| 1. |
- Bar, Laure, et al.
(författare)
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Impact of antigen density on recognition by monoclonal antibodies
- 2020
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Ingår i: Analytical Biochemistry. - : American Chemical Society (ACS). - 0003-2697 .- 1096-0309 .- 0003-2700 .- 1520-6882. ; 92:7, s. 5396-5403
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Tidskriftsartikel (refereegranskat)abstract
- Understanding antigen–antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.
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| 2. |
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| 3. |
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| 4. |
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| 5. |
- Lundberg, Peter, et al.
(författare)
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Nuclear magnetic resonance studies of cellular metabolism
- 1990
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 191:2, s. 193-222
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear magnetic resonance (NMR) spectroscopy was described in 1946 (1,2), initially as a method that had appeal only for nuclear physicists who used it to accurately determine nuclear magnetic moments. Thissituation changed rapidly, however, when it was demonstrated that the NMR frequency for the same nucleus in different chemical compounds was different (3). For example, two separate signals are observed in a 14N NMR spectrum of a solution of NH,NO,, representing the NH: and NO; ions, respectively (4). Since individual atoms within one molecule also give rise to resolved signals (5) it became clear that the NMR technique held great analytical potential, in particular since the spectra can be recorded in such a way that the area under a signal is directly proportional to its concentration. Such phenomena and various theoretical aspects of NMR are currently quite well understood (6,7). Because of these features NMR has become the foremost spectroscopic method for the analysis of all sorts of chemical compounds.
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| 6. |
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| 7. |
- Göransson, Ulf, et al.
(författare)
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Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation
- 2003
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 318:1, s. 107-117
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Tidskriftsartikel (refereegranskat)abstract
- The expression of cyclotides—macrocyclic plant peptides—was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis,V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single speciescontaining >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation ofcysteines. This overcomes a number of problems intimately associated with the cyclotide core structure—that is, their joined N and Ctermini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result,charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with trypticdigestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown bythe sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B(cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined bynanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of otherpeptides and proteins displaying similar structural problems for MS analysis.
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| 8. |
- Kussak, Anders, et al.
(författare)
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Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from Gram-negative bacteria
- 2002
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 307:1, s. 131-137
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Tidskriftsartikel (refereegranskat)abstract
- The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry. The lipid A was analyzed underivatized with ESI in negative-ion mode. With multiple stages of fragmentation (MSn), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined. It was possible to determine the degree of acylation by the MSn technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated. One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids. Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group. The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism. The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis.
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| 9. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O
- 1988
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Ingår i: Analytical Biochemistry. - : Academic Press. - 0003-2697 .- 1096-0309. ; 168:2, s. 352-357
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 m urea, 1% Triton X-100, 30% sucrose, 0.02% NaN3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 m guanidinium chloride and 3 m CsCl reduced the sensitivity of the assay to 30–50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.
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| 10. |
- Martin, Steven C., et al.
(författare)
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In vitro phosphorylation of serum albumin by two protein kinases : a potential pitfall in protein phosphorylation reactions
- 1986
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 154:2, s. 395-399
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Tidskriftsartikel (refereegranskat)abstract
- Bovine albumin was phosphorylated by both cAMP-dependent protein kinase and casein kinase I to a significant extent. Other albumins were also tested and it was found that the extent of phosphorylation varied with the species of origin of the albumin, but was between 1 and 3 mol phosphate per mole albumin for the cAMP-dependent protein kinase-catalyzed reactions. The phosphorylation occurred at and above pH 7.5 and required the presence of thiol reagents. Phosphoamino acid analyses of bovine albumin showed that it was phosphorylated on at least two serine residues. The phosphorylation could not be demonstrated in vivo.
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| 11. |
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| 12. |
- Ohlson, Sten, et al.
(författare)
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Novel Approach to Affinity Chromatography Using "Weak" Monoclonal Antibodies
- 1988
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 169:1, s. 204-208
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Tidskriftsartikel (refereegranskat)abstract
- Affinity purification generally relies on specific high-affinity recognition between two species of biological molecules: one molecular species (the ligate) dissolved in a mobile phase is selectively adsorbed to the other species (the ligand) coupled to a solid support. Desorption of the ligate often requires harsh conditions that degrade biological activity of the purified product. As an alternative to this general procedure, we have studied affinity chromatography in a weak affinity mode, where ligand-ligate interactions are in dynamic equilibrium. Ligates recognized with low affinities (dissociation constant > 104 ) elute from affinity columns under mild, isocratic conditions as retarded peaks, separated from noninteracting solutes that elute in the void volume. To illustrate the procedure, we report chromatography of an oligosaccharide on a 2-ml column containing 86 mg of a monoclonal antibody coupled to 10-μm microparticulate silica particles. Using a temperature-sensitive antibody, we observed that when the ligand-ligate dissociation constant is > 10−3 , performance of the system exceeds 300 theoretical plates/10 cm column length and approaches the efficiencies generally associated with high-performance liquid chromatography.
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| 13. |
- Rennel, Emma, et al.
(författare)
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How to make tetracycline-regulated transgene expression go on and off
- 2002
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 309:1, s. 79-84
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Tidskriftsartikel (refereegranskat)abstract
- Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.
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| 14. |
- Toomik, Reet, et al.
(författare)
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A potential pitfall in protein kinase assay: phosphocellulose paper as un unreliable adsorbent of produced phosphopeptides
- 1992
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 204:2, s. 311-314
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Tidskriftsartikel (refereegranskat)abstract
- The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.
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| 15. |
- Wang, W T, et al.
(författare)
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Analysis of a Glucose-Containing Tetrasaccharide by High-Performance Liquid Affinity Chromatography
- 1989
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 182:1, s. 48-53
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Tidskriftsartikel (refereegranskat)abstract
- In the present work we have explored conditions for using a pulsed amperometric detector for on-line analysis of oligosaccharides eluted from a high-performance liquid affinity chromatography column. A monoclonal antibody that specifically binds a glucose-containing oligosaccharide is coupled to a SelectiSphere-10-activated tresyl column. The system is eluted isocratically and easily detects 10 ng of the oligosaccharide with a linear response up to 250 ng. Analysis of both serum and urine samples from normal individuals and patients with acute pancreatitis gives a single retarded peak with a retention time identical to that of authentic (Glc)4. Retarded material pooled from several analyses of urine was positively identified as (Glc)4 by GC-MS analysis. As this method requires little cleanup and no chemical derivitization of the sample and is performed rapidly (less than 20 min) at sensitivities of at least 10μg/liter in biological fluids, it represents a substantial improvement over previous GC-MS, radioimmunoassay, and enzyme-linked immunoadsorbent assay methods used to determine (Glc)4.
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| 16. |
- Ålin, Per, et al.
(författare)
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Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing
- 1985
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 146:2, s. 313-320
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Tidskriftsartikel (refereegranskat)abstract
- Seven major isoenzymes of glutahione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W. B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539–2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.
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| 17. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 18. |
- Ahmadian, Afshin, et al.
(författare)
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Single-nucleotide polymorphism analysis by pyrosequencing
- 2000
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 280:1, s. 103-110
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Tidskriftsartikel (refereegranskat)abstract
- There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.
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| 19. |
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| 20. |
- Almstrand, Ann-Charlotte, et al.
(författare)
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Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis
- 2015
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 474, s. 50-58
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Tidskriftsartikel (refereegranskat)abstract
- Chemical reactions with unsaturated phospholipids in the respiratory tract lining fluid have been identified as one of the first important steps in the mechanisms mediating environmental ozone toxicity. As a consequence of these reactions, complex mixtures of oxidized lipids are generated in the presence of mixtures of non-oxidized naturally occurring phospholipid molecular species, which challenge methods of analysis. Untargeted mass spectrometry and statistical methods were employed to approach these complex spectra. Human bronchoalveolar lavage (BAL) was exposed to low levels of ozone, and samples with and without derivatization of aldehydes were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry. Data processing was carried out using principal component analysis (PCA). Resulting PCA scores plots indicated an ozone dose-dependent increase, with apparent separation between BAL samples exposed to 60 ppb ozone and non-exposed BAL samples as well as a clear separation between ozonized samples before and after derivatization. Corresponding loadings plots revealed that more than 30 phosphatidylcholine (PC) species decreased due to ozonation. A total of 13 PC and 6 phosphatidylglycerol oxidation products were identified, with the majority being structurally characterized as chain-shortened aldehyde products. This method exemplifies an approach for comprehensive detection of low-abundance, yet important, components in complex lipid samples.
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| 21. |
- Andersson, Henrik, et al.
(författare)
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Optimizing immobilization conditions on a two dimensional carboxylbiosensor surface : pH dependence of antibody orientation andantigen binding capacity
- 2009
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We have shown that successful immobilization is highly dependent on surface pKa, antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Generic immobilization conditions were demonstrated on a panel of antibodies which resulted in an average coefficient of variation of 4% for the immobilization of these antibodies. Antigen binding capacity as a function of immobilization pH was studied. In most cases the antigen binding capacity followed the immobilization response. However, the antigen to antibody binding ratio differed between the antibodies investigated, and for one of the antibodies, the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab antibodies on different antibody surfaces showed that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.
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| 22. |
- Andersson, R. M., et al.
(författare)
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Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo c xanthene dyes using intensity-modulated multiple-wavelength scanning technique
- 2000
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 283:1, s. 104-110
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative pH imaging using the carboxy semi-naphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using confocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same trends as observed in the in vitro studies.
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| 23. |
- André, Ingemar, et al.
(författare)
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Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software.
- 2002
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 305:2, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The complexity of Ca2+ cell signaling is dependent on a plethoria of Ca2+-binding proteins that respond to signals in different ranges of Ca2+ concentrations. Since the function of these proteins is directly coupled to their Ca2+-binding properties, there is a need for accurately determined equilibrium Ca2+-binding constants. In this work we outline the experimental techniques available to determine Ca2+-binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for least-square fitting directly to the measured quantity. The use of the software is illustrated for Ca2+-binding data obtained for two deamidated forms of calbindin D(9k), either an isospartate-56 (beta form) or a normal Asp-56 (alpha form). Here, the Ca2+-binding properties of the two isoforms have been studied using the chelator method. The alpha form shows similar Ca2+-binding properties to the wild type while the beta form has lost both cooperativety and affinity.
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| 24. |
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| 25. |
- Bally, Marta, 1981, et al.
(författare)
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Fluorescent vesicles for signal amplification in reverse phase protein microarray assays
- 2011
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Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 416:2, s. 145-151
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Tidskriftsartikel (refereegranskat)abstract
- Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning. (C) 2011 Elsevier Inc. All rights reserved.
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