Bacteriophage and impurity carryover and total organic carbon release during extended protein A chromatography

In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na₂SO₄ with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.     

Accumulation and trapping of hepatitis A virus particles by electrohydrodynamic flow and dielectrophoresis

Hepatitis A virus particles (d = 27 nm) were successfully accumulated and trapped in a microfluidic system by means of a combination of electrohydrodynamic flow and dielectrophoretic forces. Electric fields were generated in a field cage consisting of eight microelectrodes. In addition, high medium conductance (0.3 S/m) resulted in sufficient Joule heating and the corresponding spatial variation of temperature, density, and permittivity to induce electrohydrodynamic flow in the vicinity of the field cage. Flow vortices transport particles toward the center of the field cage, where dielectrophoretic forces cause permanent entrapment and particle aggregation. Spatial distribution of temperature, density, and permittivity as well as resulting flow patterns were modeled numerically and are in good agreement with experimental results. This accumulation scheme might be applicable to sample concentration enhancement in biosensor applications.     

基于拉曼光谱的PE膜包装食用农产品品质检测误差的校正方法

透明保鲜膜包装以其方便、经济、卫生的特点被广泛应用于超市卖场及日常生活中,却也给食品农产品的检测增加了难度,所以急需一种能够隔透明薄膜无损检测农产品的方法。叶绿素是影响黄瓜品质的重要因素之一,该研究主要探讨了拉曼技术检测黄瓜品质时,食品保鲜膜层数对叶绿素特征峰的影响,并通过对菠菜、油菜和芹菜对得出的结论进行验证。采用点检测拉曼光谱系统,采集无保鲜膜包装以及多层包装下黄瓜的拉曼光谱。拉曼信号经Savitzky-Golay(SG) 5点平滑和自适应迭代惩罚最小二乘法扣除荧光背景,探究不同层数(1~6层)的PE保鲜膜对黄瓜叶绿素特征峰的影响。采集被保鲜膜覆盖的黄瓜的拉曼信号并处理,即可建立叶绿素的特征峰(1 158和1 528 cm-1)减少值与保鲜膜层数之间的变化规律,并对规律的预测效果进行评价。随着保鲜膜层数的增加,透明包装层数与黄瓜叶绿素特征峰强度之间呈现良好的线性相关关系。提取出单层透明包装对叶绿素在1 158和1 528 cm-1处特征峰强度的降低值分别为81.4和103.1,分别占未加保鲜膜时叶绿素特征峰强度值的7.98%和8.56%,多组验证结果的相关系数达到0.95以上,验证的相关系数达到0.94。随着样品浓度增高,线性递减效果越明显。作为验证,通过透明保鲜膜检测菠菜、油菜和芹菜叶片中的叶绿素表明,每加一层膜叶绿素在1 158和1 528 cm-1处的特征峰强度会比覆膜前分别降低7.9%～8.6%和8.1%～8.6%;导致检测信号强度呈线性降低是由于保鲜膜使激发光分散不聚焦导致,保鲜膜成分对检测结果无影响。该研究为拉曼光谱隔透明包装检测拉曼光谱特征峰强度,进而获得农产品品质信息提供了一种新的分析方法。     

Unraveling virus identity by detection of depleted probes with capillary electrophoresis

With the emergence of new viral infections and pandemics, there is a need to develop faster methods to unravel the virus identities in a large number of clinical samples. This report describes a virus identification method featuring high throughput, high resolution, and high sensitivity detection of viruses. Identification of virus is based on liquid hybridization of different lengths of virus-specific probes to their corresponding viruses. The probes bound to target sequences are removed by a biotin–streptavidin pull-down mechanism and the supernatant is analyzed by capillary electrophoresis. The probes depleted from the sample appear as diminished peaks in the electropherograms and the remaining probes serve as calibrators to align peaks in different capillaries. The virus identities are unraveled by a signal processing and peak detection algorithm developed in-house. Nine viruses were used in the study to demonstrate how the system works to unravel the virus identity in single and double virus infections. With properly designed probes, the system is able to distinguish closely related viruses. The system takes advantage of the high resolution feature of capillary electrophoresis to resolve probes that differ by length. The method may facilitate virus identity screen from more candidate viruses with an automated 4-color DNA sequencer.     

Comparison of Electrochemical and Spectro-Photometric Detection in Hrp-Based Elisa for Tobacco Mosaic Virus

ABSTRACT

An electrochemical method was compared with a spectrophotometric method using a horseradish peroxidase (HRP)-based indirect enzyme-linked immunosorbent assay (ELISA) with direct antigen coating (DAC) technique for the determination of tobacco mosaic virus (TMV). In substrates for a spectrophotometric HRP-based ELISA method, was selected as the substrate for the electrochemical method as well as the spectrophotometric method. 2, 3-Diaminophenazine is the product of the oxidation of OPD with H₂O₂ catalyzed by HRP in the selected conditions. It is electroactive and has a sensitive voltammetric response at -0.93 V (vs. Ag/AgCl) in alkaline solution. The detection limit of free HRP, by second-order derivative linear-sweep voltammetry, is 1.1 mU/L. This method can be further used to detect TMV antigen. The peak current is linear with TMV concentration in the range of 0.25-5.0 ng/mL. The detection limit for TMV is 0.25 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1:102400. Compared with the classical OPD ELISA spectrophotometric method, the detection limits of the electrochemical method for the clarified TMV and the infected leaf sap detection are about ten and four times lower, respectively.     

Transport of Viruses Through Saturated and Unsaturated Columns Packed with Sand

Laboratory-scale virus transport experiments were conducted in columns packed with sand under saturated and unsaturated conditions. The viruses employed were the male-specific RNA coliphage, MS2, and the Salmonella typhimurium phage, PRD1. The mathematical model developed by Sim and Chrysikopoulos (Water Resour Res 36:173–179, 2000) that accounts for processes responsible for removal of viruses during vertical transport in one-dimensional, unsaturated porous media was used to fit the data collected from the laboratory experiments. The liquid to liquid–solid and liquid to air–liquid interface mass transfer rate coefficients were shown to increase for both bacteriophage as saturation levels were reduced. The experimental results indicate that even for unfavorable attachment conditions within a sand column (e.g., phosphate-buffered saline solution; pH = 7.5; ionic strength = 2 mM), saturation levels can affect virus transport through porous media.     

基于反射光谱的温室黄瓜叶片磷素含量分析与预测

以温室栽培黄瓜作物为对象,分析了叶片反射光谱与叶片磷素含量之间的相关关系,并建立了预测模型。首先利用便携式光谱辐射仪测量了自然光照条件下温室黄瓜叶片的光谱反射率,并计算了反射率光谱的一次微分光谱。相关分析表明反射率光谱与叶片含P量之间具有一定的相关性,但线性相关不显著。利用微分光谱可以部分消除系统误差、背景噪声等的影响,明显提高了相关系数,但预测模型精度仍然达不到实用程度。在理论分析的基础上,选取978,920,737和458 nm等4个波长作为特征波长,分别利用人工神经网络和支持向量机建立了黄瓜叶片P素含量对应于微分光谱特性的非线性预测模型,结果表明两种算法都获得了较好的预测效果,支持向量机模型的预测能力(R_v=0.754)优于人工神经网络模型(R_v=0.712)。     

镶嵌型乳胶粒子的合成

以苯乙烯、甲基丙烯酸甲酯、丙烯腈等单体或它们的混合物为硬单体,天然胶乳为弹性组分,经多步种子乳液聚合法制得了在天然胶乳的粒子上镶嵌硬聚合物相的互穿网络型乳胶粒子.考察了十二烷基硫酸钠、十二烷基苯磺酸钠、壬基酚聚氧乙烯醚、油酸等乳化体系,过硫酸钾、过氧化苯甲酰热引发体系及异丙苯过氧化氢-四乙烯五胺、叔丁基过氧化氢-四乙烯五胺等氧化还原引发体系对聚合反应的影响.研究了交联剂用量对互穿结合率、溶胶含量的影响及溶胀时间、硬单体组成、乳化剂种类对乳胶粒子形态的影响,确定了适宜的聚合配方和工艺条件.透射电镜观察乳粒形态结果表明,单一使用极性或非极性单体,仅得到核-壳结构乳液,而采用不同极性单体复合、溶胀、互穿,得到的是镶嵌硬聚合物型乳粒结构.     

二十面体病毒及其衣壳蛋白质

许多病毒具有二十面体结构。本文对已经发现的各种二十面体病毒进行了分类,分析了二十面体病毒衣壳的结构特征,阐述了二十面体病毒的结构蛋白。