Highly sensitive quantification of bacterial chondroitin in a culture based on ELISA techniques

Abstract

Some bacteria produce non-sulfated chondroitin (CH). Accurate, rapid, and high throughput methods to quantify CH in fermented cultures helps to improve microbial breeding and fermentation conditions efficiently. In this study, highly sensitive methods to quantify bacterial CH were developed based on ELISA techniques. An assay using an anti-K4 antiserum successfully determined the concentration of fructosylated CH in the range from 9 to 800 ng/mL. The method also enabled the determination of CH concentration exceeding 9 μg/mL. To improve the assay sensitivity for CH, hyaluronan (HA) binding protein (HABP) was applied instead of a capture antibody. HABP was bound to CH, but not to chemically desulfated chondroitin sulfate or fructosylated CH. The quantification limit of CH was 18 μg/mL in the HA assay using HABP. Replacing the HA-coated microplate with a CH-coated microplate increased the sensitivity >1000 times (assay range = 14 to 1000 ng/mL). Pretreatment with hyaluronidase enabled us to accurately quantify CH in samples mixed with HA.