Performance comparison of EasyFix G26 and HYDRASYS 2 SCAN for the detection of serum monoclonal proteins.

BACKGROUND: Serum protein electrophoresis (SPE) is a widely used laboratory technique to diagnose patients with multiple myeloma (MM) and other disorders related to serum protein. In patients with MM, abnormal monoclonal protein can be detected by SPE and further characterized using immunofixation electrophoresis (IFE). There are several semi-automated agarose gel-based systems available commercially for SPE and IFE. In this study, we sought to evaluate the analytical performance of fully automated EasyFix G26 (EFG26) and semi-automated HYDRASYS 2 SCAN (H2SCAN) for both SPE and IFE. METHODS: Both instruments were operated according to manufacturer's instructions. Samples used include a commercially available normal control serum (NCS) and patients' specimens. The following were evaluated: precision and comparison studies for SPE, and reproducibility and comparison studies for IFE. Statistical analyses were performed using Microsoft Excel. RESULTS: For SPE repeatability study, our results showed that EFG26 has higher coefficient of variation (%CV) compared with H2SCAN for both samples except for monoclonal component with %CV of 0.97% and 1.18%, respectively. Similar results were obtained for SPE reproducibility study except for alpha-1 (4.16%) and beta (3.13%) fractions for NCS, and beta fractions (5.36%) for monoclonal sample. Subsequently, reproducibility for IFE was 100% for both instruments. Values for correlation coefficients between both instruments ranged from 0.91 to 0.98 for the five classic bands. CONCLUSION: Both instruments demonstrated good analytical performance characterized by high precision, reproducibility and correlation.

Development of an electrophoretic method based on nanostructured materials for HbA1c determination.

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 µg/mg) as pseudo-stationary phase. Before the sample injection, SiO2 NP─B(OH)2 were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%-1 × HbA1c - 377.02; R2  = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t-test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.

Comparison of Fully Automated and Semiautomated Systems for Protein Immunofixation Electrophoresis.

BACKGROUND: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation. METHODS: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components. RESULTS AND CONCLUSION: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care.

Transverse gradient electrophoresis: protein homogeneity test and subfractionation technique.

Electrophoresis of protein (including enzyme) was conducted in a gel medium across which, transverse to the direction of protein migration, a continuous (p)H gradient extends. Any splitting of a resultant trace for the change of protein mobility with (p)H suggests both protein heterogeneity and the (p)H conditions under which further purification and subfractionation may best be pursued.

Analytical sensitivity and diagnostic performance of serum protein electrophoresis on the HYDRAGEL 30 PROTEIN(E) ß1-ß2 Sebia Hydrasys system.

BACKGROUND: Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are used in the diagnosis and monitoring of plasma cell dyscrasias. IFE is considered the most sensitive method for the detection of monoclonal proteins (M-proteins), but it is not quantitative. The goal of this study was to establish the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. METHODOLOGY: Patient sera with a previously identified M-protein (IgG, IgA or IgM) were serially diluted with a normal serum pool and electrophoresed on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. The SPE gels were individually interpreted by five independent observers and IFE was performed on selected samples. Limit of detection was determined as the lowest concentration of M-protein band visible on the gel. SPE diagnostic performance was evaluated against the "gold standard" IFE according CLSI EP12-A2 guidelines. RESULTS: Detection limit was comparable among all M-proteins migrating in the gamma region, IgG-κ (0.18±0.08g/L; n=6), IgG-λ (0.36±0.25g/L; n=8), IgA-κ (0.40±0.13g/L; n=7), IgA-λ (0.37±0.23g/L; n=4), IgM-κ (0.47±0.20g/L; n=13) and IgM-λ (0.29±0.24g/L; n=6). Percentage agreement with IFE for IgG and IgA in the gamma region ranged from 65% to 100%, whereas IgM migrating in the gamma region and immunoglobulins co-migrating with alpha or beta globulins, showed poor (0-38%) agreement. CONCLUSIONS: This study evaluates the analytical sensitivity and diagnostic performance of SPE on the Sebia Hydrasys using HYDRAGEL 30 PROTEIN(E) ß1-ß2. There was acceptable agreement between SPE and IFE for IgG-κ/λ and IgA-κ/λ migrating in the gamma region, suggesting that repeating IFE for samples with these isotypes, when the previous IFE and second SPE are both negative, may not be necessary.

Label-free biomolecular detection at electrically displaced liquid interfaces using interfacial electrokinetic transduction (IET).

Biosensors require a biorecognition element that specifically binds to a target analyte, and a signal transducer, which converts this targeted binding event into a measurable signal. While current biosensing methods are capable of sensitively detecting a variety of target analytes in a laboratory setting, there are inherent difficulties in developing low-cost portable biosensors for point-of-care diagnostics using traditional optical, mass, or electroanalytical-based signal transducers. It is therefore important to develop new biosensing transducer elements for recognizing binding events at low cost and in portable environments. Here, we demonstrate a novel electrokinetic liquid biosensing method for the sensitive label-free detection of a model biomolecule against a background of serum protein. The biosensor is based on the motion of a microfluidic-generated electrical liquid interface when subjected to an external alternating current electrical field. We demonstrate that the electric field-induced motion of the interface can be used as a sensitive and specific transducer for the detection of avidin at femtomolar concentrations in solution. This new detection strategy does not require surface functionalization or fluorescent labels, and has the potential to serve as a sensitive low-cost method for portable biomarker detection.

Protein and cholesterol electrophoresis of plasma samples from captive cownose ray (Rhinoptera bonasus).

Our study was undertaken to assess the application of semiautomated methods available at the reference laboratory level for the evaluation of plasma protein and cholesterol via electrophoresis in samples from cownose rays (Rhinoptera bonasus). Three groups of animals were assessed: clinically normal, clinically abnormal, and parasitized with leeches. As reported previously, the albumin band was negligible; the protein electrophoretograms were dominated by a large beta-globulin fraction. While the group of samples from the leech-parasitized rays did not show any large differences, the abnormal group exhibited significantly elevated total solids and cholesterol levels. The latter was related to a significant increase in very low density lipoprotein levels. The results demonstrate the potential application of these laboratory methods in quantitation of plasma proteins and cholesterol fractions in subclass Elasmobranchii.

Microscope slide electrophoresis of serum lipoproteins in agarose gel.

An ultramicro electrophoretic technique for the separation of serum lipoproteins is described using agarose gel on microscope slides.The interaction between the agarose gel and lipoproteins has been reduced and the separation between beta- and pre-beta-lipoproteins has been improved over existing methods. A semiquantitative assay of the lipoproteins can be done in a densitometer. It is suggested that the interaction between agarose gel and lipoproteins, which, without albumin in the system, results in distortion of the lipoprotein bands, may be mediated by free fatty acids.

The pre-albumin fraction: a useful parameter in the interpretation of routine protein electrophoresis.

A technique of protein electrophoresis suitable for routine use which ensures good separation of the prealbumin fraction is described. The clinical value of visually estimating this protein fraction from the electrophoretic strip is shown with reference to 134 cases. Low levels of prealbumin were found to be associated with liver disorders, malignancy, collagen diseases, congestive cardiac failure, and burns. Finally, pitfalls in electrophoretic technique which may cause failure to visualize the prealbumin fraction are discussed.

Sample application position and the resolution of protein separation on the Beckman Microzone Electrophoretic System.

Many clinical laboratories use the Beckman Microzone electrophoretic system (Beckman Instruments Inc. Fullerton, CA 92634) for serum protein separation. Although detailed instruction is supplied in the manufacturer's manual (1) for protein separation, the effect of different sample application positions on the cellulose acetate membrane is not discussed. We have processed the serum specimen in various application positions and found that there is a definite relationship between the resolution of fractionated protein bands and the application position, particularly with specimens from patients with monoclonal gammonpathies. The resolution of the protein bands increases as the sample is applied closer to the anode end of the cellulose acetate strip. Although studies were carried out on the Beckman electrophoretic system, a similar application effect may be anticipated with other electrophoretic systems.

Microchip-based capillary electrophoresis of human serum proteins.

The separation and relative quantitation of human serum proteins is important to the clinical diagnosis of various states of disease. Microchip-based capillary electrophoresis (CE) of human serum proteins offers several advantages over sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis and conventional CE methods, including decreased sample consumption and analysis time and the possibility of on-chip sample manipulation (dilution, labelling, etc.). The microchip used in these studies was designed to allow for on-chip, post-separation labelling of the proteins and subsequent laser-induced fluorescence detection. 2-Toluidinonaphthalene-6-sulfonate (TNS) is a virtually non-fluorescent reagent which, upon non-covalent association with the protein and excitation at 325 nm, produces a fluorescent product with an emission maximum near 450 nm. After optimization of buffer conditions (100 mM borate with 2 mM lactate, pH 10.5), individual serum proteins (IgG to mimic the gamma zone, transferrin the beta zone, alpha-1-antitrypsin the alpha 1 zone and albumin its own zone) were successfully resolved on-chip, as was a "synthetic" serum solution composed of a mixture of all four of the previously mentioned proteins. Analysis of all five protein zones in a true human serum sample, however, has not yet been achieved on-chip due to the poor sensitivity of the TNS label for several of the serum proteins.

Electrophoretic techniques in today's clinical laboratory.

The clinical laboratory today is being asked to do more tests faster than ever before. The field of electrophoresis is no different than any other part of the clinical laboratory. With these demands comes the need to get more information in a more timely fashion. Agarose gives the clinical electrophoresis laboratory a tool for resolving proteins in a more meaningful manner. These patterns, when confirmed with specific protein analysis and immunofixation electrophoresis, can lead to earlier diagnosis and treatment. Those who say they have no time to run electrophoresis probably have not run electrophoresis lately and are missing out on a very valuable and cost-effective tool.

Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.

A simple, horizontal device for rapid electrophoretic transfer of proteins from several polyacrylamide gels simultaneously is described. Up to six 'TRANS-UNITS' consisting of soaked filter paper on either side of polyacrylamide gel/nitrocellulose sheets that are separated by dialysis membranes are stacked between graphite plate electrodes. The only buffer reservoir in the apparatus is that in stacked, soaked filter paper. A special buffer system based on the isotachophoresis theory was developed for this purpose. The electrophoretic transfer was performed with equal efficiency in all TRANS-UNITS of the stack. Only traces of a few proteins remained in the polyacrylamide gel after transfer. With this apparatus, 50 protein bands from a human serum protein sample (diluted 1 : 100) were detected by immunoblotting with the retainment of the high resolution of the SDS-PAGE technique. The apparatus provided a constant current density of 0.8 mA/cm2 during the 1-h transfer time at 21 degrees C, irrespective of the number of TRANS-UNITS. The apparatus generated 1-5 W in joule heat, depending on the number of TRANS-UNITS in the stack.

Bloodstain characterization in the EAP, Hp, Hb, AK and Glo I typing systems using minigels and the PhastSystem.

Application of minigels and the PhastSystem to obtain phenotyping results from bloodstains in the EAP, Hp, AK, and Glo I typing systems was investigated. Nonequilibrium isoelectric focusing with 4-6.5 PhastGel produced readily interpretable phenotypes in the EAP typing system. Both 4-6.5 and 5-8 PhastGel produced AK typing system phenotypes using nonequilibrium isoelectric focusing conditions. The 8-25% PAG PhastGel developed by two staining techniques allowed discrimination of phenotypes for the Hp typing system. Phenotypes from the Glo I typing system were also obtained with this gel type. Variant haemoglobins could be detected on pH 5-8 PhastGel using isoelectric focusing conditions. Much potential for standardized, rapid phenotyping of bloodstains was found to exist utilizing the PhastSystem.

Sex differences in blood protein patterns: a computer-assisted electrophoretic analysis of mid-molecular weight range proteins of human serum.

We have studied the sexual dimorphism of human serum proteins between the ranges of approximately 60,000 and 150,000 daltons using computerized imaging and densitometry. Methodology is presented, based on the TEXAC computer system for the detection, segmentation and integration of protein bands. The results are discussed in terms of potential future applications in computerized medical diagnosis.

[A new approach to the diagnosis of epilepsy]. / Novyi podkhod k diagnostike épilepsii.

Clinical and biochemical investigation was performed in 49 patients with epilepsy of different etiology and clinical course, 10 patients with nonepileptic forms of neurologic and psychic disorders and 50 normal subjects. An additional blood serum protein fraction was found in epileptic patients as compared with the normals using the electrophoretic investigation on the acetate-cellulose plates and devices produced by Hélène France company. Additional biochemical study is suggested as a diagnostic tool in epilepsy.

[Use of proteins, covalently bound to agar gel, for specific sorption in electrophoresis]. / Primenenie belkov, kovalentno prisoedinennykh k geliu agara, dlia spetsificheskoi sorptsii v usloviiakh elektroforeza

A method is developed for electrophoretic fractionation of serum proteins in agar gel with simultaneous specific sorption of gamma-globulin fraction, migrating during the electrophoresis through specific immunosorbents incorporated into agar gel. The reaction was carried out in a special apparatus designed for the electrophoresis in two perpendicular directions. The sorbent was separated from impurities by passing the current in the first direction. The unspecific sorbent did not alter the electrophoretic mobility of migrating proteins, but the specific sorbents immobilized completely both antibodies and antigens. The antibodies were shown to bind repeatedly to the immunoglobulins, which had been linked to the sorbent. The method facilitated distinctly the estimation of antigens and antibodies in complicated mixtures. By analogy with affinity chromatography a term "affinity electrophoresis" was used for designation of the method described.

[Assessment of proteinograms obtained using the Beckman Paragon system]. / Otsenka proteinogrammy, vypolnennoi na sisteme Paragon firmy "Beckman".

A high resolving capacity of agarose gel in electrophoretic separation of blood serum proteins permits the isolation of 7 or 8 fractions instead of 5 routinely isolated by paper or acetate cellulose film electrophoresis. The strips on proteinograms obtained using Beckman Paragon device are identified. The authors emphasize that reference values should be determined for each laboratory and present the proteinograms of 80 normal subjects they obtained. If M-gradient appears on the proteinogram, the zone of its localization should be specified in comments.