| 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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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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| 3. |
- Račaitytė, Kristina, et al.
(författare)
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Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography–electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger
- 2000
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Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier B.V. - 0021-9673 .- 1873-3778. ; 890:1, s. 135-144
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Tidskriftsartikel (refereegranskat)abstract
- A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC–electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33×4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up–HPLC–ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 μl. Peaks due to the fragments NPY 1–36, 3–36 and 13–36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma. © 2000 Elsevier Science B.V. All rights
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