Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT

A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.     

痕量糖-8-氨基芘-1,3,6-三磺酸衍生物的毛细管电泳-激光诱导荧光分析

以芘为原料经磺化、氨化合成了８－氨基芘－１,３,６－三磺酸钠盐（ＡＰＴＳ）,建立了相应的纯化方法,考察了多种条件对还原单糖衍生效率的影响,研究单糖及葡聚糖水解寡糖－ＡＰＴＳ衍生物的毛细管电泳－激光诱导荧光分离与检测,单糖的检测限在ａｍｏｌ（１０＾－１８）水平。     

毛细管电泳分离-激光诱导荧光检测生物胺

采用新合成的6-氧-[(1-琥珀酰亚胺)氧酰甲基]-荧光素乙酯(SOFE)作为柱前衍生试剂,毛细管电泳分离-激光诱导荧光检测了乙醇胺(EOA)、组胺(His)、甲胺(MA)、乙胺(EA)、酪胺(TYR)及苯乙胺(PEA)6种生物胺。在硼酸-硼砂缓冲溶液(pH8.5)中,室温(25℃)下衍生10min。用pH8.5的含25mmol/LSDS15%(V/V)乙腈的40mmol/L硼酸盐溶液作为电泳缓冲溶液,6种衍生物在15min内完全分离,检出限在2.5×10-11～8×10-11mol/L之间。该方法应用于酱油中生物胺的测定,结果令人满意。     

532nm激光诱导荧光毛细管阵列电泳仪的研制

基于共聚焦激光诱导荧光检测技术,研制了一台旋转扫描高效毛细管阵列电泳装置。以波长为532nm的半导体二极管激光器作为激发光源,多根毛细管阵列采用圆形布局,微型高速直流电机带动旋转反射镜进行激光扫描,加快了数据采集的速度;采用旋转编码器实现了毛细管的定位与位置读出,并触发数据采集系统进行荧光信号采集。以罗丹明6G和罗丹明B为分析样品,考察了16通道毛细管阵列电泳仪的基本性能。     

532 nm激光诱导荧光毛细管阵列电泳仪的研制

基于共聚焦激光诱导荧光检测技术,研制了一台旋转扫描高效毛细管阵列电泳装置.以波长为532nm的半导体二极管激光器作为激发光源,多根毛细管阵列采用圆形布局,微型高速直流电机带动旋转反射镜进行激光扫描,加快了数据采集的速度;采用旋转编码器实现了毛细管的定位与位置读出,并触发数据采集系统进行荧光信号采集.以罗丹明6G和罗丹明B为分析样品,考察了16通道毛细管阵列电泳仪的基本性能.     

Determination of Amino Acids in an Individual Erythrocyteby Capillary Electrophoresis with Intracellular FITC-derivatization and Laser-induced Fluorescence Detection

A novel approach for analysis of amino acids in individual erythrocytes was established.In this method, the derivatization reagent was in~oduced into the living cells by electroporation.After derivatization, the amino acids in a single cell were determined by capillary electrophoresis with laser-induced fluorescence detection.     

Quantification of Neurotransmitter Amino Acids in Human Serum by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is developed as a simple and sensitive method for the quantification of arginine (Arg), tyrosine (Tyr) and glutamic acid (Glu) in human serum. The separation conditions and the derivatization conditions with fluoresceinisothiocyanate (FITC) were investigated. Regression equations revealed a linear relationship (correlation coefficients: 0.9927–0.9998) between the peak area and concentration of each analyte. For the amino acids detected, 10^–10M detection limits were reached, and the levels of these amino acids in human serums were easily determined with recoveries of 93.5–106.5%.     

毛细管电泳-激光诱导荧光测定大鼠血浆内痕量氨基酸类神经递质

用菁色素衍生物 (Cy5 )作为荧光衍生试剂 ,毛细管电泳结合激光诱导荧光分离测定痕量氨基酸类神经递质甘氨酸和谷氨酸。讨论了Cy5浓度 ,缓冲液浓度 ,pH值和反应时间等实验条件对衍生和分离的影响。衍生物的荧光强度和氨基酸的浓度在 7.5× 1 0 - 8～ 3 .5× 1 0 - 6 mol L范围内呈现良好的线性 ,线性相关系数均达到 0 .999。甘氨酸和谷氨酸的最低检出限浓度分别为 3 .3× 1 0 - 9mol L和 1 .0×1 0 - 9mol L。测定了大鼠血浆中甘氨酸和谷氨酸的浓度 ,分别为 (62± 1 4 )× 1 0 - 6 mol L和 (1 9± 2 )× 1 0 - 6 mol L。与其它氨基标记荧光试剂 ,如FITC和TRITC相比 ,Cy5与一级胺反应时间短 ,选择性高     

Capillary Electrophoretic Immunoassay with Laser-induced Fluorescence Detection for Interferon-gamma

Capillary electrophoretic immunoassay with laser-induced fluorescence detection for recombinant human interferon-gamma (IFN-γ) was established. The limits of detection for three forms of IFN-γ are 6.9ng/L, 5.7ng/L and 5.0ng/L, respectively.     

毛细管电泳免疫分析法分析雌三醇

利用一种温敏水凝胶作为毛细管电泳的填充介质,建立了毛细管电泳免疫分析血清中雌三醇的方法。研究了缓冲溶液的浓度和pH值、水凝胶的浓度、电压等因素对分析结果的影响。雌三醇的检出限和线性范围分别为31．6ng／L和50-5000ng／L。     

毛细管电泳-激光诱导荧光用于中药制剂中氨基酸成分的分析

建立了一种用于测定中药制剂中氨基酸成分的毛细管电泳-荧光检测方法. 用含有α-环糊精(α-CD)的硼砂缓冲溶液为背景电解质, 经异硫氰酸荧光素(FITC)衍生的5种氨基酸在50 min内可以得到很好的分离和测定. 考查了各个分离参数对分离的影响, 得到的优化条件为: 含45 mmol/L的α-环糊精的80 mmol/L硼砂缓冲溶液(pH值9.2)作为背景电解质, 分离电压20 kV; 柱温22 ℃. 衍生试剂FITC与单个氨基酸的化学计量比为4∶1时, 能够获得稳定荧光强度的氨基酸衍生物. 在优化条件下, 各氨基酸成分在73.5～2900 nmol/L 的浓度范围内呈良好的线性关系(相关系数r2为0.9906～0.9998). 保留时间和峰面积的相对标准偏差分别为0.8%～3.0%和0.7%～5.7%, 检测限(3倍信噪比)为3.5～35 nmol/L. 该方法准确可靠, 可用于质量控制为目的的中药制剂中氨基酸成分的定量测定.     

Derivatization of Hydrophobic Amino Acids in Nonaqueous Media and Separation by Nonaqueous Capillary Electrophoresis with Laser-Induced Fluorescence Detection

A method of nonaqueous capillary electrophoresis was established for the separation and determination of alanine, phenylalanine, isoleucine and valine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol in nonaqueous media. The derivatization and separation conditions were optimized. The optimum derivatization conditions were 70 min for the reaction time, 55 °C for the reaction temperature and 20 mM ammonium acetate in methanol for the derivatization buffer. The most suitable running buffer was composed of 60 mM ammonium acetate, 10% acetonitrile in methanol with a fused-silica capillary column (47 cm × 75 μm i.d.), 20 kV applied voltage and 20 °C capillary temperature. The correlation coefficients were better than 0.995 in the investigated concentration ranges. The relative standard deviation (R.S.D.) (n = 5) of the migration times and peak areas were 2.0–4.3% and 1.9–4.5%, respectively. The method was applied to the determination of four compounds in two compound amino acid injections, with the exception of phenylalanine, the recoveries for the other three compounds ranging between 88–116%.     

蛋白质样品的非竞争性毛细管电泳免疫分析激光诱导荧光检测

以牛血清白蛋白与其单克隆抗体为样品,初步研究了蛋白质的非竞争性毛细管电泳免疫分析方法。混合温育可以在２０ｍｉｎ内达到平衡,电泳分离在１２ｍｉｎ内完成。当示踪物浓度为３２０ｎｍｏｌ／Ｌ时,所得到的标准曲线的线性范围为８￣１５０ｎｍｏｌ／Ｌ,检出限为５ｎｍｏｌ／Ｌ,并考察了缓冲溶液的浓度、ｐＨ、分离电压等因素的影响。     

毛细管电泳发光二极管诱导荧光对免疫球蛋白G的检测

采用自行设计、组装的毛细管电泳光导纤维发光二极管诱导荧光检测装置,建立了一种直接测定免疫球蛋白G(IgG)的方法。以蓝色发光二极管(LED)为荧光检测器的激发光源,荧光素异硫氰酸酯(FITC)为柱前衍生试剂,采用毛细管区带电泳,以20 mmol/L硼砂缓冲溶液(pH9.2)为背景电解液进行分离检测。通过对衍生反应条件和电泳分离条件进行优化,确定了最佳实验条件,在该条件下,IgG的线性范围为4.5×10-8~1.2×10-6g/L,检出限为2.0×10-8g/L。该方法简单、高效、选择性好,无需前处理,可用于人血清中IgG含量的测定。     

激光诱导荧光检测微流控芯片生化分析仪的研制

研制出一种集激光诱导荧光检测、微流控芯片电泳及控制系统于一体的生化分析仪。分析仪内采用可重复使用的玻璃基质微流控芯片,利用销式固定技术实现芯片的精确定位,定位精度达到±2μm。以四触点高电压系统控制芯片上的进样和电泳分离操作。激光诱导荧光检测系统采用正交光路模式,对Cy5染料的检出限达到1.0×10-10mol/L(S/N=3)。以羟乙基纤维素为筛分介质,初步进行了ΦΧ174-HaeШdigest DNAmarker限制性片段的毛细管电泳分离。     

毛细管电泳手性分离-激光诱导荧光检测普萘洛尔对映体

用异硫氰酸荧光素衍生普萘洛尔,衍生产物用毛细管电泳分离,激光诱导荧光方法检测。在含有10mmol/L-βCD,50mmol/L硼砂(pH=9.0)的溶液中,普萘洛尔对映体得到了基线分离。本方法线性范围宽(R型:2.0×10-3~1mg/L),灵敏度高(检出限均为1.24μg/L),可用于活体中普萘洛尔对映体的药代动力学研究。     

毛细管电泳法测定尿中的姜黄素

采用高效毛细管电泳法测定人尿中姜黄素的含量,建立了一种测定体液中姜黄素的简便的分析方法。所用缓冲溶液为15 mmol/L的硼砂溶液(pH 9.24),运行电压为20 kV,检测波长262 nm。姜黄素的峰面积与其质量浓度的线性关系良好,其线性范围为10~300 mg/L,健康人尿中姜黄素的4个浓度水平的添加回收率均大于96.3%,相对标准偏差(RSD）小于2.3%。该法灵敏、快速、准确,可用于体液中姜黄素的批量检测。     

Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

A new method for rapidly detecting restriction enzyme patterns of Mycobacterium DNA using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439-bp fragment of a 65,000-kDa (M _r) heat shock protein gene (hsp65) of Mycobacterium. After digesting amplification products by BstEII and HaeIII, patterns of enzyme cleavage products were detected by both CE-LIFD and agarose gel electrophoresis (AGE), respectively. Experimental parameters of CE were optimized. Restriction enzyme patterns of Mycobacterium DNA were detected in optimum electrophoresis conditions: a coated capillary column with a length of 50 cm and an internal diameter of 100 μm, an electrophoresis buffer of 45 mmol/l Tris-boric acid-ethylenediaminetetraacetic acid, and a running voltage of 11 kV. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of DNA segments were <3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means to DNA restriction enzyme pattern analysis. Translated from the Chinese Journal of Chromatography, 2005, 23(1) (in Chinese)     

A New Immunoassay Method by Capillary Electrophoresis with Enhanced Chemiluminescence Detection

Immunoassay based on capillary electrophoresis has recently been paid a great attention due to its excellent separation power, high analysis speed, small sample volume requirements, and automation 1,2. Since the injection volume is too small, immunoassay by CE needs an extremely sensitive detector. Laser-induced fluorescence3 and electro-chemical detections 4 have been used for this purpose. In fact chemilumi- nescence (CL) is also a highly sensitive method for detection, and is widely used …