Development of capillary electrophoresis as an alternative to high resolution agarose electrophoresis for the diagnosis of multiple sclerosis.

The presence of oligoclonal bands in cerebrospinal fluid (CSF) is used as a diagnostic indicator of multiple sclerosis (MS). These bands, gamma-globulins thought to result from a restricted antibody response directed against autoantigens or viral antigens, are consistent with CSF-specific immunoglobulin synthesis when observed in the spinal fluid and not in the serum. Current methodology commonly involves electrophoresing concentrated CSF with high-resolution agarose gel electrophoresis (HRAGE) followed by protein staining in order to visualize the oligoclonal bands. Capillary zone electrophoresis (CZE) was evaluated as an alternative method. Separation of CSF and serum proteins from 54 patients in a bare silica capillary containing a high pH borate buffer allowed for resolution of the five major zones including the gamma-region and showed a 90% concordance with the results from HRAGE banding studies. Since a simple borate buffer did not provide adequate resolution of the oligoclonal bands in the gamma-region, the separation buffer was augmented with polyethylene glycol (PEG) which provided a significant enhancement in resolution of proteins in this region (24 patient study). In addition to obtaining banding information from electropherograms obtained with these separation conditions, it was feasible to calculate a CSF Index which compared favorably with the results from nephelometry. Finally, we show that zwitterionic additives such as O-phosphorylethanolamine may further enhance resolution and that capallary electrophoresis (CE) may allow oligoclonal banding information to be obtained directly from CSF without concentration.

Temporal sequence of transcription of perforin, Fas ligand, and tumor necrosis factor-alpha genes in rejecting skin allografts.

BACKGROUND: Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection. METHODS: We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes. RESULTS: Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection. CONCLUSIONS: The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection.

Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor. Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis.

The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10(-18) mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.

Effective and reproducible capillary electrophoretic separation of thiols under conditions where exceptionally high current is generated.

There is an escalating interest in the role of endogenous nitric oxide (NO) in biological systems and how this chemical regulates physiology in normal and disease states. In biological systems, the cellular concentration can be estimated, in the simplest form, by accounting for NO and its common metabolites, nitrate and nitrite. However, since NO is also known to interact with other chemical entities, such as thiols, it would be valuable to have a rapid qualitative assay that could account for thiol binding and S-N bond cleavage in the presence of different reducing agents. A separation buffer consisting of 10 mM phosphate, 10 mM HCl, and 250 mM KCl is shown to be adequate for the separation of glutathione, nitrosylated glutathione, and glutathione disulfide solubilized in 2 M HCl. The current observed under these separation conditions (249 microA at 11 kV) is extremely high by capillary electrophoresis (CE) standards, with a total power (current x voltage/capillary length) calculated to be in excess of 7 W/m. While this exceeds the approximately 1.0 W/m recommended by previous studies as a maximum for CE-based separations, we demonstrate that effective CE separation of thiols can, in fact, be accomplished under these conditions with acceptable reproducibility, provided that buffer depletion issues are addressed.

Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA.

Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.

Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis.

Attempts are frequently made to semiquantitate mRNA as a means of circumventing the laborious and time-consuming process of quantitation that is inherent in the use of competitor templates. However, semiquantitative approaches present the risk of generating non-reproducible data due to tube-to-tube variability and/or misinterpretation of quantities of product being generated during the plateau phase of PCR. Subsequently, it is difficult to compare semiquantitative data from separate experiments, and comparisons of levels of mRNA transcript from genes that amplify with different primer pairs cannot be made. Thus, reliable methods for mRNA quantitation continue to rely on the use of internal standardization. In this report, we describe a strategy for dependable quantitation of low-abundance mRNA transcripts based on quantitative competitive reverse transcription PCR (QC-RT-PCR) coupled to capillary electrophoresis (CE) for rapid separation and detection of products. Recommendations are included for the design of RNA competitors that can be paired with target RNA for cDNA synthesis primed with a gene-specific primer; these synthesized cDNAs are then co-amplified directly in the same tube using a single primer pair. We describe (i) a protocol for a single-tube RT-PCR that provides for cDNA synthesis and subsequent PCR amplification of target and competitor in identical reaction environments at each critical enzymatic step, (ii) a unique hot-start provision for optimizing precise and consistent PCR amplifications and (iii) a method for rapid PCR product separation, detection and quantitation by CE and laser-induced fluorescence.

Capillary electrophoresis of insulin-like growth factors: enhanced ultraviolet detection using dynamically coated capillaries and on-line solid-phase extraction.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35-40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction-capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.

Capillary electrophoresis in biotechnology.

Capillary electrophoresis (CE) is a versatile micro/macroanalytical technique gaining widespread usage for the separation and analysis of ionic substances. It has captured the attention of those working in a variety of arenas focused on biologically-active molecules. Its appealing characteristics include unprecedented mass sensitivity and the ability for precise, automated electrophoretic separation of microvolume samples with relatively short analysis times. Such versatility in bioanalysis makes it an inviting replacement for some of the labor-intensive, time-consuming methodologies performed via electrophoretic gels. Moreover, CE compliments the ease and speed of HPLC while eliminating the problem of excessive solvent volume usage and hazardous waste disposal. Further attractive characteristics of this technology include the analyses of a diverse spectrum of analytes, ranging from small organic ions to macromolecular protein complexes and DNA. While combining some of the most robust aspects of traditional electrophoresis, chromatography, and capillary technology, recent CE research and development has focused on avenues leading to improving detection and understanding and employing the basic chemistry of CE vis-a-vis new applications.

Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids.

Most electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor-intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laboratory.

Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis.

The structural investigation of G protein-coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid-based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrene-coated fused silica capillary. The potential for detection sensitivity to be increased at least 100-fold by use of on-line solid-phase extraction on C18-silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.

Identification of monoclonal proteins in serum: a quantitative comparison of acetate, agarose gel, and capillary electrophoresis.

A selected group of 308 sera were analyzed by capillary electrophoresis (CE), agarose gel electrophoresis (AGE), and cellulose acetate electrophoresis (CAE) and evaluated for abnormalities that would suggest the presence of a monoclonal protein. The sensitivity (an electrophoretic abnormality in sera that contained a monoclonal protein) and specificity (a normal electrophoretic pattern in sera that did not contain a monoclonal protein) was determined for each electrophoretic procedure. CAE was the most specific procedure and CE was the most sensitive. The increase in sensitivity of CE was primarily due to increased detection of cryoglobulins and free light chains. The quantitation of the gamma region and/or monoclonal antibody peaks by CE was similar to results obtained by AGE. Quantitation of very large monoclonal protein peaks (> 3.0 g/dL) by on-line absorption detection (CE) yielded higher results than quantitation by dye-binding (AGE).

Capillary electrophoresis-based separation of transferrin sialoforms in patients with carbohydrate-deficient glycoprotein syndrome.

The heterogeneity associated with protein glycoforms has been a challenge to analytical chemists and the subject of structure-function studies for biochemists since their presence in biological systems had been confirmed some three decades ago. Initial investigations led to discoveries of synthetic and degradative pathways, and brief forays into functional determination of the "glyco" portion on the protein activity in glycoproteins. Only recently has it come to our understanding that variations from the "normal" glycosylation patterns might be indicative of pathological states. The presence of certain transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human Tf has become increasingly important. It this study, we demonstrate that a simple method utilizing a DB-17-coated capillary to slow endoosmotic flow and a sieving buffer containing hydroxyethyl cellulose allows for the resolution of sialoforms of transferrin. An analysis time of less than eight minutes allows for baseline resolution of the lower sialoforms of Tf, presenting a simple, rapid test for carbohydrate-deficient transferrin (CDT). We demonstrate the utility of this methodology for the facile diagnosis of carbohydrate-deficient glycoprotein syndrome, and postulate that it may allow for the detection of other carbohydrate-deficient protein-related disease states.

Development of a nonisotopic capillary electrophoresis-based method for measuring glomerular filtration rate.

The conditions for quantitative measurement of nonisotopic iothalamate meglumine (Conray) in urine and plasma by capillary zone electrophoresis (CE) have been developed. The impetus for developing this methodology was to replace the traditional [125I]iothalamate glomerular filtration rate (GFR) marker assay, a routine tool in the measurement of kidney function. This new approach for measuring kidney function is attractive since it avoids the cost of administration of radioisotopic compounds to patients, as well as the cost associated with purchase and disposal of isotopic compounds and contaminated samples. The concentration of iothalamate in urine and plasma determined by CE can be used directly to calculate GFR. The GFR in patients injected with [125I]iothalamate and nonisotopic iothalamate simultaneously showed an excellent correlation (0.998) with between-day coefficient of variation of 2.30% and a recovery of 102% and 98%, respectively, when added to urine and plasma. Interference from drugs and other urinary compounds is eliminated with this method. Collectively, this study has shown that CE is a cost-effective alternative to the current methodology for measuring GFR.

Capillary electrophoretic detection of metabolites in the urine of patients receiving hypoglycemic drug therapy.

Micellar electrokinetic chromatography (MEKC) in tandem with diode array detection (DAD) has been exploited as an analytical method for the separation and detection of sulfonylurea drugs. The ultimate goal is the development of an assay to detect these drugs or their metabolites in urine as a means of diagnosing sulfonylurea drug abuse. Using a separation buffer consisting of 5 mM borate/5 mM phosphate/75 mM sodium cholate, separation of both the second and third generation sulfonylurea drugs can be achieved. The characteristic absorbance spectra associated with each of the third generation drugs, glipizide and glyburide, allow for their identification in mixtures. Coinjection of glyburide, its primary metabolite, hydroxy glyburide, and glipizide demonstrated that the metabolite was resolved from the parent drug but shared its absorbance spectral properties. MEKC analysis of a series of solid phase-extracted urine samples from patients prescribed glipizide or glyburide, as well as from control patients not ingesting the drug, showed that the parent compounds were difficult to detect in the urine. However, the use of DAD allowed for detection of metabolites in the urine of these patients. With glyburide patients, only primary metabolites were detected, while urine from patients on glipizide showed a series of peaks whose absorbance spectra was consistent with the presence of both primary and secondary metabolites. In addition, the intensity of the metabolite peaks corresponded reasonably well with the respective dose and in vivo time interval associated with the urine collection. This study shows that MEKC with DAD has potential for further exploration as a clinical assay for detecting surreptitious abuse of sulfonylurea drugs.

Determination of nitrite and nitrate reduction by capillary ion electrophoresis.

Production of nitrates and nitrites is a common step in many methodologies used to measure nitric oxide (NO) and NO-derived products in biological fluids. We report conditions that allow the rapid separation and quantification of nitrite from nitrate ions in biological fluids by capillary ion electrophoresis (CIE). CIE can be used to directly quantify nitrites and nitrates near the millimolar range. To detect lower levels, we have used CIE to monitor the reduction of nitrites and nitrates to NO for chemiluminescence detection. For reduction reactions, we directly compared the ability of three commonly used agents--potassium iodide (KI), mercuric chloride (HgCl2) and vanadium chloride (VCl3)--to reduce nitrite and nitrate ions to NO. Nitrites/nitrates can be efficiently reduced to NO at 37 degrees C using vanadium chloride (100%) or HgCl2 (80%). However, these CE-derived conditions cannot simply be extrapolated to chemiluminescence measurements. Vanadium (III) yields high background in the photomultiplier that diminishes the sensitivity of chemiluminescence measurement to that outside of physiological ranges. We find that reactions carried out at 37 degrees C in 2 M HCl using HgCl2 is efficient using both techniques.

Rapid capillary electrophoretic analysis of human serum proteins: qualitative comparison with high-throughput agarose gel electrophoresis.

This study details the qualitative results from a comparative evaluation of agarose gel electrophoresis (AGE) and (CZE) as a screening procedure for monoclonal proteins in serum. Three hundred and five serum samples were analyzed by the two techniques; samples suspected of containing monoclonal proteins based on abnormalities observed with AGE or CZE were confirmed by immunoelectrophoresis and/or immunofixation. CZE separation conditions were simple (requiring only a bare silica capillary and 150 mM borate buffer, pH 10.0) and separation was complete in under 120 s. The results obtained by CZE were comparable or better than those obtained with AGE. Samples displaying "point-of-application" artifacts on AGE were not problematic by CZE analysis; an abnormal profile, due to the presence of a monoclonal protein, or a normal profile were clearly observable. The rapid analysis, excellent reproducibility, automation and relatively high throughput (approximately 90 samples per 8 h on a single instrument) sets the stage for CZE analysis of serum proteins to be used routinely in a clinical setting.

Evaluation of capillary electrophoresis in polymer solutions with laser-induced fluorescence detection for the automated detection of T-cell gene rearrangements in lymphoproliferative disorders.

The rapid increase in the number of DNA-based clinical diagnostic procedures, particularly polymerase chain reaction (PCR)-based assays, has generated interest in analytical techniques that are less time-consuming and labor-intensive than traditional procedures such as polyacrylamide gel electrophoresis (PAGE). Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection, which allows for rapid and sensitive detection of DNA fragments in an automated format, is well-suited for DNA-based clinical assays. In this study, we demonstrate the potential of CE-LIF for the detection of PCR products from T-cell receptor gamma (TCR gamma) gene rearrangements present in monoclonal populations of lymphocytes. The presence of monoclonal populations of T-cells is associated (but not always synonymous) with lymphocytic malignancies. Analysis of 31 patient samples, as well as sensitivity controls, demonstrated that CE-LIF detection of monoclonal lymphocytic populations is comparable to that of PAGE-SYBR Green I staining, and that CE-LIF detection can be accomplished in less than 20 min. These preliminary results illustrate the potential feasibility of a CE-based diagnostic assay for the detection of T-cell gene rearrangements.

High-resolution glycoprotein analysis using capillary electrophoresis.

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary wall and the subsequent decrease in protein-wall interactions in known to be key in the effectiveness of these additives, much remains to be learned regarding the mechanism through which they function.

Effect of cationic buffer additives on the capillary electrophoretic separation of serum transferrin from different species.

The presence of specific transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human transferrin has become increasingly important. As a means of evaluating the potential for developing a rapid capillary electrophoresis-based assay for the analysis of carbohydrate-deficient transferrins (CDTs), the capillary zone electrophoretic (CZE) analysis of Tfs from several species was evaluated using uncoated capillaries and a separation augmented with cationic additives. With bovine Tf, five peaks (representing different sialylated forms) were partially resolved in borate and baseline-resolved when 1,4-diaminobutane was added to the buffer. These same conditions were found to be inadequate for the resolution of the sialoforms from other species. Some success was achieved using alpha, omega-bis-quaternary ammonium alkanes instead of the 1,4-diaminobutane and optimizing the pH for each of the species Tfs. Human Tf was found to be resolved in an uncoated capillary equilibrated with a borate buffer containing millimolar concentrations of decamethonium bromide as a buffer additive. Under these conditions, resolution of the various sialoforms from the iron-saturated Tf was possible and the glycoforms were found to migrate differently than their iron-depleted counterparts. Despite the resolution achievable under these conditions, the lengthy analysis time is incompatible with the requirements for a clinical CZE-based assay.

Capillary electrophoresis of DNA potential utility for clinical diagnoses.

The last few years have witnessed a tremendous shift in the use of capillary electrophoresis for clinical applications, particularly with DNA analysis. As a result of the large number of DNA-based clinical assays, there is an intense interest in making DNA analysis faster, less expensive and more automated. We describe the evaluation of CE-based single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) analysis for the detection of single-point mutations within a Mycobacterium tuberculosis-specific amplified DNA fragment. Both were found to be capable of detecting the mutation in the resistant isolate but ddF showed the most promise with respect to specificity and ease of implementation. In addition, initial results with a CE-based sizing method is shown to be competitive and, perhaps, superior to a Southern blot analysis for the detection of hepatitis C viral (HCV) infection.