| 1. |
- Lutz, Mareike, 1967-, et al.
(författare)
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Applying hollow fibres for separating free and bound label in continuous-flow immunochemical detection
- 1996
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Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier B.V. - 0021-9673 .- 1873-3778. ; 755:2, s. 179-187
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Tidskriftsartikel (refereegranskat)abstract
- On-line liquid chromatography-immunochemical detection (LC-ICD) provides the possibility to individually monitor cross-reactive compounds overcoming the need of tedious fraction collection. ICD is performed as a post-column reaction detection system and is based on a two-step immunoreaction. In the first step unlabelled antibodies are added to the LC effluent and allowed to react with antigens (analytes) eluting from the LC column. The amount of analytes bound to the antibodies is measured by adding, in a second step, labelled antigen to the reaction mixture. For quantitation, free and bound label need to be separated prior to detection. The present paper describes a hollow fibre module (HFM), which can be used for this purpose. Separation of free and bound label occurs on discrimination by size. Using biotin as a model compound, a detection limit of 30 nmol/l can be reached employing anti-biotin antibodies and a low-molecular-mass fluorescence label in the LC-ICD system. Additional to low-molecular-mass labels, the HFM allows the use of small enzyme labels. In this context, horseradish peroxidase-labelled biotin was used as a label in combination with antibodies in the immunochemical detection of biotin. This allows future implementation of commercially available enzyme immunoassay kits in continuous-flow immunochemical detection.
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| 2. |
- Lutz, Mareike, 1967-, et al.
(författare)
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Biochemical detection for direct bead surface analysis
- 1997
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Ingår i: Analytical Chemistry. - Washington : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 69:23, s. 4878-4884
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Tidskriftsartikel (refereegranskat)abstract
- A continuous-now biochemical detection system is presented which recognizes biologically active compounds immobilized to solid phases. This approach can be used to screen, for example, solid-phase combinatorial libraries for lead compounds. Biochemical detection is performed by mixing a plug of a solid-phase suspension with labeled affinity protein, During a short reaction time, the labeled affinity protein will only bind to ligands, i.e., compounds with biological activity. Hereafter, the free and bound labels are separated by means of a hollow fiber module, Quantitation of the free label is performed with a conventional now-through fluorescence detector, Total assay time amounts to less than 3 min. Biochemical detection for direct bead surface analysis was developed for two model systems. The first model system used fluorescence-labeled avidin as affinity protein and its ligands biotin and iminobiotin immobilized to agarose as analytes. The second model system used fluorescence-labeled antisheep (Fab)(2) fragments as affinity protein and different IgGs immobilized to agarose as analytes. The feasibility of this approach for recognition of solid-phase immobilized ligands was documented by screening 50 samples with a 100% hit rate.
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| 3. |
- Lutz, Mareike, 1967-, et al.
(författare)
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Implementation of affinity solid-phases in continuous-flow biochemical detection
- 1997
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Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier B.V. - 0021-9673 .- 1873-3778. ; 776:2, s. 169-178
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Tidskriftsartikel (refereegranskat)abstract
- A continuous-flow biochemical detection system is presented which allows the use of solid-phase immobilised affinity proteins. The biochemical detection is performed by mixing analyte with a labelled ligand followed by the addition of solid-phase immobilised affinity protein. After a reaction time of 85 s, free and bound label are separated by means of a hollow fibre module. Quantitation of the free label is performed with a conventional flow-through fluorescence detector. Total assay time amounts to less than 2 min. Biotin was chosen as the model compound using a range of streptavidin-coated solid-phases and an antibody-coated solid-phase as affinity material, and fluorescein–biotin as low-molecular-mass label. The relative standard deviation for twenty repetitive injections was 10.9%. A calibration curve was constructed in the concentration range between 20 and 400 nmol l−1 leading to a correlation coefficient of 0.994. A limit of detection of 8 nmol l−1 was obtained. © 1997 Elsevier Science B.V.
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| 4. |
- Lutz, Mareike, 1967-, et al.
(författare)
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On-line Coupling of Liquid Chromatography to Biological Assays
- 1997
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Ingår i: Chimica oggi. - Milan : Tekno Scienze. - 0392-839X .- 1973-8250. ; 15:1-2, s. 11-15
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Tidskriftsartikel (refereegranskat)abstract
- Combining two powerful technologies - liquid chromatography and bioassays results in analytical methodologies which are characterised by high selectivity and sensitivity. In contrast to microtitre-type bioassays, biochemical reactions proceed in a closed, continuous-flow reaction detection system coupled directly to the outlet of the chromatographic separation column. The interaction of substances eluting from the separation column with molecular targets such as antibodies or receptors is monitored directly overcoming tedious fraction collection and manual operations required to prepare the functions for batch immunoassays. important application areas are bioanalysis and drug discovery.
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