Immunocapture isotope dilution mass spectrometry in response to a pandemic influenza threat

As a result of recent advances in mass spectrometry-based protein quantitation methods, these techniques are now poised to play a critical role in rapid formulation of pandemic influenza vaccines. Analytical techniques that have been developed and validated on seasonal influenza strains can be used to increase the quality and decrease the time required to deliver protective pandemic vaccines to the global population. The emergence of a potentially pandemic avian influenza A (H7N9) virus in March of 2013, prompted the US public health authorities and the vaccine industry to initiate production of a pre-pandemic vaccine for preparedness purposes. To this end, we evaluated the feasibility of using immunocapture isotope dilution mass spectrometry (IC-IDMS) to evaluate the suitability of the underlying monoclonal and polyclonal antibodies (mAbs and pAbs) for their capacity to isolate the H7 hemagglutinin (HA) in this new vaccine for quantification by IDMS. A broad range of H7 capture efficiencies was observed among mAbs tested by IC-IDMS with FR-545, 46/6, and G3 A533 exhibiting the highest cross-reactivity capabilities to H7 of A/Shanghai/2/2013. MAb FR-545 was selected for continued assessment, evaluated by IC-IDMS for mAb reactivity against H7 in the H7N9 candidate vaccine virus and compared with/to reactivity to the reference polyclonal antiserum in allantoic fluid, purified whole virus, lyophilized whole virus and final detergent-split monovalent vaccine preparations for vaccine development. IC-IDMS assessment of FR-545 alongside IC-IDMS using the reference polyclonal antiserum to A/Shanghai/2/2013 and with the regulatory SRID method showed strong correlation and mAb IC-IDMS could have played an important role in the event a potential surrogate potency test was required to be rapidly implemented.     

Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen

We have developed a highly accurate and sensitive real-time polymerase chain reaction (PCR) assay to detect and quantify Leishmania parasites. The assay targets GP63, a highly conserved gene in Leishmania. We demonstrate that, with a single assay, we are able to detect and quantify several species of Leishmania. Our assay system detects Leishmania donovani and Leishmania major down to 0.1 parasite. The dynamic range of the assay extends over 6 log cycles of target, with an average correlation coefficient >0.988. In addition, we utilize a simple approach to distinguish between Leishmania species causing diverse spectra of disease. We have also used this assay to follow the course of cutaneous disease in CBA/CaJ mice, known to be resistant to L. major. The assay is sensitive enough to quantify parasite load in the absence of overt lesions and reveals a systemic distribution of Leishmania, which has implications for our understanding of the disease.     

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm x 30 cm, 250 A pore size, 5 microm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol-potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8+/-0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.     

Quantitative analysis of lovastatin in capsule of Chinese medicine monascus by capillary zone electrophoresis with UV-vis detector

A capillary zone electrophoresis (CZE) method was developed for the quantitative analysis of lovastatin (Lvt) in capsule of monascus-Chinese medicine. Lvt in the capsule was separated using an electrolyte system consisting of 16% ethanol (v/v) in 60 mM Gly-sodium hydroxide buffer, pH 10.5, 16 kV applied voltage, 238 nm detection wavelength with a capillary of 51 cm x 75 microm i.d (43 cm to detector). Under the optimized conditions, the linear response of Lvt concentration ranges from 4.0 to 240 microg/mL with high correlation coefficient (r=0.9998, n=9), the limits of detection (LOD) and quantification (LOQ) for Lvt are 0.73 and 2.42 microg/mL, the precision values (expressed as R.S.D.) of intra-day and inter-day are 1.40-2.12% and 1.47-3.88%, respectively. The recoveries of the analyte at three concentration levels are 90.28-100.71%. The developed method can be well used for the quantification of Lvt in the drug in commercial formulations.     

Cross-reactive pseudovirus-neutralizing anti-envelope antibodies coexist with antibodies devoid of such activity in persistent hepatitis C virus infection

Most RNA viruses have evolved mechanisms to avoid neutralizing antibody responses, and it is generally believed that variability of envelope-encoding regions is the major molecular basis of this phenomenon. However, it has been hypothesized that other mechanisms can be involved. Recent experimental data indicate that in hepatitis C virus (HCV) infection, the anti-envelope humoral response includes cross-reactive antibody clones able to neutralize vesicular stomatitis virus (VSV) pseudotypes containing HCV E1 and E2 glycoproteins (HCV/VSV pseudotype) as well as other clones devoid of such activity. In this work, we demonstrate that natural infection with a large variety of HCV isolates belonging to different genotypes elicits HCV/VSV pseudotype-neutralizing cross-reactive anti-envelope antibodies together with clones unable to neutralize this pseudovirus. This was shown by designing a novel strategy for quantitation of serum antibodies binding selectively to single viral cross-reactive conformational epitopes. These data can be useful not only for a better understanding of the virus-host interplay in important viral diseases, but also for the development of an effective anti-HCV vaccine.     

应用磁共振生理学成像定量测定兔膝关节软骨退变的研究

目的：探讨磁共振生理学成像技术在检测关节软骨退变中的应用价值。方法：20只新西兰大白兔随机分为甲、乙、丙、丁四组。甲组左膝关节行常规磁共振成像后即刻处死,取股骨髁软骨行苏木素和伊红染色（hematoxylln and eosin,HE）、阿利辛兰染色（alcian blue,AB）及蛋白多糖含量测定。乙、丙、丁各组每只兔左膝关节内注射0．2na（10U）木瓜蛋白酶,并于注射前及注射后分别于24、48、72h先行相同常规磁共振成像及磁化传递对比成像（magnetization transfer contrast。MTC）,后行磁共振延迟增强软骨成像（delayed gadolimum enhanced MRI of cartilage,dGEMRIC）,测定关节软骨磁化传递率和L驰豫时间值。扫描结束后处死动物,取左膝股骨髁部软骨行大体观察、HE、AB染色及蛋白多糖含量测定。结果：注射木瓜蛋白酶后24、48h,蛋白多糖含量与甲组比较,统计学均有差异（P=0．048和0．045,P〈0．05）,注射后72h,统计学没有差异（P=0．455,P〉0．05）。注射木瓜蛋白酶后24、48、72h的T1弛豫率分别降低了316．09ms、244．01ms和143．98ms,注射后24、48h与注射前比较有统计学差异（P=0．047和0．045,P〈0．05）。注射木瓜蛋白酶前后相比,各组关节软骨磁化传递率不同程度地降低,但均没有统计学差异。结论：本研究采用的dGEMRIC、MTC成像技术能够通过定量检测T1弛豫时间值、磁化传递率反映软骨退变早期的生化改变。