Capillary Electrophoresis Analysis Research Articles

Identification of IgH gene rearrangement and immunophenotype in an animal model of Epstein-Barr virus-associated lymphomas.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma. Because the susceptible hosts of EB virus are limited to human and cotton-top tamarins (Saguinus oedipus), there have been no appropriate animal models until the lymphoma model induced by EBV in human peripheral blood lymphocyte (hu-PBL)/SCID chimeric mice was reported. However, it is still controversial whether the EBV-associated lymphoma induced in hu-PBL/SCID mice is a monoclonal tumor. In this study, we transplanted normal human peripheral blood lymphocytes (hu-PBL) from six donors infected with EBV into SCID mice to construct hu-PBL/SCID chimeric mice. The induced tumors were found in the mediastinum or abdominal cavity of SCID mice. Microscopic observation exhibited tumor cells that were large and had a plasmablastic, centroblastic or immunoblastic-like appearance. Immunophenotyping assays showed the induced tumors were LCA-positive, CD20/CD79a-positive (markers of B cells), and CD3/CD45RO-negative (markers of T cells). A human-specific Alu sequence could be amplified by Alu-PCR. This confirmed that induced tumors were B-cell lymphomas originating from the transplanted human lymphocytes rather than mouse cells. EBER in situ hybridization detected positive signals in the nuclei of the tumor cells. Expression of EBV-encoded LMP1, EBNA-1, and EBNA-2 in the tumors was significantly positive. PCR-based capillary electrophoresis analysis of IgH gene rearrangement revealed a monoclonal peak and single amplification product in all six cases of induced tumors. This indicated that EBV can induce monoclonal proliferation of human B lymphocytes and promotes the development of lymphoma. J. Med. Virol. 88:1804-1813, 2016. © 2016 Wiley Periodicals, Inc.

Impact of capillary conditioning and background electrolyte composition on capillary electrophoresis analysis of prostate specific antigen isoforms

Glycoproteins expressed in the human body can experience modifications as result of pathological situations. Detection of those changes can be useful as disease biomarkers. As a result of these modifications, size and/or electrical charge of the glycoprotein can be altered. Migration in capillary zone electrophoresis (CZE) is governed by the size to charge ratio of the analyte and therefore this separation technique can be used to monitor those modifications. At its turn, the alteration of the electrophoretical pattern of a given glycoprotein could be used as disease biomarker. To this aim, high repeatability for separation of a large number of peaks for a given glycoprotein is desirable.For prostate cancer, new markers are needed to decrease the high number of false positive results provided by the biomarkers currently used in clinics. In this sense, CZE methods for analysis of the several prostate specific antigen (PSA) peaks which this glycoprotein exhibit, called isoforms and containing one or more glycoforms, could be useful to study the PSA pattern as prostate cancer marker.In this study two complementary strategies to achieve both lot-to-lot capillary repeatability and high resolution of a large number of PSA isoforms are developed. Better performance and precision have been obtained for capillaries conditioned with HCl than for those conditioned with NaOH. Optimization of the background electrolyte (BGE) pH value to 8.0 and inclusion of 3M urea on its composition were the two factors of highest impact for enhancing resolution of the highest number of PSA peaks. Under the optimized conditions for capillary conditioning and BGE pH and composition, long-term resolution of 10 isoforms of PSA was achieved. Inter-day (n=3) %RSD was 0.55 for the ratio tm/tEOF, 1.15 for μeff, and 5.02 for % Acorr of the PSA peaks.

The potential of SNP-based PCR-RFLP capillary electrophoresis analysis to authenticate and detect admixtures of Mediterranean olive oils.

Authentication and traceability of extra virgin olive oil is a challenging research task due to the complexity of fraudulent practices. In this context, the monovarietal olive oils of Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) require new tests and cutting edge analytical technologies to detect mislabeling and misleading origin. Toward this direction, DNA-based technologies could serve as a complementary to the analytical techniques assay. Single nucleotide polymorphisms are ideal molecular markers since they require short PCR analytical targets which are a prerequisite for forensic applications in olive oil sector. In the present study, a small number of polymorphic SNPs were used with an SNP-based PCR-RFLP capillary electrophoresis platform to discriminate six out of 13 monovarietal olive oils of Mediterranean origin from three different countries, Greece, Tunisia, and Lebanon. Moreover, the high sensitivity of capillary electrophoresis in combination with the DNA extraction protocol lowered the limit of detection to 10% in an admixture of Tsounati in a Koroneiki olive oil matrix.

Antinociceptive effect of hydroalcoholic extract and isoflavone isolated from Polygala molluginifolia in mice: evidence for the involvement of opioid receptors and TRPV1 and TRPA1 channels

PurposeThe plants of the genus Polygala (Polygalaceae) have been used for a long time in folk medicine to treat pain and inflammation. The species Polygala molluginifolia is native to southern Brazil and is popularly known as “cânfora”. The presented study analyzes the antinociceptive effect of hydroalcoholic extract from Polygala molluginifolia (HEPm) and an isoflavone (ISO) isolated from the extract, in behavioral models of pain in mice, as well as the mechanism underlying this effect. Materials and methodsThe phytochemical analysis of HEPm was performed through a capillary electrophoresis analysis and colorimetric test. The antinociceptive effects of HEPm and ISO (10–1000mg/kg, i.g.) were evaluated by applying the formalin test; mechanical and thermal hyperalgesia to postoperative pain in mice. The possible involvement of opioid receptors, TRPV1 and TRPA1 channels in the antinociceptive effect of HEPm and ISO were also evaluated. Finally, the nonspecific effects of HEPm and ISO were evaluated by measuring locomotor activity (Open-field Test) and corporal temperature. ResultsThe 5,3′,4′-trihydroxy-6″,6″-dimethylpyrano[2″,3″:7,6] isoflavone (ISO) was identified in HEPm by capillary electrophoresis analysis and selected for the experimental tests. The oral administration of HEPm or of ISO significantly inhibited the neurogenic and inflammatory phases of formalin-induced pain, edema formation and local hyperemia, without causing any change to locomotor activity. Acute and repeated treatment of animals with HEPm reduced mechanical and thermal (heat and cold) hyperalgesia in the postoperative pain. In addition, administering HEPm or ISO markedly reduced nociceptive behavior induced by the peripheral and central injection of TRPV1 and TRPA1 channels activators. Finally, the antinociception provided by the administration of HEPm or ISO was reversed by the preadministration of naloxone. ConclusionsTaken together, these results provide the first experimental evidence of the significant antinociceptive effect of HEPm and ISO in animal models of acute pain without causing sedation or locomotor dysfunction. This effect appears to be mediated, at least in part, by the activation of opioid receptors and/or by the inhibition of TRPV1 and TRPA1 channels. Moreover, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant Polygala molluginifolia in the development of phytomedicines with analgesic properties.

On‐chip quantitative‐PCR using integrated real‐time detection by capillary electrophoresis

Electrophoresis 2016, 37, 545–552. DOI: 10.1002/elps.201500298 Quantitative polymerase chain reaction (qPCR) and capillary electrophoresis (CE) were integrated into a microfluidic chip that was designed to achieve real-time amplicon sampling, separation, and quantitation without requiring various probes. A novel channel design and PCR-CE synchronization scheme allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on-chip qPCRCE method was demonstrated using model assay protocols to detect phiX174 bacteriophage and E. coli genomic DNA. By combining the separation capability of CE and the amplification power of PCR assay, this method is expected to find applications in awide variety of infectious disease detection and monitoring assays.

Quantification of major camel milk proteins by capillary electrophoresis

Proteins from dromedary camel milk (CM) produced in Europe were separated and quantified by capillary electrophoresis (CE). CE analysis showed that camel milk lacks β-lactoglobulin and consists of high concentration of α-lactalbumin (2.01 ± 0.02 mg mL−1), lactoferrin (1.74 ± 0.06 mg mL−1) and serum albumin (0.46 ± 0.01 mg mL−1). Among caseins, the concentration of β-casein (12.78 ± 0.92 mg mL−1) was found the highest followed by α-casein (2.89 ± 0.29 mg mL−1) while κ-casein represented only minor amount (1.67 ± 0.01 mg mL−1). These results were in agreement with sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns. Overall, CE offers a quick and reliable method for the determination of major CM proteins, which may be responsible for the many nutritional and health properties of CM.

Ribosomal RNA analysis in the diagnosis of Diamond-Blackfan Anaemia.

Diamond-Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a 'ribosomopathy'. We report a multi-centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool.

Typing Method for the QUB11a Locus of Mycobacterium tuberculosis: IS6110 Insertions and Tandem Repeat Analysis.

QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis of Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp) that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments (>1,200 bp) investigated contained IS6110 insertions and varied in the number of repeats (18 patterns) and location of IS6110 insertions. This method allows VNTR analysis with high discrimination.

A Systematic Procedure to Develop a Capillary Electrophoresis Method Using a Minimal Experimental Data

The choice of an appropriate background electrolyte (BGE) and its components for capillary electrophoresis analysis is the main step in capillary electrophoresis method development. The use of an inadequate co-ion component could lead to asymmetrical peaks and selecting an inappropriate counter-ion could affect the buffer capacity and the pH of the BGE, leading to unreliable analysis. In this paper, we describe a systematic procedure for the development of a capillary electrophoresis method, based on the effect of varying pH on the ion effective mobility, to optimize the BGE composition. The method was applied to the separation of L-ascorbic acid in different samples. The optimized background electrolyte composition was 40 mmol L-1 tris(hydroxymethyl)aminomethane and 20 mmol L-1 2-morpholinoethanesulfonic acid, at pH 8.1. Sorbic acid was used as the internal standard and separation was carried out in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 µm inner diameter), with a short-end-injection configuration and direct ultraviolet (UV) detection at 266 nm. The separation was performed in 26 s. The method shows good linearity (R2 > 0.999), excellent values for inter-day and intra-day precision and good recovery (in the range of 94-107%). The values obtained for limit of detection (LOD) and limit of quantification (LOQ) were 0.14 and 41 mg L-1, respectively. The systematic procedure applied shows to be a very useful tool for the first step method development for capillary electrophoresis.

Antioxidant Activity of Uva di Troia Canosina: Comparison of Two Extraction Methods

Two extraction methods: microwave-assisted extraction (MAE) and extraction by convection heating were used to assess the active antioxidant ingredients from Uva di Troia Canosina grape pomace. MAE gave higher yields in the recovery of resveratrol (70 µg/g versus 20 µg/g) as assessed by capillary electrophoresis (CE) analysis. The MAE was also advantageous as it allowed reduced extraction times: 15 minutes versus 30 minutes required by conventional heating extraction. However, both methods gave extracts endowed with comparable antioxidant activity as demonstrated by the 2,2-diphenyl-1- picrylhydrazyl radical (DPPH) scavenging assay (IC50 = 106±48 µg/mL versus 110±34 µg/mL, respectively). The observed antioxidant effect of the MAE extract appears to be correlated with the detected resveratrol concentration while other unidentified antioxidants may contribute to the radical scavenging effect of the extract obtained by convection heating. In agreement with previous results, our study underlays the possibility that the antioxidant properties of Uva di Troia Canosina grape could be attributed to resveratrol along with other polyphenols. Considering the multifunctional medicinal and health effects of resveratrol, the MAE method herein proposed may be considered as an efficient way to obtain resveratrol from Uva di Troia Canosina grapes. Keywords: Capillary electrophoresis, DPPH, environment, microwave, polyphenols, resveratrol, Uva di Troia.

Development and validation of CALR mutation testing for clinical diagnosis.

To validate a diagnostic assay for detecting CALR mutations in the clinical setting. Traditional polymerase chain reaction (PCR) was performed on DNA previously extracted from 60 specimens (30 bone marrow aspirates [BMAs] and 30 peripheral blood [PB] samples) from 55 patients. Nearly all reported CALR mutations are insertions or deletions in exon 9. Therefore, we performed amplicon sizing by capillary electrophoresis and fragment length analysis (FLA) to determine mutation status. Mutations were confirmed by Sanger sequencing. Fourteen samples from 10 patients with JAK2 and MPL wild-type myeloproliferative neoplasms were positive for CALR mutation. Detected mutations included a 52-base pair (bp) deletion (n = 6), a 5-bp insertion (n = 2), a 31-bp deletion (n = 1), and a 61-bp deletion (n = 1). Sanger sequencing of 15 samples showed 100% concordance. Matched patient PB and BMA samples (n = 5) harbored identical mutations, and samples run multiple times (n = 8) showed 100% reproducibility. We conclude that CALR mutations may be quickly and accurately detected by FLA of PCR amplicons by capillary electrophoresis. These methods are routine procedures for most molecular laboratories and should allow for straightforward incorporation of the CALR assay into the clinical diagnostic testing menu.

Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences.

Direct electrokinetic injection of inorganic cations from whole fruits and vegetables for capillary electrophoresis analysis

A novel approach for the direct injection from plant tissues without any sample pre-treatment has been developed by simply placing a small piece of the tissue into a capillary electrophoresis vial followed by application of a voltage for electrokinetic injection. Separations of sodium, potassium, calcium and magnesium were achieved using a BGE comprising 10mM imidazole and 2.5mM 18-crown-6-ether at pH 4.5. The addition of 2% (m/v) hydroxypropylmethyl cellulose to the separation buffer allowed for precise and accurate electrokinetic injection of ions from the plant material by halting the movement of tissue fluid into the capillary. This method provides both qualitative and quantitative data of inorganic cations, with quantitation in zucchini, mushroom and apple samples in agreement with Sector Field Inductively Coupled Plasma Mass Spectrometric analysis (r2=0.98, n=9). This method provides a new way for rapid, quantitative analysis by eliminating sample preparation procedures, and has great potential for a range of applications in plant science and food chemistry.

Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2 -microglobulin as a simple tool for misfolding and stability studies.

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

Single-Crystal-to-Single-Crystal Transition in an Enantiopure [7]Helquat Salt: The First Observation of a Reversible Phase Transition in a Helicene-Like Compound.

Here it is reported that crystals of an enantiopure [7]helquat salt undergo reversible thermal solid-solid phase transition at 404 K. Differential scanning calorimetry (DSC), capillary electrophoresis (CE), and X-ray diffraction analysis were used to unravel the mechanistic details of this process. The single-crystal-to-single-crystal course enabled direct monitoring of the structural changes by in situ variable-temperature X-ray diffraction, thus providing the first direct evidence of a solid phase transition in a helicene-like compound.

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection

We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.

Advances in Capillary Electrophoresis-Based Enzyme Assays

Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.

Salt De-Emulsification Dispersive Liquid-Liquid Microextraction and Back-Extraction Combined with Sweeping Micellar Electrokinetic Capillary Chromatography for Detection of Triazine Herbicides in Honey

A rapid, simple, and sensitive method has been developed for the analysis of triazine herbicides in honey samples by using salt de-emulsification dispersive liquid-liquid microextraction and back-extraction (SD-DLLME-BE) coupled with sweeping in micellar electrokinetic chromatography (MEKC). In the newly developed method, chloroform can be generated by the reaction of chloral hydrate and sodium hydroxide, and the chloroform droplets dispersed into bulk aqueous solution can be swiftly carried to the bottom of the centrifuge tube with the addition of calcium chloride. Then, back-extraction was utilized to extract the analytes into an aqueous solution prior to capillary electrophoresis (CE) analysis. The advantages of the new in situ DLLME lie in dispersive solvent-free and no centrifugation operation compared with the conventional DLLME. Several important parameters influencing the preconcentration and extraction efficiency of SD-DLLME-BE such as sodium dodecyl sulfate (SDS) concentration, pH, chloral hydrate and sodium hydroxide content, extraction time, and calcium chloride content were optimized. Under the optimized conditions, the enrichment factors were achieved in the range from 459 to 636. The proposed method provided a good linearity, low limits of detection (8.56 ng/mL), and good repeatability of the extractions (relative standard deviations (RSDs) below 8.8 %, n = 6). Honey samples were analyzed by the proposed method, and obtained results indicated that the proposed method provides acceptable recoveries and precisions.

Characterization of hydrophilic coated gold nanoparticles via capillary electrophoresis and Taylor dispersion analysis. Part II: Determination of the hydrodynamic radius distribution – Comparison with asymmetric flow field-flow fractionation

In the first paper of this series we have shown for hydrophilic coated Au nanoparticles that capillary electrophoresis in combination with Taylor dispersion analysis in fused silica capillaries with an inner diameter of 75μm allows for the unbiased precise determination of the number-weighted mean hydrodynamic diameter, the zeta potential and the effective charge number, although mobility corrected double layer polarization has to be taken into account.In this second paper we investigate whether the modified approximate analytic expression developed by Ohshima (2001) permits the calculation of calibration lines and the concomitant conversion of electropherograms into number-weighted particle radius distributions. We show that with the method developed size distributions are obtained which are independent of the measurement conditions. These size distributions are much narrower than those obtained via dynamic light scattering and data evaluation by the CONTIN algorithm. Capillary electrophoresis together with the proposed data evaluation method reveals that the analyzed nanoparticle populations have very narrow size distributions with a width of 2–4nm. The hydrodynamic radius distributions of the coated NPs are only slightly broader than the solid particle radius distribution of the Au-NP cores. The presence of monomodal/bimodal size distributions is confirmed by asymmetric flow field-flow fractionation.

Influence of main whey protein components on the mechanism of complex coacervation with Acacia gum

Complex coacervation between whey proteins isolate (WPI) and Acacia gum was investigated in order to disclose the roles and the contributions of each component of WPI to the formation of the complex coacervate. The main aim was to establish the balance of the main components of WPI, beta-lactoglobulin and alpha-lactalbumin, during the phase separation of complex coacervate. The complex coacervation of Acacia gum and pure beta-lactoglobulin, pure alpha-lactalbumin, and WPI have been investigated and compared together by means of macroscopic observations and capillary gel electrophoresis analyses performed along pH scans from basic to acidic medium. Coacervate composition was influenced by the protein/polysaccharide (Pr: Ps) ratio and pH. An optimum pH for best coacervation yield was found for each Pr: Ps ratio. Non-negligible concentrations of beta-lactoglobulin and alpha-lactalbumin remain in solution in most instances. beta-lactoglobulin undergoes complex coacervation stronger than alpha-lactalbumin in their competitive coacervation of WPI and Acacia gum.