Quantitation of acidic capsular polysaccharides by Alcian blue binding

Abstract

Alcian blue is a cationic dye used to stain acidic polysaccharides, including bacterial capsules, and as a biochemical reagent to quantitate glycosaminoglycans and glycoproteins. Alcian blue capsular polysaccharide (CP) complex formation can be quantitated by measuring the absorption at 620 nm of Alcian blue released from the complex in 40% manoxol-1B. Optimal complex formation, which is dependent on the pH of the buffer used as well as MgCl 2 concentration, is determined for purified CPs of Neisseria meningitidis group A, Hemophilus influenzae type b, and Streptococcus pneumoniae type III. Under optimal electrolyte conditions, the amount of Alcian blue released from the complex shows a linear relationship to the concentration of CP ( r = 0.99). Quantitation of H. influenzae type b CP recovered from the complex by solubilization with 40% manoxol-1B followed by precipitation with ethanol also correlates with the absorption of Alcian blue ( r = 0.99). CPs could also be recovered (66–92% of applied CP) from the complex by salt extraction.