共查询到20条相似文献,搜索用时 15 毫秒
1.
A general method for the analysis of asparaginyl-linked (N-linked) carbohydrate moieties of an IgG1 monoclonal antibody is described here. The antibody, rituximab, is a mouse/human chimeric antibody to human CD20 antigen. The glycans present on rituximab are neutral complex biantennary oligosaccharides with zero, one, and two terminal galactose residues (G0, G1, and G2, respectively). To monitor the variation of the glycosylation during manufacture, the glycans were first enzymatically released from the antibody via digestion with peptide-N-glycosidase F, then derivatized with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonic acid and further separated by capillary electrophoresis with laser-induced fluorescence detection. All observed glycans were fully resolved, including the positional isomers of G1. The exact nature of the isomers in terms of the location of the terminal galactose was further characterized via multiple enzymatic digestion steps including mannosidase with activity toward specific Man(alpha 1,3) linkage. The optimization and several key parameters, i.e., enzymatic digestion and derivatization, in the assay development will be discussed. Moreover, to ensure that the assay can be used in routine lot release testing, the assay was validated and found to be accurate and precise. The analytical approach described is suitable for characterization as well as routine testing of the N-linked glycan content in any IgG1 monoclonal antibody and glycoproteins in general. 相似文献
2.
Zhu C He X Kraly JR Jones MR Whitmore CD Gomez DG Eggertson M Quigley W Boardman A Dovichi NJ 《Analytical chemistry》2007,79(2):765-768
In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300. 相似文献
3.
Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange. 相似文献
4.
A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data. 相似文献
5.
The sheath-flow cuvette is a key component in a high-sensitivity post-column laser-induced fluorescence detector for capillary electrophoresis. Most designs are based on commercial cuvettes originally manufactured for use in a flow cytometer. In these devices, a quartz flow chamber is held in a stainless-steel fixture that is difficult to machine and subjects the cuvette to a torque when sealed, which frequently leads to damage of the flow chamber. In this report we present a design for a cuvette that may easily be constructed. This design uses compression to hold and seal the quartz flow chamber without applying torque. The system produces detection limits (3sigma) of 115 yoctomoles (70 copies) for FQ-labeled carbonic anhydrase. 相似文献
6.
The violet (415 nm) diode laser is used for indirect laser-induced fluorescence detection in capillary electrophoretic separations of inorganic anions and chemical warfare agent degradation products. Inorganic anions were detected using 8-hydroxypyrene-1,3,6-trisulfonic acid as the indirect probe and achieved submicromolar (40-80 ppb) detection limits in a 2-min separation. The chemical warfare agent degradation products methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate were detected using the porphyrin tetrakis(4-sulfophenyl)porphine as the indirect probe and achieved detection limits of 0.1 microM (9 ppb), which are 1 order of magnitude better than that achieved using indirect UV detection. Baseline stability achieved with the violet diode laser was excellent, with dynamic reserve (DR) values of > 1000, which are 15 times better than that achieved using an unstabilized HeCd laser. 相似文献
7.
Nichkova M Feng J Sanchez-Baeza F Marco MP Hammock BD Kennedy IM 《Analytical chemistry》2003,75(1):83-90
An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 microm) produced by a piezoelectric generator system with 10-microm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 microg L(-1) reaching a LOD of 0.04 microg L(-1). The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 microg L(-1) in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 ,g L(-1), with a dynamic range between 4 and 149.5 microg L(-1) in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment. 相似文献
8.
Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis. 相似文献
9.
Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization 总被引:3,自引:0,他引:3
Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA. 相似文献
10.
Duffy CF Gafoor S Richards DP Admadzadeh H O'Kennedy R Arriaga EA 《Analytical chemistry》2001,73(8):1855-1861
Individual liposome measurements by capillary electrophoresis with postcolumn laser-induced fluorescence detection facilitated the determination of liposome property distributions, two-dimensional plots, and an improved characterization of a liposomal preparation. This advancement in liposome analysis was feasible by using a high-sensitivity postcolumn laser-induced fluorescence detector wired for millisecond response. For each individual liposome containing fluorescein, peak height and migration time were determined. From these measurements the individual entrapped volumes and electrophoretic mobilities were determined. Distribution analysis of these properties facilitated comparison of various liposome dilutions and indicated that the method is reproducible and unaffected by the density of liposomes (10(7)-10(9) liposomes/mL) in the suspension. Furthermore, liposomes showed entrapped volumes that vary from 0.3 to 13 fL with apparent radius varying from 370 nm to 1.8 microns. Two-dimensional plots of reduced mobility versus kappa R (Debye parameter x liposome radius) revealed that the liposomes resuspended from a dried film of phospholipids are heterogeneous in regard to the surface charge density of individual liposomes. The described method has the potential of becoming a new tool for characterization of commercial liposomal preparations and theoretical studies. 相似文献
11.
Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis 总被引:1,自引:0,他引:1
Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells. 相似文献
12.
Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies. 相似文献
13.
On-line focusing of flavin derivatives using Dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection 总被引:1,自引:0,他引:1
Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes. 相似文献
14.
This technical note describes a detector capable of simultaneously monitoring scattering and fluorescence signals of individual particles separated by capillary electrophoresis. Due to its nonselective nature, scattering alone is not sufficient to identify analyte particles. However, when the analyte particles are fluorescent, the detector described here is able to identify simultaneously occurring scattering and fluorescent signals, even when contaminating particles (i.e., nonfluorescent) are present. Both fluorescent polystyrene particles and 10-nonyl acridine orange (NAO)-labeled mitochondria were used as models. Fluorescence versus scattering (FVS) plots made it possible to identify two types of particles and a contaminant in a mixture of polystyrene particles. We also analyzed NAO-labeled mitochondria before and after cryogenic storage; the mitochondria FVS plots changed with storage, which suggests that the detector reported here is suitable for monitoring subtle changes in mitochondrial morphology that would not be revealed by monitoring only fluorescence or scattering signals. 相似文献
15.
Essaka DC White J Rathod P Whitmore CD Hindsgaul O Palcic MM Dovichi NJ 《Analytical chemistry》2010,82(23):9955-9958
The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique. 相似文献
16.
Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection 总被引:3,自引:0,他引:3
Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values. 相似文献
17.
Kuningas K Ukonaho T Päkkilä H Rantanen T Rosenberg J Lövgren T Soukka T 《Analytical chemistry》2006,78(13):4690-4696
We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches. 相似文献
18.
3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde has been utilized as a precolumn derivatization agent for various amino sugars. Constituents of various biological mixtures can be converted to highly fluorescent isoindole derivatives, separated by high-performance capillary electrophoresis and determined at attomole (10(-18) mol) levels by a laser-induced fluorescence detector. This method has been applied to the analysis of monosaccharides and acid-hydrolyzed polysaccharides. Carbohydrate moieties derived from a glycoprotein were also tagged and determined. 相似文献
19.
Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [...GGCGCGCAG...] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with (32)P end labeling. 相似文献
20.
Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A0, and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A0 reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A0 for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage. 相似文献