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Concept: Electrophoresis

173

The novel heavy/light chain (HLC) assay was used for the detection and measurement of monoclonal immunoglobulins, response evaluation and prognostication. This test allows identification and quantification of the different light chain types of each immunoglobulin class (for example, IgGκ and IgGλ) and enables calculation of ratios of monoclonal/polyclonal immunoglobulin (HLC ratio). Sequential sera of 156 patients with IgG or IgA myeloma started on first-line therapy and followed for a median of 46.1 months were analyzed. Results were compared with those obtained with conventional techniques (serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), nephelometry (NEPH), and the free light chain test (FLC)). Our data show that the HLC assay allowed quantification of monoclonal proteins not accurately measurable by SPEP or NEPH. When both HLC and FLC testing were applied for response assessment, clonal excess was noted in 14/31 patients with complete response (CR). HLC ratio indicated presence of disease in 8/31 patients who achieved CR and, in sequential studies indicated evolving relapse in three patients before IFE became positive. Highly abnormal HLC ratios at presentation were significantly associated with shorter overall survival (40.5 months vs median not reached, P=0.016). Multivariate analysis revealed HLC ratio (P=0.03) and β(2)-microglobulin (P<0.01) as independent risk factors for survival.

Concepts: Immune system, Protein, Multiple myeloma, Electrophoresis, Protein electrophoresis, Immunoglobulin light chain, Paraprotein, Serum protein electrophoresis

28

High resolution digital imaging systems were recently introduced to capture and visualize serum protein electrophoresis results. In this study, we compared the performance of five, experienced interpreters using digital images and physical gels to identify and characterize monoclonal gammopathies by immunofixation.

Concepts: Gel electrophoresis, Computer graphics, Electrophoresis, Digital photography, Monoclonal gammopathy of undetermined significance, Protein electrophoresis, Digital image, Serum protein electrophoresis

28

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.

Concepts: Protein, Molecular biology, Western blot, Gel electrophoresis, Electrophoresis, Laboratory techniques, Two-dimensional gel electrophoresis, Isoelectric focusing

28

We present a comprehensive review and comparison of the methodologies for increasing sensitivity and resolution of capillary electrophoresis (CE) using online transient isotachophoresis (tITP). We categorize the diverse set of coupled tITP and CE (tITP-CE) methods based on their fundamental principles for disrupting isotachophoretic preconcentration and triggering electrophoretic separation. Based on this classification, we discuss important features, advantages, limitations, and optimization principles of various tITP-CE methods. We substantiate our discussion with original simulations, instructive examples, and published experimental results.

Concepts: Sensitivity and specificity, Gel electrophoresis, Debate, Capillary electrophoresis, Electrophoresis, Binary classification, Affinity electrophoresis, Isotachophoresis

28

The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration, and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

Concepts: Monoclonal antibodies, Gel electrophoresis, Mixture, Chromatography, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis

28

In this contribution we present an innovative way to easy, fast and highly sensitive analyses by capillary electrophoresis with electrospray ionization mass spectrometric (ESI-MS) detection. The new method is designed to be applied to ESI-compatible electrolytes (e.g. ammonium acetate) and offers advanced tuning of selectivity conditions within a wide range of analyte mobilities. We use a full capillary isotachophoresis (ITP) format to provide powerful on-line analyte stacking at the ITP boundary all the way to detection and introduce the model of extended ITP where a controlled concentration of the leading ion is added to the terminating zone. Such systems preserve all properties of an ITP system and the velocity of the stacking ITP boundary can be tuned by the composition of both the leading and terminating zone. In this way the system properties can be controlled flexibly and the mobility window of stacked analytes can be tailored to actual needs. The presented theory and the newly defined concept of zone-related boundary mobility allow easy assessment of system selectivity using simple diagrams. We demonstrate the model and its potential on the example of simple acidic cationic systems composed of only two substances (ammonium and acetate) including the example of thiabendazole analysis with a detection limit of 10(-10) M (20 ng/L) and its determination in orange juice by direct sampling after filtration, selective stacking by a tuned extended ITP system and ESI-MS detection.

Concepts: Mass spectrometry, Electrophoresis, Electrospray ionization, Taylor cone, Ion source, Electrospray, Protein mass spectrometry, John Bennett Fenn

28

A new, sensitive, and robust analytical method based on capillary zone electrophoresis with on-line capillary isotachophoresis sample pretreatment (ITP-CZE) using a column-coupling (CC) arrangement of automated capillary electrophoretic analyzer was developed for determination of bromate in different type of drinking water samples. Both columns were provided with contact-less conductivity detectors and in CZE step UV detection at 200nm wavelength was used. Electroosmotic flow of the buffer solutions was suppressed with the addition of 0.1% or 0.05% (m/v) methylhydroxyethylcellulose into the leading and terminating electrolyte, respectively. Hydrodynamic and electroosmotic flows of the buffer solutions were successfully suppressed and therefore, only the electrophoretic transport of ions was significant. Limit of detection for bromate approaching 0.6μg/L was achieved. Good repeatabilities of migration time (RSD less than 0.3%) and peak area (RSD less than 4.0%) at concentration level 1μg/L were obtained. Robustness of proposed ITP-CZE method and validation parameters were evaluated. Developed automated ITP-CZE method was applied to the determination of bromate in drinking water samples with different content of inorganic macroconstituents without the need of further sample preparation.

Concepts: Water, Gel electrophoresis, Capillary electrophoresis, Electrophoresis, Micellar electrokinetic chromatography, Affinity electrophoresis, Capillary fringe, Isotachophoresis

28

Cumin (Cuminum cyminum L.), Fennel (Foeniculum vulgare L.) and Longleaf (Falcaria vulgaris Bernh) that all belong to Apiaceae family as medicinal plants are very important in many countries. Study of genetic diversity for medicinal plant is important for researches in future. One of the methods to evaluate plant genetic diversity and classification of them is the electrophoresis of seed storage proteins. This research was conducted in order to evaluate seed protein variability in different Iranian Cumin, Fennel and Longleaf accessions and grouping them based on these proteins as a biochemical marker. For this purpose, the samples were first powdered in liquid nitrogen and seed protein was extracted with extraction buffer. Then total soluble proteins were resolved on 12.5 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. The electrophoretic protein pattern showed 38 bands that were low polymorphism among the accessions. The result of cluster analysis showed that the accessions were classified in three groups (all 29 Cumin accessions in the first group, three Fennel ecotypes in second group and three Longleaf accessions in the last one).

Concepts: Protein, Molecular biology, Gel electrophoresis, Electrophoresis, Apiaceae, Medicinal plants, Gel, Caraway

28

Portable and field deployable analytical instruments are attractive in many fields including medical diagnostics, where point of care and on-site diagnostics systems capable of providing rapid quantitative results have the potential to vastly improve the productivity and the quality of medical care. Isotachophoresis (ITP) is a well known electrophoretic separation technique previously demonstrated as suitable for miniaturization in microfluidic chip format (chip-ITP). In this work, a purpose-designed ITP chip compatible with a commercial end-used targeted microfluidic system was used to study different injection protocols and to evaluate the effect of the length of the separation channel on the analytical performance. The in-house ITP chips were made from Corning glass and compared to the commercial DNA chip for the ITP separation of anions from the hydrodynamic injection of human serum. Using the in-house ITP chip the isotachophoretic step of lactate from human serum was approximately two times longer. The results of this research suggested that microfluidic ITP with indirect fluorescence detection is a viable technique for separation of organic acids in human serum samples, especially when a chip with suitable design is used.

Concepts: Acetic acid, Carboxylic acid, Electrophoresis, Lactic acid, Microfluidics, Organic acid, Isotachophoresis, Lab-on-a-chip

28

A neutral marker of the electroosmotic flow (EOF) can gain a nonzero effective mobility because of its possible interaction with a charged complexing agent, such as a chiral selector in capillary electrophoresis. We determined effective mobilities of four compounds often used as EOF markers (dimethyl sulfoxide, mesityl oxide, nitromethane and thiourea) in the background electrolyte (BGE) containing sulfated β-cyclodextrin (60g/L). All the compounds studied were measurably mobilized by their interaction with the selector. The highest effective mobility (-3.0·10(-9) m(2) s(-1) V(-1) ) was observed for thiourea and the lowest (-1.5·10(-9) m(2) s(-1) V(-1) ) for dimethyl sulfoxide and nitromethane. The mobilities were determined by a new two-detector pressure mobilization method (2d method), which we propose, and the results were confirmed by standard capillary electrophoresis measurements. In the 2d method, one marker zone is situated in the BGE containing the charged selector, while the second marker zone is surrounded with a selector free BGE, which prevents its complexation. The initial distance between the two marker zones equals the capillary length from the inlet to the first detector. After a brief voltage application, the final distance between the marker zones is determined based on known capillary length from the first to the second detector. The difference between these two distances determines the effective mobility.

Concepts: Gel electrophoresis, Ligand, Capillary electrophoresis, Electrophoresis, Affinity electrophoresis, Length, Distance, Gadolinium