In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.
The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H3 O+ and OH- ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pKa values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.
In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( μLeff(x)$\mu _{\rm{L}}^{{\rm{eff}}}( x )$ / κ(x)$\kappa ( x )$ values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, 675-88) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on. In this paper, we demonstrate some of the operational features of the tbdITP model using the new ITP/IEF version of Simul 6.
Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pKa values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pKa values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pKa and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download).
Electrophoretic focusing on an inverse electromigration dispersion (EMD) profile is based on a principle different from those of other electrophoretic separation methods. It has already proved its applicability in analytical practice by offering competitive separation performance and sensitivity and specific selectivity. It can be classified as an intermediate between field-driven and equilibrium gradient methods and is therefore interesting from the viewpoint of theory of separation methods. This work presents a comprehensive theoretical description of electrophoretic focusing on an inverse EMD profile comprising properties of the electrolyte system, formed gradients, and focused analyte zones. The separation properties are described in terms of resolution and peak capacity and their dependence on system and analyte properties is discussed from the viewpoint of how the counteracting phenomena of electromigration and dispersion are affected by electric current, voltage, and hydrodynamic and electroosmotic flow. The overall performance of the present method is shown to be comparable with other electrophoretic separation methods like zone electrophoresis or isoelectric focusing.
Úvod: Nemoc těžkých řetězců gama je vzácné onemocnění dosud popsané přibližně u 150 případů. Cílem práce byla laboratorní diagnostika nemoci těžkých řetězců imunoglobulinu. Materiál a metody: Do FN Ostrava byl referován 60letý pacient pro suspektní lymfom marginální zóny z biopsie žaludku. U pacienta byla doplněna stagingová vyšetření vč. trepanobiopsie kostní dřeně a PET/CT. Ze speciálních vyšetření byla požadována elektroforéza sérových proteinů, imunofixační elektroforéza, stanovení polyklonálních imunoglobulinů, volných lehkých řetězců a párů těžkých/lehkých řetězců imunoglobulinů. Z důvodu nejasného nálezu byla doplněna izoelektrická fokusace v agarózovém gelu s následným afinitním imunoblottingem a SDS elektroforéza. Výsledky: V kostní dřeni bylo nalezeno 0,1 % plazmatických buněk, z toho 87 % klonálních (patologických) plazmocytů, s následujícím imunofenotypem: cyt LAMBDA+ CD38+ CD138+ CD45+ CD19+ CD56– CD27+ CD81– CD117–. V séru pacienta byly nalezeny monoklonální těžké řetězce gama. V moči monoklonální těžké ani lehké řetězce imunoglobulinu detekovány nebyly. PET/CT vyšetření prokázalo generalizovanou lymfadenopatii, splenomegalii a nehomogenní akumulaci fluorodeoxyglukózy v axilárním i apendikulárním skeletu, nicméně bez přítomnosti typických osteolytických ložisek. Závěr: Monoklonální těžké řetězce imunoglobulinů jsou vzácným onemocněním. Pro jejich potvrzení je nutné použít na rozdíl od průkazu kompletní molekuly paraproteinu další pomocné metody. Nález monoklonálního těžkého řetězce gama v séru studovaného pacienta souvisí s přítomností lymfomu marginální zóny, který byl prokázán z biopsie žaludku.
Background: Gamma-heavy chain disease is a rare disease, described so far in approximately 150 cases. The aim of this work was laboratory diagnostics of immunoglobulin heavy chain disease. Materials and methods: A 60-year-old patient was referred to the University Hospital in Ostrava for suspected marginal zone lymphoma from gastric biopsy. Staging examinations including bone marrow trepanobiopsy and PET/CT were added; special examinations required serum protein electrophoresis, immunofixation electrophoresis, determination of polyclonal immunoglobulins, free light chains, and immunoglobulin heavy/light chain pairs. Isoelectric focusing in agarose gel followed by affinity immunoblotting and SDS electrophoresis was added due to unclear findings. Results: 0.1 % of plasma cells were found in the bone marrow, of which 87 % were clonal (pathological) plasma cells, followed by the cyt cytotype LAMBDA + CD38 + CD138 + CD45 + CD19 + CD56- CD27 + CD81- CD117-. Monoclonal heavy chains were found in the patient‘s serum. No monoclonal immunoglobulin heavy or light chains were detected in urine. The PET/CT examination showed generalized lymphadenopathy, splenomegaly and inhomogeneous accumulation of fluorodeoxyglucose in axillary and appendicular skeleton, but without the presence of typical osteolytic lesions. Conclusion: Monoclonal heavy chains of immunoglobulins are a rare disease. In contrast to the detection of a complete paraprotein molecule, additional methods must be used to confirm them. The finding of monoclonal heavy chain gamma in the serum of the study patient is related to the presence of marginal zone lymphoma, which was proven from a gastric biopsy.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- elektroforéza metody MeSH
- isoelektrická fokusace metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoc z těžkých řetězců * diagnóza patologie MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
The study aimed to investigate free light chain (FLC) monoclonality in patients with an abnormal free kappa/lambda ratio (FLC ratio). Seventy serum samples with abnormal FLC ratio were examined using an immunoturbidimetry (Binding Site, SPA) and the two different enzyme-linked immunosorbent assays (1. Sebia diagnostic kit; 2. in house methods), the monoclonal or oligoclonal bands of (FLC) by immunofixation electrophoresis (IE) and isoelectric focusing followed by affinity immunoblotting (IEF/AIB). The reference interval was calculated by non-parametric percentile method. 5.7% of samples examined by IE were suspected of monoclonal character of FLCs, but subsequently monoclonality was refuted by more sensitive IEF/AIB method; 7%, resp. 2.9% of samples showed FLC kappa, resp. FLC lambda oligoclonal character of bands. A statistically significant dependence was found between FLC ratio (Sebia) and FLC ratio (SPA), rs = 0.510, p = .001. Kappa statistic evaluated a fair conformity between the FLC ratio (Sebia) and IEF/AIB (kappa = 0.468) and between FLC ratio (in house) and IEF/AIB (kappa = 0.300). The verified reference interval for FLC ratio (Binding Site) is between 0.35 and 2.18. The IEF/AIB is the most sensitive method to discriminate between monoclonal and oligoclonal bands of FLC. The Binding Site and Sebia diagnostic kits do not give consistent results. The Binding Site diagnostic kit provides more results above reference interval of FLC ratios. For routine decision on monoclonality of the FLC ratio (SPA) it is advisable to use a verified reference interval.
- MeSH
- dospělí MeSH
- imunoglobuliny - lambda-řetězce analýza imunologie MeSH
- isoelektrická fokusace metody MeSH
- krevní proteiny analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- monoklonální protilátky analýza imunologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL-1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.
- MeSH
- Escherichia coli izolace a purifikace MeSH
- infekce močového ústrojí mikrobiologie moč MeSH
- isoelektrická fokusace metody MeSH
- lidé MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Staphylococcus aureus izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy.
BACKGROUND AND AIMS: Detection of oligoclonal IgG (o-IgG) in the cerebrospinal fluid (CSF) not found in serum is the principal laboratory test to support a diagnosis of multiple sclerosis. The aim of this study was to compare chemiluminescent and chromogenic detection of oligoclonal immunoglobulins in the cerebrospinal fluid and serum after their separation by means of isoelectric focusing followed by immunoblotting. METHODS: A set of experiments was designed to detect oligoclonal immunoglobulins by means of alkaline phosphatase BCIP/NBT substrate and chemiluminescent peroxidase substrate. RESULTS: Based on visual evaluation of signals, chemiluminescent detection requires about a 4 times lower amount of applied protein than very sensitive BCIP/NBT chromogenic detection. Very good correlation between methods has been shown for oligoclonal IgG. Antigen-specific oligoclonal IgG could be demonstrated by both methods although the pattern was clearer using chemiluminescence. In one patient, oligoclonal IgD bands barely visible by BCIP/NBT were convincingly demonstrated by chemiluminescence. CONCLUSION: Chemiluminescent detection is a feasible option for oligoclonal immunoglobulin detection and could be used in cases when the sensitivity needs to be improved. Further studies and method optimisation are warranted.
