Development and validation of an NMR-based identity assay for bacterial polysaccharides

Citation
C. Abeygunawardana et al., Development and validation of an NMR-based identity assay for bacterial polysaccharides, ANALYT BIOC, 279(2), 2000, pp. 226-240
Citations number
43
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
279
Issue
2
Year of publication
2000
Pages
226 - 240
Database
ISI
SICI code
0003-2697(20000315)279:2<226:DAVOAN>2.0.ZU;2-K
Abstract
A method utilizing NMR spectroscopy has been developed to confirm the ident ity of bacterial polysaccharides used to formulate a polyvalent pneumococca l polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generate d for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysac charide and is sensitive to any structural alteration of the repealing unit s. The similarity of any two spectral profiles is evaluated using a correla tion coefficient (rho), where rho greater than or equal to 0.95 between a s ample and reference profile indicates a positive identification of the samp le polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharid e powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be rea dily adapted for use with other bacterial polysaccharide preparations as we ll. (C) 2000 Academic Press.