| 61. |
- Hartmann, Michael, et al.
(författare)
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Expanding assay dynamics : A combined competitive and direct assay system for the quantification of proteins in multiplexed Immunoassays
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:6, s. 956-963
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. METHODS: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible. RESULTS: We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation. CONCLUSIONS: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.
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| 62. |
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| 63. |
- Helander, A, et al.
(författare)
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Improved HPLC method for carbohydrate-deficient transferrin in serum
- 2003
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Ingår i: Clinical Chemistry. - : American Association for Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 49:11, s. 1881-1890
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms. Methods: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron-transferrin complex at 470 run. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration. Results: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo- and trisialotransferrin were almost baseline separated. The intra- and interassay CV for disialotransferrin were <5%. Serum and the pretreated samples were stable for at least 2 days at 22 or 4 degreesC. Sera from 132 healthy controls contained [mean (SD)] 1.16 (0.25)% disialotransferrin, 4.77 (1.36)% trisialotransferrin, 80.18 (2.01)% tetrasialotransferrin, and 13.88 (1.69)% pentasialo- + hexasialotransferrin. In some cases of a high (>6%) trisialotransferrin, monosialotransferrin was detected at <0.25%. Asialotransferrin was not detected in control sera, but was detected in 57% of chronic heavy drinkers and in 62% of sera with greater than or equal to2% disialotransferrin. Conclusions: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases. (C) 2003 American Association for Clinical Chemistry.
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| 64. |
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| 65. |
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| 66. |
- Helander, A, et al.
(författare)
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Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS
- 2009
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Ingår i: Clinical chemistry. - : Oxford University Press (OUP). - 1530-8561 .- 0009-9147. ; 55:7, s. 1395-1405
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Tidskriftsartikel (refereegranskat)abstract
- Background: The alcohol biomarker phosphatidylethanol (PEth) comprises a group of ethanol-derived phospholipids formed from phosphatidylcholine by phospholipase D. The PEth molecular species have a common phosphoethanol head group onto which 2 fatty acid moieties are attached. We developed an electrospray ionization (ESI) LC-MS method for qualitative and quantitative measurement of different PEth species in human blood.Methods: We subjected a total lipid extract of whole blood to HPLC gradient separation on a C4 column and performed LC-ESI-MS analysis using selected ion monitoring of deprotonated molecules for the PEth species and phosphatidylpropanol (internal standard). Identification of individual PEth species was based on ESI–tandem mass spectrometry (MS/MS) analysis of product ions.Results: The fatty acid moieties were the major product ions of PEth, based on comparison with PEth-16:0/16:0, 18:1/18:1, and 16:0/18:1 reference material. For LC-MS analysis of different PEth species in blood, we used a calibration curve covering 0.2–7.0 μmol/L PEth-16:0/18:1. The lower limit of quantitation of the method was <0.1 μmol/L, and intra- and interassay CVs were <9% and <11%. In blood samples collected from 38 alcohol patients, the total PEth concentration ranged between 0.1 and 21.7 μmol/L (mean 8.9). PEth-16:0/18:1 and 16:0/18:2 were the predominant molecular species, accounting for approximately 37% and 25%, respectively, of total PEth. PEth-16:0/20:4 and mixtures of 18:1/18:1 plus 18:0/18:2 (not separated using selected ion monitoring because of identical molecular masses) and 16:0/20:3 plus 18:1/18.2 made up approximately 13%, 12%, and 8%.Conclusions: This LC-MS method allows simultaneous qualitative and quantitative measurement of several PEth molecular species in whole blood samples.
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| 69. |
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