High-performance reverse phase chromatography with fluorescence detection assay for characterization and quantification of pneumococcal polysaccharides

A methods using high-performance reverse phase (RP) chromatography with fluorescence detection, has been developed to determine the composition and identity of Streptococcus pneumoniae capsular polysaccharide used in formulating conjugate vaccine for prevention of pneumococcal infection. For the monosaccharide composition, the polysaccharides were subjected to hydrofluoric acid (HF) hydrolysis followed by trifluoroacetic acid (TFA). After acid hydrolysis, the released monosaccharides were re-N-acetylated and labeled with 2-aminobenzamide (2AB) by reductive amination reaction. High-performance RP chromatography was performed on C18 TSKODS 120T column. Nuclear magnetic resonance was used to confirm chemical structure and purity of pneumococcal capsular polysaccharides.     

Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection

A new method for the quantitative determination of Neisseria meningitidis group A (MenA) capsular polysaccharide (CPS) has been developed. The method is based on trifluoracetic acid (TFA) hydrolysis of the CPS (2 M at 80 degrees C for 3 h), followed by chromatographic separation and quantification of the liberated mannosamine-6-phosphate from the area of the peak obtained using an IonPac AS11 column coupled to the sensitive pulsed amperometric detector ED40. The highly selective nature of this method circumvents the interference problems associated with the classical method based on a colorimetric assay for phosphorus. Provided that suitable hydrolysis conditions can be found, this chromatographic approach might be applicable to the quantification of other bacterial antigens containing phosphorylated sugars such as meningococcal groups H, L, X and Z, and pneumococcal serotypes 6, 10A and 19.     

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by use of high-performance anion-exchange chromatography with pulsed amperometric detection

Capsular polysaccharides of Streptococcus pneumoniae are used to formulate polyvalent pneumococcal vaccines. A sensitive method, using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), has been developed to quantify the contamination of pneumococcal capsular polysaccharides (PnPs) with the C-polysaccharide (C-Ps). As this polysaccharide is highly immunogenic, and since anti C-Ps antibodies are not protective, the need to monitor and reduce its level is of uppermost importance. The method is based on the quantification by HPAEC-PAD of ribitol, which is released by a two-step hydrolysis of the PnPs using aqueous hydrofluoric acid (HF) followed by trifluoroacetic acid hydrolysis (TFA). This simple method has been shown to provide both qualitative and quantitative information about the purity of polysaccharide preparations.     

Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD)

The O-acetyl groups in meningococcal A and typhoid Vi polysaccharides (PSs) are functional immunogenic epitopes in humans. To quantify and determine the extent of O-acetylation in these and other bacterial vaccine PSs, anion-exchange HPLC methods have been developed for quantification of O-acetyl, N-acetyl, and phosphate groups in the PSs after these groups were hydrolyzed into anions. The O-acetylation in meningococcal A, C, Y and W-135, pneumococcal 9 V and 18C and typhoid Vi PSs were analyzed. The O-acetyl group was selectively released from a PS as acetate by mild alkaline hydrolysis in 10 or 20 mM NaOH at 37 degrees C until maximum release. The acetate in the hydrolysate was then quantified by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD) after removal of the PS by filtration with a 10,000 molecular-weight-cut-off membrane. Since the extent of O-acetylation on the PSs depends on bacterial species, strains and growth conditions, the N-acetyl group of amino-sugars, phosphate or monosaccharide components of the PSs were also quantified using HPAEC with conductivity or amperometry detection to determine the molar ratios of the O-acetyl group to these components. The average numbers of O-acetyl molecules in one PS repeating unit of the PSs were obtained from the molar ratios. Besides the O-acetyl determination, the pyruvate component in non-O-acetylated pneumococcal type 4 PS was analyzed by the HPAEC method. The HPAEC method can quantify the O-acetyl content in 0.2 microg of the meningococcal C PS and has a sensitivity at least 10 times higher than that of the colorimetric Hestrin assay. The method can be used for routine analysis of O-acetylation of PSs for quality control of vaccine PSs.     

Validation of a high-performance liquid chromatography/fluorescence detection method for the simultaneous quantification of fifteen polycyclic aromatic hydrocarbons.

A high-performance liquid chromatography/fluorescence method using multiple wavelength shift for simultaneous quantification of different PAH compounds was developed. The new method was superior to the methods of DONG and GREENBERG [J. Liquid Chromatogr. 11, 1887-1905 (1988)] and WISE et al. [Polycyclic aromat. Hydrocarb. (in press)] with respect to sensitivity of detection of the majority of 15 PAH compounds, and in particular of naphthalene, acenaphthene, fluorene and benzo(b)fluoranthene. The method of validation analysis employed showed that the new method is in statistical balance meaning that no systematic errors, and only small unsystematic errors, could be demonstrated. Furthermore, the method had a good reproducibility and a high sensitivity.     

二极管阵列检测离子色谱法测定水中NO2^-、Br^-、NO3^-

目的:建立直接紫外检测离子色谱法测定水中NO2-、Br-、NO-3的方法,并用于水中NO2-、Br-、NO3-的检测.方法:采用IonPae ASl4-2mm型阴离子分离柱,以3.5 mmol/L Na2CO3 1.0 mmol/L NaHCO3为淋洗液,二极管阵列检测法,NO2-、Br-、NO3-的检测波长分别为210、199、205 nm.结果:直接紫外检测法不受多种共存离子的干扰,可选择每一种组分的最佳吸收波长进行分析.于分离柱与检测池之间添加抑制器可降低背景吸收,消除氯离子和硫酸根离子的负峰.各阴离子的线性好(r>0.9990),相对标准偏差均小于5%(n=7),平均加标回收率均在97.7%～101.5%之间,NO2-、Br-、NO3-的检出限分别为0.20、0.26、0.21μg/L.结论:本文所用方法灵敏、快速,适用于所有的饮用水分析.     

Quantitative determination of sugars and myo-inositol in citrus fruits grown in Japan using high-performance anion-exchange chromatography

Fifteen commercial samples of citrus fruits grown in Japan were analyzed for their sugar contents by high-performance anion-exchange chromatography (HPAEC) and electrochemical detection (ED) coupled with a stationary phase D10 column prepared using chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyldiaminodecane. Myo-inositol, glucose, fructose, and sucrose in all of these various citrus fruits could be successfully separated within 25 min using 0.5 M NaOH eluent at a flow-rate of 0.4 mL/min. Myo-inositol, as a better nutritional source, was found in all the citrus fruits grown in Japan (0.7 +/- 0.04-2.1 +/- 0.2 g/L). The sugar contents of twelve citrus fruits grown in other countries were also determined.     

Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides

Background

Two electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35?µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease.

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.     

啤酒中玉米赤霉烯酮的高效液相色谱紫外检测法

目的 建立啤酒中玉米赤霉烯酮的高效液相色谱紫外检测法。方法 啤酒中的玉米赤霉烯酮用乙酸乙酯提取,50℃水浴中氮气吹干,用乙腈溶解,过滤,以Zorbax SB-18为分离柱,用甲醇+0.020 mol/L乙酸铵(75+25)溶液为流动相,检测波长236nm。结果 玉米赤霉烯酮在0 μg/ml～10.0 μg/ml范围内,线性关系良好(r=0.999 97);方法检出限5.0μg /kg。方法相对标准偏差(RSD)为2.3%～2.7%,加标回收率为86.0%～100.0%,平均回收率为93.6%。啤酒样品中玉米赤霉稀酮含量为51.6μg/kg～584.0μg/kg。结论 本法具有简便、经济、灵敏、准确的特点,适宜在基层检测机构推广使用。     

Immunogenicity of a combination vaccine containing pneumococcal conjugates and meningococcal PorA OMVs

The pre-clinical immunogenicity of a combination vaccine containing 13-valent pneumococcal conjugate (13vPnC) vaccine (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM197) and nine-valent meningococcal B PorA vaccine (NonaMen; serosubtypes P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1 and P1.18-1,3,6), and any potential immunological interference between pneumococcal and MenB components of the vaccine were evaluated. NIH mice were immunized twice subcutaneously with the vaccines combined in one syringe, or given individually. Combining 13vPnC vaccine with NonaMen vaccine in one syringe had no negative effect on the induced antibody response against any MenB serosubtypes compared to separate injection of the vaccines, and the anti-pneumococcal antibody responses were enhanced. Furthermore, co-administration of the combination vaccine with a combined diphtheria/tetanus/acellular pertussis/inactivated poliomyelitis vaccine/Haemophilus influenzae type b-TT conjugate (DTaP/IPV-Hib) vaccine to New Zealand white rabbits at a different injection site did not affect the anti-pneumococcal polysaccharide and anti-PorA antibody titres. We conclude that no immunological interference was observed by combined administration of pneumococcal conjugate and meningococcal B vaccines in one syringe.     

高效液相色谱荧光法测定尿中二元有机酸研究

目的:建立尿中丙二酸、丁二酸、戊二酸3种二元有机酸的高效液相色谱荧光测定方法。方法:采用荧光检测器,利用Waters C18(250 mm×4.6 mm i.d.,5μm)色谱柱,以70%(v/v)乙腈为淋洗液,流速1.0 ml/min,采用激发波长345 nm、发射波长480 nm进行检测,外标法定量。结果:丙二酸、丁二酸、戊二酸在相应的试验浓度范围内均具有良好的线性关系,其相关系数均大于0.999,最低检出限在0.02~0.2 mg/L范围,平均回收率在80.2%~109.6%之间,RSD均小于5.0%。结论:该法具有简单、准确、灵敏等优点,适合尿中丙二酸、丁二酸、戊二酸含量的检测。     

Development and evaluation of a monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of rinderpest virus antibodies

A competitive enzyme-linked immunosorbent assay has been standardised for the detection of antibodies to rinderpest virus in sera from cattle, sheep and goats. The test uses a neutralising monoclonal antibody (MAb) directed against the haemagglutinin protein of rinderpest virus. The test is specific for rinderpest, as it failed to detect antibodies to peste des petits ruminants virus in convalescent goat sera. A 45% inhibition of the binding of the MAb to the antigen was used as the cut-off point for deciding the rinderpest status of the test samples. The specificity and sensitivity of the test and the stability of the test reagents were determined and compared to the results obtained using a commercial kit with approximately 1,200 serum samples from cattle, sheep and goats in India. The current test compared very well with the commercial kit. The test is expected to be extremely useful for sero-monitoring and sero-surveillance of rinderpest in countries which are actively pursuing a rinderpest eradication programme.     

High throughput quantification of capsular polysaccharides for multivalent vaccines using precipitation with a cationic surfactant

The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsular polysaccharides results in a precipitation reaction wherein the suspension turbidity is proportional to polysaccharide titer. The turbidity can be quickly measured as absorbance across a range of wavelengths that resolve scattering light. Carbohydrates comprised of repeating units of one to seven monosaccharides with phosphodiester groups, uronic acids, and sialic acids all reacted strongly and there does not appear to be specificity with respect to the particular anionic moiety. The assay is compatible with an array of common buffers across a pH range of 3.0–8.75 and with NaCl concentration exceeding 400 mM. Interference from DNA can be eliminated with a short incubation step with DNase. With these treatments, the assay has been employed in samples as complex as fermentation broth. A two-log dynamic range has been established with a mean relative standard deviation less than 10% across this range although inferior performance has been observed in fermentation broth.     

The immunogenicity of pneumococcal polysaccharides in infants and children: a meta-regression

The immunogenicity of plain (not conjugated) pneumococcal polysaccharides in children and infants was reviewed using a systematic literature search. Immunogenicity was defined as the fold-increase in serotype specific antibody concentration after a single dose of plain polysaccharide vaccine in unprimed subjects. Meta-regression was used to calculate the influence of study treatments including subject age, sampling time, dosage, immunization route, vaccine composition and study location. Immunogenicity increased with age for all serotypes, and the increase was more rapid in the first 11 months of life. Study location was the next most significant study variable, with higher responses in countries with lower GDP. A flat dose-response curve was observed over a range from 5 to 50 μg polysaccharide. Serotypes 6A, 6B, 14, 19F and 23F were significantly less immunogenic than serotypes 2, 3, 4, 7F, 8, 9N, 9V and 18C in 11 month old children, but continued to increase in immunogenicity with age until reaching similar levels at 6 years. Some proposed T-independent immune mechanisms could explain the differences in serotype immunogenicity.     

Bromide space determination using anion-exchange chromatography for measurement of bromide

A high-pressure liquid chromatographic method for bromide measurement is used to determine extracellular water volume in humans. The method uses 5 microL serum ultrafiltrate and has a sensitivity of 7.5 pmol. Because of the extreme sensitivity of this method, relatively small quantities of Br can be administered and small amounts of blood are needed for the analysis. By this method, the mean corrected Br space in 82 healthy adults representing a wide range of body weights was 0.218 +/- .034 L/kg (mean +/- 1 SD) with a range of 0.153-0.295 L/kg, which is consistent with reported values. There was a significant, inverse relationship between corrected Br space per kilogram and obesity as measured by body mass index. The corrected Br space in six children aged 3-36 mo was 0.335-0.394 L/kg, which is also consistent with reported values in children of this age. This method for Br measurement can easily and readily be applied for the determination of extracellular water volume.     

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.     

Rapid and simple analysis of urinary vanilmandelic acid by high-performance liquid chromatography with electrochemical detection.

We examined an analytical method for urinary vanilmandelic acid (VMA) by high-performance liquid chromatography with electrochemical detection from the viewpoint of practical analysis and application. The sample pretreatment in our method is only the dilution of urine samples with citrate buffer. The calibration curve for VMA was linear within the range 0.2 to 20 ng. The detection limit was 50 pg at a signal-to-noise ratio of 3 and the coefficients of variation were 2.30-4.30%. Variations in the urinary levels of VMA, noradrenaline (NA) and adrenaline (Ad) during and after exercise were investigated for 5 males (mean +/- SD, 42.4 +/- 4.3 years of age). The high levels of urinary NA and Ad during 1 hr exercise rapidly decreased after exercise, whereas urinary VMA increased after exercise rather than during exercise and decreased later. The correlation of the urinary levels of VMA and NA for 71 salesman (mean +/- SD, 40.6 +/- 11.7 years of age) in a field study was significantly positive (r = 0.426, p < 0.001). These results suggest that urinary VMA mostly reflects NA, but the excretion rate of VMA is slower than that of NA.