Direct and indirect methods for molar-mass analysis of fragments of the capsular polysaccharide of Haemophilus influenzae type b.

Two methods are described for direct molar-mass measurement of low-molar-mass fragments obtained by oxidative cleavage of the capsular polysaccharide of Haemophilus influenzae type b. Absolute molar masses were determined by size-exclusion chromatography (SEC) with detection by multiangle laser-light-scattering photometry (MALLS) and differential refractometry (RI). The end-group structure of the polysaccharide fragments allowed the direct measurement of average chain length by quantitative 1H NMR, from which molar masses were derived. Variation between the molar masses obtained by the two methods ranged from 5 to 7%. When molar masses estimated by indirect methods were compared to SEC-MALLS/RI data, significant deviations were observed. Analysis by SEC with secondary calibration with dextran standards gave molar masses that exceeded the SEC-MALLS/RI data by as much as 2.5-fold. Molar masses estimated by a combination of colorimetric assays varied from the SEC-MALLS/RI data by as much as 50%. These results demonstrated the applicability and superior accuracy of the direct methods of molar-mass determination of the polysaccharide fragments.

N-Propionylated group B meningococcal polysaccharide mimics a unique bactericidal capsular epitope in group B Neisseria meningitidis.

The N-propionylated group B meningococcal polysaccharide (NPrGBMP) mimics a unique protective epitope on the surface of group B meningococci (GBM) and Escherichia coli K1. Using a series of monoclonal antibodies (mAbs) induced by the NPrGBMP-monomeric tetanus toxoid (TT) conjugate vaccine it was demonstrated that mAbs having specificities for both extended and conventional short segments of the NPrGBMP were formed, but only the former were bactericidal, and/or gave passive protection against live challenge by GBM. The failure of mAbs specific for short epitopes to protect was further established when (NeuPr)4-TT was used as the vaccine. Of all the mAbs produced that were specific for short internal segments of the NPrGBMP, none were protective, despite the fact that most of them cross-react with the GBM capsular polysaccharide. In contrast, most of the protective mAbs produced by NPrGBMP- TT did not recognize the group B meningococcal polysaccharide (GBMP) unless it was present in its aggregated high molecular weight form. The bactericidal epitope mimicked by the NPrGBMP was shown to be ubiquitous in the capsule of both GBM and E. coli K1 using immunogold labeling techniques and, because of its unique properties, its identification could be significant in the development of a comprehensive conjugate vaccine against group B meningococcal meningitis. This is because most known human alpha(2-8)-polysialic acid self-antigens can be accommodated in 30-50 alpha(2-8)-linked sialic acid residues, which is roughly equivalent to an 11-kD length of the GBMP. It has been hypothesized that the formation of the protective epitope on the surface of GBM is due to the interaction of helical segments of the GBMP with another molecule and that the protective epitope is mimicked by the NPrGBMP. Support for the above hypothesis is provided by the fact that the protective NPrGBMP epitope has a similar unusual length dependency to that of the GBMP epitope.

The detection of chemical impurities by high pressure liquid chromatography and the genetic activity of medical grades of pyrvinium pamoate in Saccharomyces cerevisiae and Salmonella typhimurium.

The genetic activity of several medical grades of the anthelmintic drug pyrvinium pamoate, which is a dipyrvinium salt, was studied in a diploid mitotic recombination and gene conversion assay (strain D5 of Saccharomyces cerevisiae), and in several haploid yeast reversion assays (strains XV185-14C, XY718-1A, and 7854-1A). All of the samples were recombinogenic in strain D5 and mutagenic in the haploid strains, however, the degree of genetic activity varied considerably among the medical grades of pyrvinium pamoate that were tested. Similarly, these samples varied in degree of mutagenicity when they were tested in strains TA97, TA98, TA100, and TA102 of Salmonella typhimurium, but some of the medical grades of pyrvinium pamoate were mutagenic both in the presence and in the absence of the metabolic transformation system, whereas other medical grades of the drug required such activation to be mutagenic. In addition, the medical grades and dosage forms of several brands of pyrvinium pamoate were examined for purity by fluorescence high pressure liquid chromatography (HPLC) using a methanol:water (90:10) solvent system. The HPLC data indicate that monopyrvinium salts are the major contaminants in these pharmaceuticals. In general, there is a correlation between the degree of genetic activity and toxicity, and the number and relative quantity of impurities found in each sample.