速率比浊法检测ACYW135群脑膜炎球菌结合疫苗中各群多糖含量

目的建立速率比浊法准确检测ACYW135群脑膜炎球菌结合疫苗(group ACYW135 meningococcal conjugate vaccine)的各群多糖含量。方法制备多糖参考品和抗血清,建立检测ACYW135群脑膜炎球菌结合疫苗各群多糖含量的速率比浊法,对建立的方法进行验证及初步应用。结果①建立的方法具备高度的特异性,各群血清仅与本群多糖抗原发生特异性反应,与非本群多糖抗原无交叉反应;②在质量浓度为0.5～5.0μg/mL多糖参考品检测区间线性良好,相关系数r>0.98;③各群血清的最低检测限均低于质量浓度为0.35μg/mL;④批内和批间变异系数(CV)值分别为0.83%～5.34%和2.33%～13.88%,加样回收率为90.5%～118.4%,精密度和准确度均符合常规质量控制要求;⑤初步应用结果显示,建立的方法可准确检测疫苗的各群多糖含量,且与火箭电泳法检测结果差异无统计学意义(P>0.05)。结论建立的方法简便快捷、灵敏准确、自动化程度高,可有效检测ACYW135群脑膜炎球菌结合疫苗的各群多糖含量。     

核磁共振波谱法在肺炎球菌荚膜多糖检定中的应用

目的 建立1 H-NMR波谱法检定23价肺炎球菌荚膜多糖结构的方法.方法 用1H-NMR方法分析肺炎球菌荚膜多糖,选择(5.89～4.64) ppm区作为‘指纹’鉴定区,以默克公司公布的肺炎球菌荚膜多糖波谱为参照谱图,将检定样品的波谱与其相比较.对1 H-NMR波谱进行专属性和耐用性验证.结果 实验中应用1H-NMR波谱分析法对23价肺炎球菌多糖疫苗包含的所有血清型多糖的检定结果与默克公司公布的结果完全一致,该方法有很好的专属性和耐用性.结论 实验中建立的1H-NMR波谱法适用于肺炎球菌荚膜多糖的检定,与传统方法相比,该方法有检测速度快,样品易于准备的优点等.     

Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.     

Immunochemical studies on the capsular polysaccharides of pneumococcal type IX

1. In addition to the previously identified components, d-glucose, N-acetyl-d-glucosamine and d-glucuronic acid, the immunologically specific capsular polysaccharide of pneumococcal type IX (polysaccharide S IX) is now found to contain mannosamine and galactosamine, probably as their N-acetyl derivatives. 2. Partial hydrolysis yields a complex mixture of oligosaccharides, several of which have been separated. 3. One of these is alpha-d-glucuronopyranosyl-(1-->3)-d-glucose. It is an inhibitor of the precipitation of antipneumococcal type IX serum by polysaccharide S IX. 4. Two others, d-glucuronopyranosyl-(1-->3)-d-glucosamine and d-glucuronopyranosyl-(1-->3)-d-galactosamine have been identified and a trisaccharide and pentasaccharide partially characterized.     

Molecular sizing of individual polysaccharides in polyvalent pneumococcal and meningococcal vaccines by column chromatography and immunoelectrophoresis

We determined the molecular size of meningococcal and pneumococcal polysaccharides in final vaccines, using a combination of column chromatography and simple rocket immunoelectrophoresis. We found a good correlation between the distribution coefficients obtained with UV monitoring or ELISA and immunoelectrophoresis.     

The extraction of imperialine and imperialine-3 beta-glucoside from Fritillaria pallidiflora Schrenk and quantitative determination by HPLC-evaporative light scattering detection

The extraction procedure and quantitative determination by HPLC-evaporative light scattering detection (ELSD) of the main bioactive components, namely, imperialine (1) and imperialine-3 beta-glucoside (2), of bulbs of Fritillaria pallidiflora Schrenk have been investigated. The most efficient method for the simultaneous extraction of 1 and 2 involved pre-treatment of the bulb powder with ammonia, followed by reflux with dichloromethane:methanol at 90 degrees C for 4 h. Simultaneous determination of non-chromophoric 1 and 2 by HPLC-ELSD employed a Kromasil C18 column eluted with acetonitrile:water:diethylamine. The assay was accurate and reproducible with an overall variation lower than 4% and a sample recovery higher than 98%. The methods described have been successfully used to evaluate the quality of three batches of the crude traditional Chinese medicinal herb derived from the bulbs of F. pallidiflora.     

Quality control of polyvalent pneumococcal polysaccharide-protein conjugate vaccine by nephelometry.

A nephelometric method was used for quantitative analysis of individual polysaccharides (PSs) in a polyvalent pneumococcal conjugate vaccine using CRM(197) as carrier protein. Using this method, the individual types 4, 6B, 9V, 14, 18C, 19F and 23F PSs were found to range between 82.3 to 119% of the manufacturer's indicated values.During conjugation using reductive amination, pneumococcal PS was first oxidized to introduce aldehyde groups. Higher or lower levels of antigen-antibody reaction were observed in periodate activated and then reduced PS of some serotypes compared to non-treated PS. Use of oxidized and reduced PS may provide an early indication of change in conjugation process. Furthermore, since the final monovalent and polyvalent conjugate vaccines gradually change during the storage period, the nephelometry provides an useful analytical method for stability study of these vaccines.     

Erythrocyte aggregation: approach by light scattering determination

Erythro-aggregometer is a Couette viscometer which was developed to measure aggregation parameters of red blood cells. The system is based on the analysis of the light intensity backscattered by a blood suspension. It allows to approach aggregation phenomenon in terms of kinetics, structural and rheological parameters. The measurement system designed for use with a microcomputer is suitable for both research and clinical investigations.     

Structural determination and immunochemical characterization of the type V group B Streptococcus capsular polysaccharide

The type V capsular polysaccharide of group B Streptococcus has been isolated and purified, and its repeating unit structure determined. The native type V polysaccharide contains D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and sialic acid in a molar ratio of 3:2:1:1. Methylation analysis and 1H NMR and 13C NMR analysis of the native type V polysaccharide and of its specifically degraded products permitted the determination of the repeating unit structure of the type V polysaccharide: [formula: see text] The type V polysaccharide has certain structural features in common with other group B streptococcal capsular polysaccharides but is antigenically distinct: no immunologic cross-reactivity was observed between type V and types Ia, Ib, II, III, or IV polysaccharides. Studies of antibody binding to the partially degraded forms of the type V polysaccharide indicated that the native epitope is complex, involving most if not all of the sugar residues of the repeating unit.     

Determination of meningococcal polysaccharides by capillary zone electrophoresis

Meningococcal polysaccharides are medically important molecules and are the active components of vaccines against Neisseria meningiditis serogroups A, C, W135, and Y. This study demonstrates that free solution capillary zone electrophoresis (CZE) using simple phosphate/borate separation buffers is capable of separating intact, native polysaccharides from these four serogroups. Separation appeared to be robust with respect to variations in test conditions and behaved in expected ways with respect to changes in temperature, ionic strength, and addition of an organic modifier. Serogroups W135 and Y are composed of sialic acid residues alternating with either galactose or glucose, respectively. Separation of these serogroups could be achieved using phosphate buffer and was therefore not dependent on differential complexation with borate. Addition of sodium dodecyl sulfate to the separation buffer (i.e., MEKC) resulted in peak splitting for all four serogroups. Changes in polysaccharide size did not affect migration time for the size range examined, but serogroup C polysaccharide (a sialic acid homopolymer) was separable from sialic acid monosaccharide. CZE quantification of multiple lots of each of the four serogroups was compared to wet chemical determination by phosphorus or sialic acid measurement. Results from CZE determination showed good agreement with the wet chemical methods.     

The determination of polar lipid solubilization boundaries in dilute solutions by light scattering

An apparatus and procedure have been developed which facilitate the acquisition of particle free water by recirculation through a fine membrane filter in a closed system. The system also permits one to vary, independently, composition of two solutes over a few hundred mg/l range, with ± 1 mg/l accuracy, while monitoring scattering intensities.Using these techniques it has been possible to define the phase boundary governing the solubilization of distearoylphosphatidyl choline (DSPC), above its chain melting temperature, by three homologous (C₁₆, C₁₈, C₂₀) ammoniohexanoate (AH) surfactants. The data suggest that negligible (< 1 mg/l) solubilization occurs up to an onset concentration of surfactant, C_on, which for C₁₆AH is 0.77 of the cmc. Beyond C_on finite solubilization occurs with an appreciable efficiency, which peaks at C₁₈AH at 1.35 g/g.     

The anti-tumour effect of Klebsiella pneumoniae capsular polysaccharides

K24 capsular polysaccharide (K24-CPS), with a known structure of a repeating unit, was isolated from the capsule of Klebsiella pneumoniae serotype K24. The polysaccharide was found to suppress the proliferation of Ehrlich ascites tumour (EAT) cells in vitro, but did not alter the cell cycle distribution of cells. K24-CPS treatment reduced the tyrosine phosphorylation of some proteins in EAT cells. Furthermore, the treatment also decreased the expression of c-JUN, but had no effect on the levels of c-FOS and c-MYC. It is speculated that the growth suppression effect of K24-CPS may be related to its effect in down-regulating c-JUN expression.     

Rapid quantitative characterization of protein interactions by composition gradient static light scattering

Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.