ABSTRACT
Non-instrumental immunoassay methods based on immunofiltration and microtiter particle agglutination (MPA) techniques have been developed using coloured polyacrolein latex. These methods have been applied to the quantification of the group-specific polysaccharide, A-PS, of S.pyogenes (group A Streptococcus) and compared to the standard ELISA tests. The assay with the ability to detect the lowest concentration of antigen was MPA; as little as 0.05 ng A-PS/ml or 10(4) cells/ml could be detected in 1.5 h. In comparison to ELISA test the sensitivity of MPA was 10 times higher and the procedure of the assay was much more simple. The sensitivity of the immunofiltration assay using both enzyme and latex markers was shown to be the same (50 ng A-PS/ml) and the duration of the assay 3-5 min. No cross-reactions of latex conjugates with non A Streptococcus cell lysates have been observed. The developed methods are easy to perform and require neither sophisticated equipment nor specially trained personal.
Subject(s)
Acrolein , Chromogenic Compounds , Immunoassay/methods , Latex , Polymers , Polysaccharides, Bacterial/analysis , Agglutination Tests , Animals , Antigen-Antibody Reactions , Enzyme-Linked Immunosorbent Assay , Equidae , Immunoglobulin G/immunology , Sensitivity and Specificity , Streptococcus pyogenes/immunologyABSTRACT
The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.
Subject(s)
Lipid A/chemistry , Neisseria meningitidis/chemistry , Fatty Acids/chemistry , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Molecular Structure , Neisseria meningitidis/pathogenicity , Oligosaccharides/chemistry , Phosphates/chemistryABSTRACT
The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic beta 1,6-linked D-glucosamine disaccharide [beta-D-GlcpN-(1----6)-D-GlcpN], phosphorylated in positions 4' and 1. Position 6' of the beta-D-GlcpN-(1----6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.
Subject(s)
Lipid A/chemistry , Lipopolysaccharides/chemistry , Mutation , Pseudomonas aeruginosa/immunology , Carbohydrate Conformation , Carbohydrate Sequence , Fatty Acids/analysis , Gas Chromatography-Mass Spectrometry , Lipid A/isolation & purification , Lipopolysaccharides/isolation & purification , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Pseudomonas aeruginosa/geneticsABSTRACT
T-cell growth factor (interleukin 2) was partially purified from the conditioned medium from Con A-stimulated rat splenocytes. It had the molecular weight of 23,000 according to gel filtration and in the range of 17,000-18,000 daltons according to PAGE in the presence of SDS. The molecular forms of IL 2 exhibited isoelectric points (pI) of 5.4-5.8 (major peak), 4.3-4.5 and 6.5-6.8 (minor peaks). T-cell growth-promoting activity was remarkably stable to temperature, pH, SDS and 2-ME treatments. Rat IL 2 did not bind to immobilized Con A, WGA, RC 1, and fucose-binding protein. The conditioned medium also contained the inhibitor of IL 2-dependent T-cell proliferation.
