| 1. |
- Lönnberg, Maria, et al.
(författare)
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Rapid detection of erythropoiesis-stimulating agents in urine and serum
- 2012
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 420:2, s. 101-114
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Tidskriftsartikel (refereegranskat)abstract
- A rapid and easy-to-use test kit, EPO WGA MAIIA, which can be used for distinguishing various endogenous human erythropoietins (hEPO) and several recombinant hEPOs and EPO analogues, has been evaluated. The test is based on chromatographic separation of the glycosylated isoforms of EPO using wheat germ agglutinin (WGA), and a sensitive immunoassay utilizing anti-EPO carbon black nanostrings and image scanning for quantification. All the reactions take place along the porous layer of a lateral flow micro-column containing WGA and anti-EPO zones. The presence of molecules resembling hEPO, like Mircera, was detected by the aberrant affinity interaction with the antibody zone on the strip. It was possible to distinguish nine recombinant hEPO expressed in hamster and human cell-lines, and also Aranesp and Mircera, from endogenous urine hEPO. The required amount of EPO in the samples, a few pg, is very low compared to other methods for EPO isoform identification. This EPO isoform determination method opens the possibility to monitor recombinant EPO therapy for clinical research and seems to be a valuable candidate to the arsenal of EPO doping control tests.
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| 2. |
- Franco Fraguas, L, et al.
(författare)
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Lectin affinity chromatography as a tool to differentiate endogenous and recombinant erythropoietins
- 2008
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1212:1-2, s. 82-88
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Tidskriftsartikel (refereegranskat)abstract
- This work exploits the combination of the lectin affinity chromatography (LAC) with an ultra-sensitive immunochromatographic assay to differentiate several types of erythropoietin (EPO). The chromatographic behaviours of different commercial types of recombinant human EPO (rhEPO), EPO analogues (Aranesp) and urine human EPO (uhEPO) from healthy individuals on eight lectin-Sepharose columns, have been worked out. Results show that when using wheat germ agglutinin (WGA)-Sepharose columns, a careful desorption regime starting with very low concentration (2mM) of the competitive sugar N-acetylglucosamine (GlcNAc) makes it possible to efficiently distinguish endogenous EPO from recombinant EPO and EPO analogues.
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| 3. |
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| 4. |
- Lönnberg, Maria, et al.
(författare)
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Chromatographic performance of a thin microporous bed of nitrocellulose
- 2001
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Ingår i: JOURNAL OF CHROMATOGRAPHY B. - 0378-4347. ; 763:1-2, s. 107-120
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Tidskriftsartikel (refereegranskat)abstract
- Chromatography along thin (125 mum) porous beds of nitrocellulose, layered on top of an polyester backing, shows good separation efficiency with plate heights of 10-20 mum. Flow is controlled by capillary forces and shows low rate variations between the i
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| 5. |
- Lönnberg, Maria, et al.
(författare)
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Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test
- 2012
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 403:6, s. 1619-1628
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Tidskriftsartikel (refereegranskat)abstract
- Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg −1 day −1 to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2–5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).
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| 6. |
- Lönnberg, Maria, et al.
(författare)
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Lab-on-a-chip technology for determination of protein isoform profiles
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1127:1-2, s. 175-182
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Tidskriftsartikel (refereegranskat)abstract
- A novel lab-on-a-chip technique for rapid (<15 min) and quantitative isoform-profile determination is presented. Ion-exchange chromatographic separation of protein-isoforms and a sensitive immunoassay detection are combined in a porous monolith chip. Thin lines of immobilized antibodies are used for specific capturing of target molecules, which can be detected by the reaction with antibodies bound to carbon black nano-strings. The bound carbon black is quantified by the use of an image scanner. As demonstrated with transferrin isoforms, differing only by 0.1 pH unit in their pI, this technology can distinguish minor differences in protein carbohydrate structure and enable specific determination of proteins in a complex environment, requiring only a few picogram of isoform for detection.
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| 7. |
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| 8. |
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| 9. |
- Lönnberg, Maria, et al.
(författare)
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Rapid affinity purification of erythropoietin from biological samples using disposable monoliths
- 2010
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1217:45, s. 7031-7037
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Tidskriftsartikel (refereegranskat)abstract
- Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods In the purification step high or low molecular weight substances can be removed by size exclusion filters and high abundant proteins can be removed or low abundant proteins can be enriched by specific capturing tools In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens The kit can be used as a pre-step in the EPO doping control procedure as an alternative to the commonly used ultrafiltration for detecting aberrantly glycosylated isoforms The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 mu L volume theta 7 mm length 0 15 mm) together with all required buffers A 24-channel vacuum manifold was used for simultaneous processing of samples The column concentrated EPO from 20 mL urine down to 55 mu L eluate with a concentration factor of 240 times while roughly 997% of non-relevant urine proteins were removed The recoveries of Neorecormon (epoetin beta) and the EPO analogues Aranesp and Mircera applied to buffer were high 76% 67% and 57% respectively The recovery of endogenous EPO from human urine was 65% High recoveries were also obtained when purifying human mouse and equine EPO from serum and human EPO from cerebrospinal fluid Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO Aranesp Mircera or endogenous EPO The kit should be particularly useful for applications in which it is essential to avoid carry-over effects a problem commonly encountered with conventional particle-based affinity columns The encouraging results with EPO propose that similar affinity monoliths with the appropriate antibodies should constitute useful tools for general applications in sample preparation not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research and for sample preparation prior to in vitro diagnostics.
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| 10. |
- Lönnberg, Maria, et al.
(författare)
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Ultra-sensitive immunochromatographic assay for quantitative determination of erythropoietin
- 2008
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 339:2, s. 236-244
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Tidskriftsartikel (refereegranskat)abstract
- An ultra-sensitive quantitative EPO (erythropoietin) lateral flow immunochromatographic test with a detection limit of 1.2 fM (10(-15) M), 0.035 ng EPO/L, which is 50-100 times more sensitive than a corresponding enzyme based immunoassay, is presented. In comparison with commercial lateral flow tests for other analytes, like cardiac troponins that also require high sensitivity, the detection limit achieved in the presented test is about three orders of magnitude lower. The thin zone for capture and concentration of the analyte, the carbon black nano-strings used as label and the use of a conventional image scanner for the quantitative determination are the key components that enable the high sensitivity obtained. The convective flow in the lateral flow monolith creates short diffusion distances between immobilised antibody, analyte and labelled antibody thus enhancing the binding efficiency. This rapid and sensitive EPO test procedure can be used both to process hundreds of samples in 1 h and be utilized as a 15-minute dipstick test for single determinations. The technique is demonstrated by measuring EPO in urine. EPO, like many of the other urine proteins, is often found in the urine precipitates and the specimens are therefore treated with a urine precipitate dissolvation buffer before analysis. It is shown that EPO in urine from normal individuals occurs in low concentration in a wide range between 1.7 and 51 ng/L. The concentration is however subjected to a wide variation during the day due to the EPO production variation and the urine concentration by the kidneys. It is also shown that the presented lateral flow device can be used as a miniaturized affinity column to distinguish an analyte (EPO) from its analogue (darbepoetin), directly by comparing the affinity profiles obtained after interaction with the immobilised antibody. The method for measuring the amount of EPO present in urine, the possibility to rapidly check the amount of EPO after a pre-treatment concentration step, and the potential to identify affinity differences between EPO and its analogues should make the presented method a valuable tool in the fight against EPO doping.
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