| 1. |
- Aldén, Anna, et al.
(författare)
-
HPLC analysis of carbohydrate deficient transferrin isoforms isolated by the Axis-Shield %CDT method
- 2005
-
Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 356:1-2, s. 143-146
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Carbohydrate-deficient transferrin (CDT) is elevated during prolonged overconsumption of alcohol and CDT is considered to be the most specific biochemical marker for alcohol overconsumption. However, an accurate method for analysing CDT is necessary because the test is frequently used for example in legal matters. Methods: Patient serum samples were analysed with the Axis-Shield %CDT and eluates were pooled together. Transferrin was purified from the pool by affinity chromatography and further analysed with HPLC to determine the ratios of different transferrin isoforms. Results: In the eluates using the Axis-Shield %CDT method, a substantial amount of trisialo transferrin was found, which is generally not considered a CDT isoform. Conclusions: The fact that trisialo transferrin is present may generate falsely elevated CDT results and it could at least partly explain the discrepancy between results of the Axis-Shield %CDT assay and HPLC in routine analysis. © 2005 Elsevier B.V. All rights reserved.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
- Bergström, Maria, et al.
(författare)
-
Cholera toxin inhibitors studied with High-performance liquid affinity chromatography: arobust method to evaluate receptor-ligand interactions
- 2009
-
Ingår i: Chemical Biology and Drug Design. - : Wiley. - 1747-0277 .- 1747-0285. ; 73:1, s. 132-141
-
Tidskriftsartikel (refereegranskat)abstract
- Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K(D) values of galactose and meta-nitrophenyl alpha-d-galactoside were determined with weak affinity chromatography to be 52 and 1 mm, respectively, which agree well with IC(50) values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K(D) values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K(D) values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.
|
|
| 7. |
- Bergström, Maria, et al.
(författare)
-
Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography
- 2012
-
Ingår i: Journal of chromatography. B. - : Elsevier. - 1570-0232 .- 1873-376X. ; 885, s. 66-72
-
Tidskriftsartikel (refereegranskat)abstract
- Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coil as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked alpha 1-3 to GIcNAc interacted with higher affinity compared to fucose linked alpha 1-6 or alpha 1-4 and the obtained dissociation constants (K-d) were in the range of 10 mu M for all AAL forms. Tetra- and pentasaccharides with fucose in alpha 1-2, alpha 1-3 or alpha 1-4 had K-d values ranging from 0.1 to 7 mM while a large alpha 1-6 fucosylated oligosaccharide had a K-d of about 20 mu M. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.
|
|
| 8. |
- Bergström, Maria, et al.
(författare)
-
Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique
- 2004
-
Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 809:2, s. 323-329
-
Tidskriftsartikel (refereegranskat)abstract
- The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of α1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a "partial filling technique" with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks, the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (KD) between Con A and a competing sugar. © 2004 Elsevier B.V. All rights reserved.
|
|
| 9. |
|
|
| 10. |
|
|
| 11. |
|
|
| 12. |
- Bornberger-Dankvardt, Sten, et al.
(författare)
-
Arbetsmiljöarbete i småföretag : samlad kunskap samt behov av forskning och utvecklingsinsatser
- 2005
-
Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Föreliggande rapport behandlar specifikt behov av och möjligheter för arbetsmiljöarbete i småföretag och är ett delarbete i tema SMARTA. SMARTA – strategier, metoder och arbetssätt för fungerande arbetsmiljöarbete – ingår i Arbetslivsinstitutets temaverksamhet. SMARTA ska bidra till ett hållbart arbetsliv på arbetsplatser där arbetsmiljöarbetet ses som en resurs för både arbetsplatsen och individen. För arbetsplatsen kan det handla om konkurrenskraft, lönsamhet samt attraktivitet och för individen om hälsa, välbefinnande, kreativitet och förnyelseförmåga. SMARTA tar ett helhetsperspektiv på arbetsmiljöarbete inom olika regioner och branscher. Temat sammanställer inledningsvis kunskapsläget och ger exempel på hur arbetsmiljöarbete kan bedrivas och vidareutvecklas. Ambitionen för FoU-projekt i tema SMARTA är att besvara frågor som: • Hur bör arbetsmiljöarbete integreras i organisationers kärnverksamhet? • Hur bör arbetsmiljöarbete bedrivas? • Hur bör interna och externa aktörer agera för att få till stånd ett hållbart och fungerande arbetsmiljöarbete? Rapporten vänder sig till praktiker och forskare som är intresserade av att få en översiktsbild över arbetsmiljösituationen i småföretag och förutsättningar som krävs för fungerande arbetsmiljöarbete.
|
|
| 13. |
|
|
| 14. |
- Bratt, T, et al.
(författare)
-
The Development of a C1q ANTI-C1q Immunoadsorbent for Removal of Immune Complexes from Plasma
- 1988
-
Ingår i: Journal of Clinical Laboratory Immunology. - 0141-2760. ; 27, s. 191-195
-
Tidskriftsartikel (refereegranskat)abstract
- An immunoadsorbent based on immobilized C1q has been developed to remove possibly pathogenic immune complexes from plasma deriving from patients suffering from systemic lupus erythematosus or rheumatoid arthritis. Traditional immobilization procedures based on, e.g., cyanogen bromide activation could not be used to produce an efficient adsorbent. However, by using antibodies directed towards C1q as handles for the immobilization of C1q it was possible to make an adsorbent that efficiently bound immune complexes in plasma. The capacity of the C1q anti-C1q adsorbent to bind artificial immune complexes such as aggregated human globulins or immune complexes from various plasma samples was evaluated. Both batch and column experiments were conducted. The typical capacity in batch was about 1 mg immune complexes/ml gel when incubated with patient plasma samples with high titers of immune complexes. Special attention has to be paid to leakage of undesirable components from the adsorbent. It was found that leakage of C1q occurred but it was not more than after covalent immobilization procedures such as cyanogen bromide.
|
|
| 15. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Comparison of weak affinity chromatography and surface plasmon resonance in determining affinity of small molecules
- 2014
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 461, s. 57-59
-
Tidskriftsartikel (refereegranskat)abstract
- In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P < 0.0001). (C) 2014 Elsevier Inc. All rights reserved.
|
|
| 16. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
High-Throughput Fragment Screening by Affinity LC-MS
- 2013
-
Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 18:2, s. 160-171
-
Tidskriftsartikel (refereegranskat)abstract
- Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in < 4 h (corresponding to > 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K-D) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.
|
|
| 17. |
- Duong-Thi, Minh-Dao
(författare)
-
Introducing weak affinity chromatography to drug discovery with focus on fragment screening
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening.WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds.In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened.Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution.In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures. In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology.
|
|
| 18. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins
- 2016
-
Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 141:3, s. 981-988
-
Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.
|
|
| 19. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins
- 2016
-
Ingår i: The Analyst. - 0003-2654 .- 1364-5528. ; 141:3, s. 981-988
-
Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.
|
|
| 20. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Weak affinity chromatography as a new approach for fragment screening in drug discovery
- 2011
-
Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 414:1, s. 138-146
-
Tidskriftsartikel (refereegranskat)abstract
- Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–uM in dissociation constant (KD)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23 compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10uM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.
|
|
| 21. |
- Duong-Thi, Minh-Dao, et al.
(författare)
-
Weak Affinity Chromatography for Evaluation of Stereoisomers in Early Drug Discovery
- 2013
-
Ingår i: Journal of Biomolecular Screening. - : Sage Publications. - 1087-0571 .- 1552-454X. ; 18:6, s. 748-755
-
Tidskriftsartikel (refereegranskat)abstract
- In early drug discovery (e.g. in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.
|
|
| 22. |
|
|
| 23. |
|
|
| 24. |
|
|
| 25. |
- Engström, Henrik, 1975-
(författare)
-
Development of Flourescence-based Immunosensors for Continous Carbohydrate Monotoring : Applications for Maltose and Glucose
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail.
|
|
