Development of a determination method for free polysaccharide content in serotype 6B pneumococcal polysaccharide conjugate

LIU Ai-ping; REN Zhen-yun; LIU Qian; LUO Shu-quan; CHEN Xiao-hang; LI Xian-lin; DU Jiao; XIE Gui-lin

Abstract：Objective:To develop and validate a method for determination of free polysaccharide in serotype 6B pneumococcal polysaccharide conjugate with TT as carrier protein (PS6B-TT).Methods:NaDC/HCl method was adopted.1％ NaDC was mixed with different concentrations of TT,supernatants were collected by centrifugation,and protein content was determined by Lowrry assay to determine the concentration range of carrier protein precipitated with NaDC.The mixtures of free polysaccharide and TT were diluted with different ionic strengths of sodium chloride solution,and the supernatants were collected and tested for polysaccharide content by anthrone-sulfuric acid method to determine the condition for separation of free polysaccharide.The serotype 6B standard for determination of polysaccharide content was precipitated with NaDC/HCl and the standard curve was compared with the normal standard to evaluate the influence of presence of NaDC on determination result of polysaccharide content.The specificity,accuracy and precision were verified.Results:The carrier protein concentration for NaDC precipitation was controlled at less than 500 μg· mL-1.0.3 mol· L-1 sodium chloride served as the diluent for determination of free polysaccharide in serotype 6B pneumococcal conjugate.NaDC/HCl method showed little effect on determination of polysaccharide content by anthrone-sulfuric acid method.After NaDC/HCl precipitation,the recovery rates of mixture of free polysaccharide and TT were 98.25％ and 0,respectively.The recovery rates of free polysaccharide in spike test were between 90％～ 100％.The intro-and inter-assay CVs of three batches of determination results were less than 10％.Conclusion:The developed NaDC/HCl precipitation combined with anthrone-sulfuric acid method is effective for separation of 6B free polysaccharide from conjugate,accurate and stable for determination of free polysaccharide content.