One-pot analysis of sulfated glycosaminoglycans.

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

Preliminary Evaluation of a Point-of-Care Testing Device (SickleSCAN™) in Screening for Sickle Cell Disease.

Sickle cell disease affects about 150,000 births annually in Nigeria. Early diagnosis is hampered by factors such as centralized and urban localization of laboratories, high cost of diagnostic equipment and inadequate skilled manpower to operate them. The need for a low-cost, portable, easy-to-use diagnostic test for sickle cell disease is critical, especially in resource-poor countries. In this study, we evaluated the performance characteristics of a novel point-of-care testing device (SickleSCAN™), and its acceptability and feasibility, as a possible screening tool for sickle cell disease. In the first phase, we assessed the performance characteristics of SickleSCAN™ by evaluating 57 subjects comprising both children and adults attending a primary health center, for Hb SS (ßS/ßS; HBB: c.20A>T), Hb SC (ßS/ßC; HBB: c.19G>A) and Hb AS (ßA/ßS) using SickleSCAN™, cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC). Performance characteristics such as diagnostic sensitivity and specificity were compared to HPLC as a standard method. We subsequently undertook a second phase wherein the acceptability and feasibility of the device for sickle cell disease screening, was evaluated using semi-structured and structured questionnaires among 197 healthcare personnel and 221 subjects, respectively. Sickle cell disease was carried by 3.4% of the subjects. The diagnostic sensitivity, specificity and test efficiency of SickleSCAN™ for sickle cell disease (Hb SS and Hb SC), were 100.0, 98.2 and 98.2%, respectively. Findings from this study showed SickleSCAN™ to be a viable screening tool that can easily be applied in community-based screening for early diagnosis of sickle cell disease with little expertise and low cost.

Evaluation of usefulness of a commercial agarose gel electrophoresis kit (QuickGel SP) for bovine serum protein electrophoresis.

The aim of this study was to show the usefulness of a commercial agarose gel electrophoresis (AGE) kit (QuickGel SP) for separating bovine serum protein fractions in comparison with conventional cellulose acetate electrophoresis (CAE). Serum protein bands were verified using five reference reagents corresponding to albumin and α1-, ß1-, ß2-, and γ-globulins. AGE clearly revealed six separated fractions of albumin and α1-, α2-, ß1-, ß2-, and γ-globulin fractions in 100% and 77.8% in serum samples of dairy cows from the healthy (n=27) and diseased groups (n=27), respectively. The α1- and α2-globulins were not separated by CAE in 14.8% and 96.3% of the samples from the healthy and diseased groups, respectively, whereas ß2- and γ-globulin were not separated by CAE in 96.3% and 100% of the samples from the healthy and diseased groups, respectively. More than 94% of the points for the α-globulin fractions (α1- and α2-globulins), the ß-γ-globulin fractions (ß1-, ß2-, and γ-globulins), and the albumin/globulin ratio between AGE and CAE were within agreement on the Bland-Altman plots. However, the mean biases were not near zero in the albumin and ß-γ-globulin fractions. These results suggest that the high-resolution commercial AGE kit can be utilized to separate bovine serum protein fractions.

Electrophoretic separation of alginic sodium diester and sodium hexametaphosphate in chondroitin sulfate that interfere with the cetylpyridinium chloride titration assay.

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in cormbination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived fromn CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use methodfor detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CSby CAME and that exhibit CPC titration behavior similar to CS.

A simple cellulose acetate membrane-based small lanes technique for protein electrophoresis.

Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis.

Electrophoretic serum protein fraction profile during the different physiological phases in Comisana ewes.

The aim of this study was to evaluate the influence of different physiological phases on serum total proteins and their fractions of ten Comisana ewes housed in Mediterranean area. From each animal, blood samples were collected at different physiological phases: late pregnancy, post-partum, early, mid-, end lactation and dry period. On all samples serum total proteins were determined by the biuret method, and albumin, α-globulins, ß(1) -globulins, ß(2) -globulins and γ-globulins concentrations were assessed using an automated system. One-way repeated measures analysis of variance was applied to determine the significant effect of different physiological phases on the parameters studied. During the late pregnancy and post-partum, total proteins, ß1- and ß2-globulins and γ-globulins showed the highest values. Starting from post-partum, α-globulins increased to reach their peaks in mid-lactation. Early lactation was characterized by low γ-globulins values. The increase in serum albumin concentration and the drop in some globulin fractions determined the significant increase in albumin/globulin ratio. The obtained results contributed to improve the knowledge on electrophoretic profile during the different physiological phases in ewes, confirming that pregnancy and lactation periods affect the protein metabolism. Particularly, serum protein fractions pattern could give information about dehydration, plasma volume expansion and hepatic function, which occur during the different physiological phases. Dynamics of the protein profile - from pregnancy to dry period - which are provided by our results, could be considered as guidelines for the management strategies to guarantee the nutritional needs of these animals during the different physiological phases and to avoid a decline of productive performance and consequently an economic loss.

[The comparative analysis of electrophoretic fractionating of blood serum proteins in diagnostics of multiple plasma cell myeloma].

The choice of technology of electrophoretic fractionating of blood serum proteins is determined, besides the analytical characteristics, by its economic component. The electrophoresis technologies developed by the R&D production facility "Astra" (Russia) and the firm "PZ Cormay S.A." (Poland) are compared from a viewpoint of applicability in routine laboratory, practice and diagnostics of multiple plasma cell myeloma in particular. It is established that under the comparable economic, "consumer" and analytic characteristics of technologies in the diagnostic process the application of the technology in agarose gel ("PZ Cormay S.A.") is more preferable.

One-dimensional cellulose acetate plate electrophoresis--a feasible method for analysis of dermatan sulfate and other glycosaminoglycan impurities in pharmaceutical heparin.

A cellulose acetate plate electrophoresis method for analysis of pharmaceutical heparin and its potential glycosaminoglycan impurities, e.g. dermatan sulfate, chondroitin sulfate and oversulfated chondroitin sulfate, is presented. Heparin is chemically degraded by application of nitrous acid and residual glycosaminoglycans are electrophoretically separated thereafter. After staining using Alcian blue 8GS, these glycosaminoglycan impurities can be quantified by means of comparison to a dermatan sulfate standard. Results of a validation study of this analytical method are shown, demonstrating its feasibility for routine use in analytical quality control labs under GMP conditions.

Cellulose Acetate Electrophoresis of Hemoglobin.

The electrophoresis on cellulose acetate membrane is most widely used because of its simplicity, and is without the use of any sophisticated instrument other than electrophoresis apparatus and the cellulose acetate strip. Here we describe a modified version of cellulose acetate membrane electrophoresis for hemoglobin separation from blood sample. Sharp, clear bands without tailing effects can be obtained with this method. The method and apparatus described here would be appropriate to separate protein fractions under 1 h at voltages up to 60 V/cm measured between the electrodes.

Assessment of serum protein fractions in rainbow trout using automated electrophoresis and densitometry.

BACKGROUND: Although rainbow trout (Oncorhynchus mykiss, Walbaum) are one of the most-studied fish, electrophoretic techniques and classification of serum protein fractions have not been standardized, such that clinically useful values are lacking. OBJECTIVE: The aim of the present study was to evaluate preliminarily the serum protein fractions of rainbow trout using automated cellulose acetate electrophoresis and densitometry. METHODS: Serum samples from 25 rainbow trout (Oncorhynchus mykiss, Walbaum) were electrophoresed on cellulose acetate plates and quantified using densitometry. RESULTS: A maximum of 6 fractions were identified and numbered, in order of decreasing mobility, as I, II, III, IV, V, and VI. In 3 of 25 (12%) samples, 6 fractions were identified; in 18 (72%) samples, 5 fractions were identified; and in 4 (16%) samples, 4 fractions were identified. Fractions I, V, and VI were always clearly identifiable, whereas fractions II and IV were frequently fused and indistinguishable from fraction III. The pattern with 5 fractions was the most probable type (chi(2), P<.01). The mean (+/-SEM) protein concentrations of the 6 fractions were I, 0.8+/-0.1 g/dL; II, 0.3+/-0.0 g/dL; III, 1.6+/-0.1 g/dL; IV, 0.3+/-0.1 g/dL; V, 0.6+/-0.0 g/dL; and VI, 0.2+/-0.0 g/dL. Based on comparison of serum and plasma electrophoretic patterns from 8 fish, fibrinogen was found in fraction V. CONCLUSION: Automated cellulose acetate electrophoresis and densitometry appear to be a practical method for estimation of serum protein fractions in rainbow trout.

Improved method for analysis of glycosaminoglycans in glycosaminoglycan/protein mixtures: application in Cohn-Oncley fractions of human plasma.

BACKGROUND: Glycosaminoglycans are found in human tissues including plasma. They encompass chondroitin sulphates, heparan sulphate/heparin, hyaluronic acid, and keratan sulphate. Glycosaminoglycans, in particular heparan sulphate and heparin, are strongly associated with plasma proteins, so that their purification results quite difficult. METHODS: In order to study the distribution of glycosaminoglycans in plasma subfractions, we developed a novel method that allows their identification even if they were still associated with proteins or peptides. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated with proteases. After centrifugation, glycosaminoglycan/protein complexes in the supernatant were analysed using a modified cellulose acetate electrophoresis which allowed identification of glycosaminoglycans in mixtures of glycosaminoglycans/proteins. RESULTS: Chondroitin sulphate was recovered in cryoprecipitate and in all Cohn-Oncley fractions. Glycosaminoglycans belonging to the class of heparan sulphate/heparin, however, were recovered in the cryoprecipitate and in fractions I and IV-1, and, in smaller amount, in fraction II+III. CONCLUSIONS: Since the largest amount of plasma proteins is partitioned in Factions II+III and V, these results demonstrate that heparan sulphate/heparin are not randomly distributed in Cohn-Oncley fractions and are associated with certain plasma proteins. This association might play a role in the physiological function of heparan sulphate/heparin, regulating hemostasis and atherogenesis.

Rapid identification of clinical Trichoderma longibrachiatum isolates by cellulose-acetate electrophoresis-mediated isoenzyme analysis.

Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorphism among ten clinical and 11 non-clinical isolates of Trichoderma. Initial testing of 13 enzyme systems for activity and resolution of bands showed that seven were appropriate for identifying the different species. Each of the enzyme systems investigated (glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidases A, B and D, and phosphoglucomutase) was diagnostic for at least one species. On the basis of the results of isoenzyme analysis, several isolates identified originally as Trichoderma pseudokoningii, T. koningii or T. citrinoviride were re-identified as T. longibrachiatum, in agreement with sequence analysis data for the internal transcribed spacer region of the isolates. The availability of a quick, inexpensive and reliable diagnostic tool for the identification of T. longibrachiatum isolates is important, as most clinical Trichoderma isolates belong to T. longibrachiatum. Furthermore, as many different enzyme systems are available, the method may also be suitable for the identification of other clinically relevant fungal species.

Ion-imprinted silica adsorbent modified diffusive gradients in thin films technique: Tool for speciation analysis of free lead species.

A new diffusive gradients in thin films (DGT) device, using Pb(II) ion-imprinted silica (IIS) as the binding agents and commercial cellulose acetate dialysis (CAD) membrane as the diffusion layer (CAD/IIS-DGT), has been developed and evaluated for sampling and measurement of free Pb(II) species. The CAD/IIS-DGT devices were successfully applied to the measurement of free Pb(II) species in synthetic solutions, in natural freshwaters and in industrial wastewaters. The CAD/IIS-DGT provides reliable results over pH range of 4.5-6.5 and a wide range of ionic strength from 1.0×10(-3) to 0.7 mol L(-1). The concentrations of the free Pb(II) species in synthetic solution containing different concentrations of ligands measured by CAD/IIS-DGT showed a good agreement with the value measured by Pb-ion selective electrode. Field deployments of the CAD/IIS-DGT devices allowed accurate measurements of the concentrations of free Pb(II) species.

Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel electrophoresis.

Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.

Molecular taxonomic study of Chagas disease vectors from the phyllosoma, lecticularia, and rubrofasciata complexes.

The Triatiominae (Hemiptera: Reduviidae) are hematophagous hemipters of importance because they transmit Trypanosoma cruzi, the causal agent of Chagas disease. The aim of this study was to define the possible relationships between species of the Phyllosoma complex (Triatoma mazzottii, Triatoma pallidipennis, and Triatoma longipennis) and species of other complexes present in Mexico that have not been previously analyzed (Triatoma lecticularia and Triatoma rubida). In addition, it was determined the inclusion of Triatoma bassolsae in the Phyllosoma complex by using 10 isoenzymatic systems (corresponding to the 14 loci). Results of isoenzymatic study show that between the species of the Phyllosoma complex including Triatoma bassolsae, the polymorphism of the analyzed enzymes ranges from 14% to 50% (P < or = 0.95) and the species from external complexes showed polymorphism values of 43% (Triatoma lecticularia), 43% (Triatoma rubida), and 36% (Triatoma infestans). The genetic tree shows a clear difference between species of the Phyllosoma complex and the other complexes.

Atypical clinical variants in New World cutaneous leishmaniasis: disseminated, erysipeloid, and recidiva cutis due to Leishmania (V.) panamensis.

In recent times, there has been an increase in the number of reports for new and rare variants of cutaneous leishmaniasis (CL). Here, we describe three unusual clinical forms of CL identified in Ecuadorian children. A total of 131 patients with CL were diagnosed over a 2-year period of active search. In 3 (2.29%), the lesions were very unusual; these included erysipeloid, recidiva cutis (LRC), and disseminated leishmaniasis (DL). The erysipeloid case is characterized by erythematous and indurated plaque seen on the face of a 5-year-old boy; the LRC one is differentiated by slowly progressing red-brown papules around large scars of healed sores in a 6-year-old girl, and the DL case is characterized by dozens of cutaneous ulcers distributed in the whole body of a 1-year-old girl. Leishmania parasites were isolated by lesion aspirate and analyzed by the technique multilocus enzyme electrophoresis (MLEE). All three isolates were identified as Leishmania (Viannia) panamensis. These distinct clinical variants rarely have been reported previously in the American cutaneous leishmaniasis, and for the first time L. (V.) panamensis was identified as the etiologic agent. Our cases extend the spectrum of clinical presentations in New World leishmaniasis.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes.

Alcian blue staining has been widely used to visualize acidic mucins and mucopolysaccharides in supported molecular matrix electrophoresis (SMME) and on membrane transferred from electrophoresis gels. Mucins with low acidic glycan content, however, cannot be stained with Alcian blue, which is one of the major drawbacks of this staining method. On the other hand, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content; however, periodic acid-Schiff staining decomposes glycans. Here, we introduce succinylation-Alcian blue staining as an alternative staining method to visualize mucins, regardless of the acidic residue content, and without glycan decomposition.

Instantaneous roll-blot from cellulose acetate after electrophoresis. A versatile tool for monoclonal antibody characterization.

An instantaneous blotting method from cellulose acetate to nitrocellulose was developed using a high pressure roll apparatus. This method was applied to the early characterization of monoclonal antibody specificity and to monoclonal immunoglobulin typing in mouse hybridoma supernatants, human sera or unconcentrated urines. Immunoglobulins were then revealed using, successively, anti-isotype specific monoclonal or polyclonal antibodies, avidin-biotinylated peroxidase complexes and cobalt-enhanced diaminobenzidine substrate.

Thin layer gel filtration immunofixation: identification of abnormal molecular weight immunoglobulins or related fragments.

A simple procedure is described to identify immunoglobulin (Ig) fragments without isolation from biological fluids. It involves an initial separation based on molecular weight (MW) by thin layer gel filtration (TLG) followed by immunofixation (IF) in cellulose acetate strips with monovalent antisera. TLG-IF permits detection of differences about 10,000 Da MW and specific immunological typing, making it a useful tool in the accurate identification of protein fragments.

Trypanosoma cruzi: long-term sub-cultures in two different culture media do not confirm the existence of highly versatile multilocus genotypes.

Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T. cruzi IIb and T. cruzi IId, respectively. Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media. We tried to reproduce this observation, but used a different cloning method of individual organisms. Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only. The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes. The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.