 0.2 ng ml −1 ) than ours. The standard curve generated with our method showed a 50-fold linear dynamic range, which was wider than any reported to date; 20-fold (Lee et al 2012), 10-fold (Mizuno et al 2009), 18-fold (Tokita et al 2010).…”

“…On the other hand, the sandwich ELISA method described by Tokita et al (2010) showed higher sensitivity (LOD, 0.2 ng ml −1 ) than ours. The standard curve generated with our method showed a 50-fold linear dynamic range, which was wider than any reported to date; 20-fold (Lee et al 2012), 10-fold (Mizuno et al 2009), 18-fold (Tokita et al 2010). …”

“…Several methodologies for fucoidan quantification have been reported, and include competitive enzyme-linked immunoassay (ELISA; Irhimeh et al 2005;Mizuno et al 2009), sandwich ELISA assay (Tokita et al 2010), and methylene blue staining of fucoidan immobilized on a filter (Lee et al 2012). A semiquantitative method using toluidine blue staining of fucoidan separated by agarose or polyacrylamide gel electrophoresis (Pereira et al 1999) has also been reported.…”

“…A semiquantitative method using toluidine blue staining of fucoidan separated by agarose or polyacrylamide gel electrophoresis (Pereira et al 1999) has also been reported. A feature of ELISA methods is that they can be used to quantify specific substructures recognized by each of the antibodies; therefore, the specificity of antibodies affects quantification (Tokita et al 2010;Torode et al 2015). Another feature is that the availability of the antibodies directly affects the generality of the methods.…”

Detection of Fucoidan in Urine after Oral Intake of Traditional Japanese Seaweed, <i>Okinawa mozuku</i> (<i>Cladosiphon okamuranus</i> Tokida)

“…It has been long believed that fucoidan, with a high molecular weight, is hardly absorbed in the human digestive system due to a lack of digestive enzymes for fucoidan. Intestinal absorption of fucoidan from Okinawa mozuku has been demonstrated in humans and rats (18,19); however, it is unclear whether fucoidan in mozuku can be absorbed after digestion of mozuku.…”