| 1. |
- Hjerten, Maria, et al.
(författare)
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Renewable enzyme reactors based on beds of artificial gel antibodies.
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1188-1191
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach is described for the synthesis of beds for enzyme reactors. The method is based on the use of artificial antibodies in the form of polyacrylamide gel particles with diameters around 0.1-0.3 mm. These gel particles mimic protein antibodies, raised in experimental animals, in the sense that they selectively recognize and adsorb only the protein present during the preparation of the "antibodies". The gel antibodies have several advantages over conventional protein antibodies, which can be taken advantage of in the design of enzyme reactors; for instance, if upon prolonged use the immobilized enzyme loses its activity it can easily be replaced by an active enzyme, which is not possible when the enzyme is immobilized via a conventional protein antibody (a new bed with immobilized protein antibodies must be prepared); and equally or more remarkable: the enzyme can be applied in the form of a non-purified extract since the selectivity of the artificial gel antibodies is so high that they will "fish-out" the enzyme, but no other proteins in the extract. In addition, no preconcentration of the enzyme solution is required prior to the immobilization, since the enzyme is enriched at the top of the column upon the application. These unique properties make enzyme reactors based on artificial gel antibodies very attractive, also in process chromatography. The potential application range of the artificial gel antibodies is enormous since the same method for their synthesis can be used independent of the structure and the size of the "antigen"; for instance, renewable biosensors based on gel antibodies for the selective detection of protein biomarkers, as well as pathogenic viruses, bacteria, and spores (for instance Anthrax) should not be difficult to design.
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| 2. |
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| 3. |
- Bacskay, Ivett, et al.
(författare)
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Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria). III : Gel antibodies against cells (bacteria)
- 2006
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:23, s. 4682-4687
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Tidskriftsartikel (refereegranskat)abstract
- Artificial antibodies in the form of gel granules were synthesized from the monomers acrylamide and N,N'-methylenebisacrylamide by the imprinting method in the presence of Echerichia coli bacteria as template. The electrophoretic migration velocities of the gel antibodies (i) saturated with the antigen (Escherichia coli MRE-600), (ii) freed of the antigen, and (iii) resaturated with bacteria, were determinated by electrophoresis in a rotating narrow-bore tube of 245 mm length and the 2.5 and 9.6 mm inner and outer diameters, respectively. Removal of bacteria from the gel antibodies was made by treatment with enzymes, followed by washing with SDS and buffer. Gel granules becoming charged by adsorption of bacteria move in an electrical field. We obtained a significant selectivity of gel antibodies for E coli MRE-600, since the granules did not interact with Lactococcus lactis; and when E coli BL21 bacteria were added to the gels selective for E coli MRE-600, a significant difference in the migration rate of the complexes formed with the two strains was observed indicating the ability of differentiation between the two strains. The gel antibodies can be used repeatedly. The new imprinting method for the synthesis of artificial gel antibodies against bioparticles described herein, and the classical electrophoretic analysis technique employed, thus represent - when combined - a new approach to distinguish between different types and strains of bacteria. The application area can certainly be extended to cover other classes of cells.
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| 4. |
- Drevinskas, Tomas, et al.
(författare)
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Chromatographic Data Segmentation Method : A Hybrid Analytical Approach for the Investigation of Antiviral Substances in Medicinal Plant Extracts
- 2019
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Ingår i: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 91:1, s. 1080-1088
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Tidskriftsartikel (refereegranskat)abstract
- The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) beta-cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) alpha-thujone, beta-thujone, cis-p-menthan-3-one, and estragole show cytotoxic effects.
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| 5. |
- Farkas, Viktor, et al.
(författare)
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General approach for certain quantitative calculations for Instance of the variance of reversible adsorption to the capillary wall in CE
- 2009
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:1, s. 343-348
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Tidskriftsartikel (refereegranskat)abstract
- Miniaturization of analytical separation methods offers several advantages, including short run times, high resolution, and high recovery of the sample constituents. To optimize these parameters, the reversible adsorption (to minimize loss in resolution), as well as the irreversible adsorption (to minimize loss of analytes) must be quantified. However, no useful equation is available for the calculation of the variance of reversible adsorption. Therefore, we have taken another approach to quantify the reversible interaction. The method is unique and important since no equation for calculation of this variance is required. Instead, two experiments are required, which are run under such conditions that the variance of a certain parameter has the same numerical value in the two experiments (one with and without EOF), except for the variance of reversible adsorption. The approach is universal in the sense that it can be used for many different mathematical concepts and be modified to also cover certain functions other than a sum of parameters. We have also introduced a simple expression for the irreversible adsorption, which shows that the hydrophobic interaction from only two methyl groups in the coating gives rise to as much as 40-50% loss of protein, and the width of the zones in the capillary with this coating was 8-15% larger compared to the zone width in the polyacrylamide-coated capillaries. The reproducibility in migration time, peak area, and peak width in two consecutive runs in capillaries with two methyl groups in the coating was very low, but in EOF-free polyacrylamide-coated capillaries extremely high, indicating that the reversible and irreversible adsorption of proteins to this coating is negligible. The scanning detector, frequently used in free zone electrophoresis in the 1960s-1970s, gives true separation parameters and is, therefore, much preferable to the stationary detector used in most CE experiments, because this detector gives apparent separation parameters.
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| 6. |
- Ghasemzadeh, Nasim, et al.
(författare)
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Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples : I. Spectrophotometric approach to design the calibration curve for the quantification
- 2008
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 31:22, s. 3945-3953
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Tidskriftsartikel (refereegranskat)abstract
- High selectivity of a biomarker is a basic requirement when it is used for diagnosis, prognosis and treatment of a disease. The artificial gel antibodies, which we synthesise by a molecular imprinting method, have this property not only for proteins, but also for bioparticles, such as viruses and bacteria. However, diagnosis of a disease requires not only that the biomarker can be "fished out" from a body fluid with high selectivity, but also that its concentration in the sample can rapidly be determined and preferably by a simple technique. This paper deals primarily with the development of a spectrophotometric method, which is so simple and fast that it can be used with advantage in a Doctor's Office. The development of this method was not straight-forward. However, by modifications of the performance of these measurements we can now design standard curves in the form of a straight line, when we plot the true (not the recorded "apparent" absorption) against known protein concentrations. In an additional publication (see the following paper in this issue of JSS) we show an application of such a plot: determination of the concentration of albumin in serum and cerebrospinal fluid from patients with neurological disorders to investigate whether albumin is a biomarker for these diseases.
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| 7. |
- Ghasemzadeh, Nasim, et al.
(författare)
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Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples : II. Albumin in body fluids of patients with neurological disorders
- 2008
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 31:22, s. 3954-3958
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Tidskriftsartikel (refereegranskat)abstract
- We have previously used the molecular-imprinting method for the synthesis of artificial gel antibodies, highly selective for various proteins. In the present work, we have synthesized artificial gel antibodies against human albumin with the aim to develop a simple and rapid procedure to measure the concentration of this protein in samples of clinical interest. The procedure, based on the design of a standard curve (see the preceding paper), was applied on a quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF). We found that our technique permitted detection of albumin in these body fluids with high precision and that the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. This finding is in agreement with results from earlier studies, which confirms the validity of our analysis technique and suggests that the barrier permeability may be affected in ALS, perhaps also for other proteins. No enhancement in plasma levels of albumin was seen in patients with ALS, but rather a decrease. The results further indicate that our approach might also apply well to other biomarkers for the actual neurological disease and other disorders.
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| 8. |
- Ghasemzadeh, Nasim, et al.
(författare)
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Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics
- 2010
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 31:16, s. 2722-2729
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Tidskriftsartikel (refereegranskat)abstract
- Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii-vii) and how to overcome them, which also may help to overcome the difficulty (ti), which in turn minimizes difficulty (i).
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| 9. |
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
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| 17. |
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| 18. |
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| 19. |
- Rezeli, Melinda, et al.
(författare)
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A new approach for on-line enrichment in electrophoresis of dilute protein solutions.
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1098-1103
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Tidskriftsartikel (refereegranskat)abstract
- A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.
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| 20. |
- Rezeli, Melinda, et al.
(författare)
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Monolithic beds of artificial gel antibodies
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1109:1, s. 100-102
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Tidskriftsartikel (refereegranskat)abstract
- The introductions of the continuous beds, now often called monoliths [S. Hjertén, J.-L. Liao, R. Zhang, J. Chromatogr. 473 (1989), 273–275] and the artificial, highly selective gel antibodies against antigens as large as proteins, viruses and cells [J.-L. Liao, Y. Wang, S. Hjertén, Chromatographia 42 (1996), 259–262] were breakthroughs in the design of chromatographic beds. This paper deals with a combination of these two methods, i.e., the artificial gel antibodies have been synthesized in the monolithic mode. As antigen we have used human hemoglobin. A comparison of the ion-exchange chromatograms of the eluates from the monolithic columns shows that the monolith prepared in the presence of hemoglobin adsorbed this protein, but not the other proteins in the sample (ribonuclease A and cytochrome c), i.e., this monolith was selective for hemoglobin, whereas the blank column (prepared in the absence of hemoglobin) had no selective properties, since none of the applied proteins were adsorbed. The diameter of the column was 6 mm, but the same approach to synthesize a monolithic selective bed can very likely also be used for capillaries and microchips.
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| 21. |
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| 22. |
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| 23. |
- Takatsy, Aniko, et al.
(författare)
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Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria) : Ia. Gel antibodies against proteins (transferrins)
- 2006
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 29:18, s. 2802-2809
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Tidskriftsartikel (refereegranskat)abstract
- Artificial antibodies in the form of gel granules were prepared by the molecular imprinting technique from the monomers acrylamide and N,N-methylene-bis-acrylamide. Gel granules, freed from the selectively adsorbed protein (the antigen), are neutral and, accordingly, do not migrate in an electrical field. However, upon selective interaction with the antigen at a pH different from its pI, the granules become charged. The selectivity of the gel antibodies was studied by free zone electrophoresis in a tube with inside diameter larger than the size of the granules. Such electrophoretic analyses showed that gel antibodies against iron-free transferrin had a high selectivity for this protein, although some crossreaction took place with iron-saturated transferrin, indicating that these artificial antibodies can easily distinguish the minute differences in the 3-D structure of the transferrins. Analogously, gel antibodies against iron-saturated transferrin were highly selective for this protein with some crossreaction with iron-free transferrin. The mobilities of iron-free and iron-saturated transferrin are very similar, and, therefore, capillary free zone electrophoresis cannot distinguish between these structurally related proteins. However, significant differences in the mobilities of the selective gel granules can be observed depending on their interaction with iron-free or iron-saturated transferrin, i.e., the artificial gel antibodies may become powerful analytical tools.
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| 24. |
- Takatsy, Aniko, et al.
(författare)
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Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria) : II. Gel antibodies against virus (Semliki Forest Virus)
- 2006
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 29:18, s. 2810-2815
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Tidskriftsartikel (refereegranskat)abstract
- Artificial and highly selective antibodies (in the form of gel granules) against proteins can easily be synthesized by a simple, cost-effective imprinting technique [Liao, J.-L. et al., Chromatographia 1996, 42, 259-262]. Using the same method for synthesis of gel antibodies against viruses in combination with analysis by free zone electrophoresis in a rotating narrow bore tube we have shown that artificial gel antibodies against Semliki Forest Virus (wild type) can sense the difference between this virus and a mutant, although they differ in their chemical composition only by three amino acids in one of the three proteins on the surface of the virus particle. The reason for this extremely high resolution is explained by the fact that we use three types of selectivity: (i) shape selectivity (created by the close fit between the antigen and its imprint in the gel), (ii) bond selectivity in the contact area between the antigen and its imprint in the gel antibody, and (iii) charge selectivity, originating from slightly different structures or/and conformations of the antigens.
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| 25. |
- Takatsy, Aniko, et al.
(författare)
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Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins viruses and cells, (bacteria). : lb. Gel antibodies against proteins (hemoglobins)
- 2007
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 28:14, s. 2345-2350
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Tidskriftsartikel (refereegranskat)abstract
- Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper la, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.
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| 26. |
- Végvári, Ákos, et al.
(författare)
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A Hybrid Microdevice for Electrophoresis and Electrochromatography Using UV-Detection
- 2002
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Ingår i: Electrophoresis. - 0173-0835 .- 1522-2683. ; 23:20, s. 3479-3486
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Tidskriftsartikel (refereegranskat)abstract
- We have designed a new class of microdevices composed of a supporting plastic (polyvinyl chloride, PVC) plate integrated with a groove for a piece of fused silica capillary (the separation channel), a slit for on-tube detection, an "islet" for the application of sample, electrode vessels and platinum electrodes. The design permits electrophoretic, electrochromatographic and chromatographic separations with on-tube UV detection. The efficient heat dissipation allows relatively high field strengths. This article is the first one dealing with microdevices where polymer solutions are replaced by homogeneous gels. A new type of gels synthesized from acrylamide and 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) as a cross-linker was employed for electrophoresis and electrochromatography. 2-Acrylamido-2-methylpropanesulfonic acid was added to the monomer solution to create a high electroendosmotic flow in electrochromatographic runs. These gels have excellent electrochromatographic and electrophoretic properties for low-molecular-weight compounds and DNA, as shown previously, namely high resolution combined with high stability. The unique cross-linker can be used for specific interaction with the alkyl and phenyl groups. The tripeptide glutathione (gamma-L-glutamyl-L-cysteinyl-glycine) and its benzyl conjugates were selected as model compounds to study the resolving power of the gel because they are difficult to separate by free zone electrophoresis. The limit of detection (LOD) for S-benzyl-glutathione was determined (ca. 7 microM). Run-by-run reproducibility was high (the separation factor of glutathione in the gel was 0.3 with 2.5% coefficient of variation, CV). Neutral compounds (acetone, acetophenone, propiophenone and butyrophenone) were separated electrochromatographically in the gel. The influence of organic solvent (acetonitrile) on the electroendosmotic mobility was similar to that in reversed-phase separations, although the separation mechanism is different. ATP, ADP and AMP were separated in less than 10 s by free-zone electrophoresis.
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| 27. |
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| 29. |
- Végvári, Ákos, et al.
(författare)
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High-resolution capillary zone and gel electrophoresis of structurally similar amphipathic glutathione conjugates based on interaction with beta-cyclodextrins
- 2002
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Ingår i: ChemBioChem. - 1439-4227 .- 1439-7633. ; 3:11, s. 1117-1125
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Tidskriftsartikel (refereegranskat)abstract
- The tripeptide glutathione is a prominent intracellular constituent that provides protection against genotoxic and carcinogenic electrophiles and is also a component of several biological signal substances. Glutathione conjugates, free glutathione, and glutathione disulfide contain charged amino acid residues, which contribute to solubility in aqueous media. However, the amphipathic nature of glutathione conjugates and the small differences that may distinguish the S substituents, pose analytical problems in their resolution. The present study demonstrates how homologous S-alkyl and S-benzyl conjugates of high structural similarity can be efficiently resolved by capillary electrophoresis. Inclusion of beta-cyclodextrins in the buffer or in a polyacrylamide gel affords baseline separation of the analytes. The separation methods described are applicable to enzyme assays in vitro and to the identification and quantification of glutathione conjugates of importance in toxicology and physiology. The contribution of beta-cyclodextrin to the separation is primarily based on interactions between its hydrophobic cavity and the S-alkyl and S-benzyl groups of the analytes.
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