Capillary Electrophoresis Assay Research Articles

Reference change values for lens culinaris agglutinin-reactive α-fetoprotein and des-γ-carboxy prothrombin in patients with chronic hepatitis C

Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3) and des-γ-carboxy prothrombin (DCP) have been routinely used as serological tumor markers of hepatocellular carcinoma (HCC) for surveillance. The aims of this study were: (i) to determine the biological variation of AFP-L3 and DCP in patients with chronic hepatitis C; and (ii) to calculate the reference change values (RCVs) of AFP-L3 and DCP. Ten patients with cirrhosis due to hepatitis C virus (HCV) infection and without HCC were enrolled in the study. Serum samples were collected at 14-day intervals, and 10 samples in total were obtained for each patient. AFP-L3 and DCP levels were measured by microchip capillary electrophoresis and liquid-phase binding assay. Intra-individual (CV(I)) and inter-individual (CV(G)) biological variations and RCVs were estimated from the data generated. The CV(I) was 29.0% for AFP-L3 and 24.6% for DCP, and CV(G) was 63.5% for AFP-L3 and 40.4% for DCP. The RCVs for AFP-L3 and DCP were 68.3% and 58.5%, respectively. Increases in values for AFP-L3 and DCP within 68.3% and 58.5% may be biological variations. Clinician should take these variations into consideration for the management of patients with HCV infection under surveillance of HCC.

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease. MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC50 and Ki values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot. ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC50 (29nM) and Ki (15nM) values. TF inhibited only seven out of 61 human kinases tested (>70% inhibition). Incubation of LNCaP cells with 50μM TF for 48h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis. General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.

Characterization of interactions between organotin compounds and human serum albumin by capillary electrophoresis coupled with inductively coupled plasma mass spectrometry

Thermodynamic data such as the binding constants are vital parameters describing interactions between exotic trace compounds and biomolecules in biochemical property modeling. In this study, the stability constants of organometallic compound and protein complexes were studied by using capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CE-ICP-MS), considering its low detection limits and low sample demand. Four organotin compounds (trimethyltin (TMT), tripropyltin (TPrT), tributyltin (TBT), triphenyltin (TPhT)) and human serum albumin (HSA) were used as model organometallic compounds and protein, respectively. Affinity capillary electrophoresis (ACE) and nonequilibrium capillary electrophoresis assays of equilibrium mixtures (NECEEM) were performed and compared by using ICP-MS as the detector to determine the binding constants of organotin compounds and HSA in 1:1 molar ratio assumption. Constant measurements of the two methods were both simple, however, ACE assays were more accurate and more appropriate for the constant determination of the organotin–HSA complexes, considering the errors of the NECEEM method. A good precision of the binding constants (logKb) using the ACE method was proved by different mathematical calculations, and the values were 6.13±0.51 (TMT), 5.72±0.38 (TPrT), 5.68±0.34 (TBT), 6.05±0.38 (TPhT) respectively for each of the organotin–HSA complexes, showing non-covalent interaction between organotin compounds and HSA. Meanwhile, this study also confirms the suitability of CE-ICP-MS method for further studies on organometallic complexation.

Analysis of casein biopolymers adsorption to lignocellulosic biomass as a potential cellulase stabilizer.

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

FLT3 ITD DeTecTIon: A cLoser Look AT The opTIons

The presence of internal tandem duplication (ITD) mutations in FLT3 has prognostic value in AML patients with normal cytogenetic profiles. There are several methods that can be utilized to detect these mutations with the tendency these days towards using expensive capillary analyzers and real-time amplification instruments, which prohibit ITD detection in more basic molecular facilities. We evaluated the use of a modified agarose gel electrophoresis assay for the detection of FLT3ITD mutations, in comparison to capillary electrophoresis and high resolution melt (HRM) assays, to assess the clinical diagnostic potential of this simpler technique. Each technique was evaluated on detection sensitivity, accuracy, reagent costs and processing time. Overall, in spite of the lure of advanced technology to provide faster and more sensitive analysis, in the case of FLT3ITD detection, basic molecular methodology proved to be the most sensitive, the most cost-effective and was able to deliver a reportable result in a time comparable to capillary electrophoresis. Under our test conditions, the HRM assay was neither reliable nor clinically sensitive enough for the routine detection of these mutations. This study showed that basic agarose electrophoresis methodology can successfully be employed to generate clinically relevant data regarding the FLT3ITD status of AML patients, allowing smaller molecular laboratories to offer this important prognostic assay.

UNC569 As Novel Small Molecule Mer Receptor Tyrosine Kinase Inhibitor for Treatment of ALL

Abstract 2589 Introduction:Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Pediatric cure rates for ALL have dramatically increased in recent years as intensified therapeutic regimens have been developed. However, intensified therapy is associated with a significant and increased risk of short- and long-term toxicities. In addition, patients who relapse, do not reach remission, or have certain cytogenetic abnormalities have a poor prognosis and limited treatment options. Therefore, novel and less toxic therapies are needed. Tyrosine kinases are frequently abnormally regulated in cancer cells. Mer receptor tyrosine kinase is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, dramatically increases the sensitivity of leukemia cells to cytotoxic agents, and significantly delays development of leukemia in a mouse model. Thus, Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule selective for Mer TK (UNC569) and characterization of its biochemical and anti-tumor activities in cell culture models of ALL. Methods:UNC569 is a substituted pyrazolopyrimidine that has been developed by a structure-based design and iterative medicinal chemistry from the known Mer/C52 cocrystal structure. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed through a LabChip EZ Reader. Western blot analysis of phosphorylated and total Mer protein was used to determine Mer inhibition by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cell lines. UNC569-mediated anti-leukemia activity was determined by detection of metabolically active cells using MTT reagent after 48 hours of exposure and by determining colony-formation in methylcellulose medium in the presence of UNC569. To investigate interactions with standard ALL therapies, apoptotic and dead cells were identified by flow cytometric analysis of cells stained with YO-PRO-1 and propidium iodide dyes after treatment with a chemotherapeutic agent alone or in combination with UNC569. Results:UNC569 is a novel small molecule Mer TKI with potent activity against Mer kinase (IC50 = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in both 697 and Jurkat ALL cells (IC50 <100 nM). Reduced proliferation and/or survival of ALL cells was also observed in 697 (IC50 = 0.91 ± 0.16 μM) and Jurkat (IC50 = 1.55 ± 0.19 μM) cultures. Treatment with UNC569 resulted in a statistically significant, dose-dependent decrease in colony-formation in Jurkat and 697 cell lines (60 ± 13.8 % (p = 0.01) and 29 ± 12.1 % (p = 0.01), respectively, relative to untreated cultures). Treatment of 697 cells with UNC569 in combination with etoposide resulted in a statistically significant increase in apoptotic and dead cells when compared with etoposide alone (43.3 ± 4.6 % vs 33.03 ± 0.73 %, p = 0.02). Treatment of Jurkat cells with a combination of UNC569 and methotrexate also resulted in a statistically significant increase in apoptotic and dead cells relative to methotrexate alone (26.9 ± 9.5 % vs 18.5 ± 5.7 %, p = 0.03). Conclusion:UNC569 is a highly effective Mer TKI that inhibits accumulation of the active phosphorylated form of Mer in B- and T-ALL cells. UNC569 mediates anti-leukemia activity against B- and T-ALL cells in culture and decreases colony-forming potential in methylcellulose. In addition, treatment with UNC569 sensitizes ALL cells to cytotoxic agents that are currently used as standard ALL therapies. Taken together, these data suggest that treatment with UNC569 may be a novel and effective ALL therapy and may be particularly effective in combination with cytotoxic therapies, thereby allowing for dose reduction and decreased incidence of toxic side effects. Disclosures:No relevant conflicts of interest to declare.

Development and validation of a capillary electrophoresis assay for the determination of the stereoisomeric purity of chloroquine enantiomers

A stereoselective CE assay for the determination of the enantiomeric purity of (R)-(-)-chloroquine and (S)-(+)-chloroquine was developed and validated. The separations were performed in a 50.2/40 cm uncoated fused silica capillary at 20°C using a 100 mM sodium phosphate buffer, pH 2.5, containing 30 mg/mL sulfobutylether(VII)-β-cyclodextrin as background electrolyte operated at an applied voltage of -25 kV and 20°C. The detection wavelength was 225 nm. Carbamazepine was used as internal standard. The assay was validated in the range of 0.05-1.0% for the respective minor chloroquine enantiomer based on a concentration of 3 mg/mL of the major enantiomer, either (R)-(-)-chloroquine or (S)-(+)-chloroquine. The method was applied to analyze the stereoisomeric purity of synthetic samples of the chloroquine enantiomers.

Detection of Nucleophosmin 1 Mutations by Quantitative Real-Time Polymerase Chain Reaction Versus Capillary Electrophoresis: A Comparative Study

Nucleophosmin 1 (NPM1) is the most commonly mutated gene in acute myeloid leukemia. Detection of NPM1 mutations is useful for stratifying patients for therapy, predicting prognosis, and assessing for minimal residual disease. Several methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or messenger RNA specimens. To directly compare a quantitative real-time polymerase chain reaction (qPCR) assay with a widely used capillary electrophoresis assay for detecting NPM1 mutations. We adopted and modified a qPCR assay designed to detect the 6 most common NPM1 mutations and performed the assay in parallel with capillary electrophoresis assay in 207 bone marrow aspirate or peripheral blood samples from patients with a range of hematolymphoid neoplasms. The qPCR assay demonstrated a higher analytical sensitivity than the capillary electrophoresis 1/1000 versus 1/40, respectively. The capillary electrophoresis assay generated 10 equivocal results that needed to be repeated, whereas the qPCR assay generated only 1 equivocal result. After test conditions were optimized, the qPCR and capillary electrophoresis methods produced 100% concordant results, 85 positive and 122 negative. Given the higher analytical sensitivity and specificity of the qPCR assay, that assay is less likely to generate equivocal results than the capillary electrophoresis assay. Moreover, the qPCR assay is quantitative, faster, cheaper, less prone to contamination, and well suited for monitoring minimal residual disease.

Substrate Promiscuity of N-Acetylhexosamine 1-Kinases

N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180–185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

Quantitative Analysis of MicroRNA in Blood Serum with Protein-Facilitated Affinity Capillary Electrophoresis

MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.

Detection of the G&gt;C SNP and Rare Mutations in the 28-bp Repeat of TYMS Using Gel-Based Capillary Electrophoresis

Polymorphisms in the 5' regulatory region of the thymidylate synthase gene (TYMS) have been shown to modulate thymidylate synthase expression and are associated with resistance to fluoropyrimidine-based therapies. These polymorphisms include a two repeat (2R) or three repeat (3R) of a 28-bp sequence and a G>C SNP in the second repeat of the 3R allele (TSER*3 G>C). Genotyping methods for the TYMS 5'-UTR polymorphisms have typically involved visualizing PCR and RFLP products on agarose gels. This article describes the use of a robust capillary electrophoresis assay for TYMS 5'-UTR genotyping. As part of pharmacogenetic studies, we performed TYMS genotyping for the 5'-UTR polymorphisms in 314 colorectal cancer patients. A gel-based capillary electrophoresis method, employing a high-resolution gel cartridge on a QIAxcel(®) system, was developed to detect PCR products and RFLP fragment sizes. The high resolution of the capillary electrophoresis technique allowed identification of a 6-bp insertion in the second repeat of the 3R allele in three patients. The frequency of the insertion allele was 0.4% in Caucasians and 1.3% in African-Americans. We also found 3.3% of Caucasian patients were heterozygous for a G>C SNP in the first repeat of the 2R allele, but this allele was not observed in the African-American patients. We describe a robust RFLP genotyping technique that employs size discrimination by capillary electrophoresis to genotype the TYMS TSER*3 G>C SNP. The technique also allows identification of a 6-bp insertion in the 3R allele, and we report the allelic frequencies for two uncommon TSER alleles.

Kinetics of Aspartic Acid Isomerization and Enantiomerization in Model Aspartyl Tripeptides under Forced Conditions

The aim of the present study was the determination of the isomerization and enantiomerization of aspartic acid (Asp) in tripeptides. Capillary electrophoresis (CE) assays were developed and validated allowing the simultaneous determination of the diastereomeric alpha-D/L-Asp and beta-D/L-Asp peptides. Rapid isomerization and enantiomerization were noted for peptides with the Phe-Asp-GlyOH sequence at pH 10 and 80 degrees C while Gly-Asp-PheOH proved to be more stable due to the steric influence of the phenyl side chain. A kinetic model assuming a central role of the succinimide intermediate was used to fit the concentration versus time data. In incubations of L-Phe-alpha-L-Asp-GlyOH the ratio of alpha-Asp/beta-Asp peptides was about 1:4 in agreement with literature data. With regard to L-Asp and D-Asp peptides an alpha-Asp/beta-Asp ratio of about 1:3 and 1:5, respectively, was observed. The stereochemistry of Phe at the X-1 position affected the ratio of L-Asp/D-Asp implying an effect of the stereochemistry of neighboring amino acids on Asp enantiomerization. Modeling only overall Asp enantiomerization rate constants in accordance to literature data were observed for Asp peptides. In case of the asparagine (Asn) peptide the data could only be fitted to the models considering a direct conversion of L-Asn to a D-configured succinimide via an alternative pathway.

Expression and function of heregulin-α and its receptors in the mouse mammary gland

Heregulin-alpha (HRGalpha) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGalpha and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGalpha and its receptors ErbB2 and ErbB3. We also determined the role of HRGalpha in the development of mammary glands, beta-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGalpha signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGalpha, indicating that HRGalpha positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGalpha enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of beta-casein. During lactation, HRGalpha enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of beta-casein in mammary epithelial cells. During involution, HRGalpha induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of beta-casein, resulting in increased levels of beta-casein in mammary epithelial cells.

Evaluation of capillary electrophoresis assay for CDT on SEBIA's Capillarys System: Intra and inter laboratory precision, reference interval and cut-off

Background Carbohydrate-deficient transferrin (CDT) measurement on the multicapillary system Capillarys™ is characterized by high throughput and an on-line sample pre-treatment. This study evaluates the linearity and the precision of this technique, the correlation with the candidate reference method and the effect of genetic transferrin variants. Upper reference limit and cut-off values were calculated. Methods The precision study was carried out following CLSI EP5-A protocol. The between laboratory variation was calculated from an eight-site study. The upper reference limit (URL) was conventionally calculated from a reference population of 225 samples and verified by a Bhattacharya analysis in a large (n = 19,129) population. A population of 314 heavy consumers was used for calculation of the cut-off limit. Additionally the measurement uncertainty was calculated according to EDMA (European Diagnostic Manufacturers Association) and IRMM. Results The imprecision found was less than 5%, linearity was excellent for CDT values ranging from 2% to 20%. The between site variation around the cut-off value (CDT ranging from 1.68% to 1.79%) was clinically not significant. The upper reference limit (95th percentile) was calculated at 1.3% by the conventional IFCC method and confirmed by Bhattacharya calculations. The optimum cut-off for this CZE method was 1.6%, taken into account the measurement uncertainty. The regression equation with the candidate reference method was Disialotransferrin(Capillarys2) = Disialotransferrin(HPLC) ∗ 0.968 − 0.248. Genetic variants and abnormal profiles were well recognized. Conclusions These results demonstrate that the Capillarys CDT method is robust and reliable in routine use with a high degree of homogeneity from one system to another and is highly correlated with the candidate HPLC reference method.

Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

The discovery of small molecules targeting the >80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.

Multiplexed Detection of Small Analytes by Structure-Switching Aptamer-Based Capillary Electrophoresis

Affinity probe capillary electrophoresis (APCE) assays, combining the separation power of CE with the specificity of interactions occurring between a target and a molecular recognition element (MRE), have become important analytical tools in many application fields. In this report, a rationalized strategy, derived from the structure-switching aptamer concept, is described for the design of a novel APCE mode dedicated to small molecule detection. Two assay configurations were reported. The first one, developed for the single-analyte determination, was based on the use of a cholesteryl-tagged aptamer (Chol-Apt) as the MRE and its fluorescein-labeled complementary strand (CS*) as the tracer (laser-induced fluorescence detection). Under micellar electrokinetic chromatography (MEKC) conditions, free CS* and the hybrid formed with Chol-Apt (duplex*) were efficiently separated (and then quantified) through the specific shift of the electrophoretic mobility of the cholesteryl-tagged species in the presence of a neutral micellar phase. When the target was introduced into the preincubated sample, the hybridized form was destabilized, resulting in a decrease in the duplex* peak area and a concomitant increase in the free CS* peak area. The second format, especially designed for multianalyte sensing, employed dually cholesteryl- and fluorescein-labeled complementary strands (Chol-CS*) of different lengths and unmodified aptamers (Apt). The size-dependent electrophoretic separation of different Chol-CS* forms from each other and from their corresponding duplexes* was also accomplished under MEKC conditions. The simultaneous detection of multiple analytes in a single capillary was performed by monitoring accurately each target-induced duplex-to-complex change. This method could expand significantly the potential of small solute APCE analysis in terms of simplicity, adaptability, generalizability, and high-throughput analysis capability.

Investigation of the [−/A]8 and C1236T genetic variations within the human toll-like receptor 3 gene for association with multiple sclerosis

Multiple sclerosis (MS) is a serious cause of neurological disability among young adults. The clinical course remains difficult to predict, and the pathogenesis of the disease is still modestly understood. Autoimmunity is thought to be a key aspect of the disease, with autoreactive T cells thought to mediate central nervous system (CNS) inflammation to some extent. Toll-like receptors are known to mediate cellular recognition of pathogens by way of patterns of molecular presentation. Toll-like receptor 3 is coded by the gene TLR3 and is recognized as an important factor in virus recognition and is known to be involved in the expression of neuroprotective mediators. We set out to investigate two variations within the TLR3 gene, an 8 bp insertion–deletion [−/A]8 and a single base-pair variation C1236T, in subjects with MS and matched healthy controls to determine whether significant differences exist in these markers in an Australian population. We used capillary gel electrophoresis and TaqMan genotyping assay techniques to resolve genotypes for each marker, respectively. Our work found no significant difference between frequencies for TLR3 [−/A]8 by genotype (χ2=1·03, p=0·60) or allele (χ2=1·09, p=0·30). Similarly, we found no evidence for the association of TLR3 C1236T by genotype (χ2=0·35, p=0·84) or allele frequency (χ2=0·31, p=0·58). This work reveals no evidence to suggest that these markers are associated with MS in the tested population. Although the role of TLR3 and the wider toll-like receptor family remain significant in neurological and CNS inflammatory disorders, our current work does not support a role for the two tested variants in this gene with regard to MS susceptibility.

Measuring aptamer equilibria using gradient micro free flow electrophoresis.

Gradient micro free flow electrophoresis (muFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the muFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of muFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer-IgE complex was estimated to be 48 +/- 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HIVRT), 2:1, and 1:1 stoichiometries.

Automated Enzyme-Based Diagonal Capillary Electrophoresis: Application to Phosphopeptide Characterization

Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have different migration times in the second capillary and will generate spots that fall off the diagonal in the electropherogram. We demonstrate the system with immobilized alkaline phosphatase to monitor the phosphorylation status of a mixture of peptides. This enzyme-based diagonal capillary electrophoresis assay appears to be generalizable; any post-translational modification can be detected as long as an immobilized enzyme is available that reacts with the modification under electrophoretic conditions.

Capillary electrophoretic development of aptamers for a glycosylated VEGF peptide fragment

The emergence of functional genomics and proteomics has added to the growing need for improved analysis methods that can detect and distinguish between protein variants resulting from allelic variation, mutation, or post-translational modification. Aptamers, single-stranded DNA or RNA molecules that fold into three-dimensional structures conducive to binding targets, have become an attractive alternative to antibodies for this type of analysis. Although aptamers have been developed for a wide range of target species, very few sequences have been identified that bind selectively to proteins with specific post-translational modifications. Using capillary electrophoresis-based selection, we have developed DNA aptamer sequences that selectively bind an N-glycosylated peptide fragment of vascular endothelial growth factor (VEGF). The selection method incorporates alternating positive- and counter-selection steps in free solution in order to obtain aptamers with both high affinity toward the glycosylated target and high selectivity versus a non-glycosylated variant. Affinity capillary electrophoresis and surface plasmon resonance binding assays indicate these sequences have low-µM dissociation constants and preferentially bind the glycosylated peptide with as much as 50-fold specificity. Such aptamers could serve as tools for rapid and simple monitoring of disease-linked functional changes in proteins, with potential applications in drug screening and disease diagnosis.