Establishment of a double antibody sandwich ELISA for determination of group C polysaccharide in meningococcal polysaccharide vaccine

Abstract

Objective To establish a double antibody sandwich ELISA for determination of group C polysaccharide in groups A,C,Y,W135 meningococcal polysaccharide vaccine (MPV4).Methods The prepared polyclonal antibodies against group C polysaccharide were purified by octanoic acid-ammonium sulfate precipitation method,and then labeled with horseradish peroxidase by sodium periodate method.The polyclonal antibodies against group C polysaccharide were used as coated antibodies and the second enzyme-labled antibodies to establish a double antibody sandwich ELISA.The reaction conditions of the established ELISA were optimized,and specific quantitative determination of group C polysaccharide was carried out by the established ELISA.Results The specificity of the double antibody sandwich ELISA for determination of group C polysaccharide was high,and no cross reactions with groups A,Y and W135 were detected.The best linearity in dose-response curve of group C polysaccharide was found in a range of 2.5-20.0 ng/ml (r ＞ 0.99).The precision and accuracy of the established ELISA were good.Coefficients of variation and recovery rates of intraand inter-assay were 0.6％-9.1％ and 87.5％-100.0％,respectively.Quantitation limit was identified as 4.0 ng/ml.Group C polysaccharide contents,molecular dimension KDvalue and recovery rates of three batches of MPV4 detected by the established ELISA were in accordance with those of previous determination and temporary quality control standards.Conclusion The double antibody sandwich ELISA can be applied to detecting the key quality indexes of group C polysaccharide in MPV4. Key words: Enzyme-linked immunosorbent assay; Meningococcal vaccines; Polysaccharides,bacterial; Methods