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INQUISAL 20936

INSTITUTO DE QUIMICA DE SAN LUIS "DR. ROBERTO ANTONIO OLSINA"

Unidad Ejecutora - UE

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Título:

Immunoaffinity capillary electrophoresis using magnetic microbeads for the determination of IgG anti-Helicobacter pylori in human serum with laser-induced fluorescence detection.

Autor/es:

PATRICIA W. STEGE; ROBERTO A. OLSINA; JULIO RABA; GERMÁN A. MESSINA

Lugar:

Sevilla, España

Reunión:

Simposio; 15th Latin American Symposium on Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2009

Resumen:

Helicobacter pylori is a pathogenic microorganism which colonizes the stomach of the half of people all over the word. Current methods using for the diagnostic of H. pylori infection can be divided into invasive and noninvasive (or minimally invasive) methods [1]. Most of the noninvasive tests are based on serological procedures which detect immunoglobulin G 1 (IgG1) against H. pylori in human serum. The IgG anti-H. pylori present in the serum has a considerable value in the diagnosis of active infection due to the reliable correlation between the presence of this antibody and gastric mucosal colonization [2]. Commonly the measurements of IgG in serum are carried out using enzyme-linked immunosorbent assay (ELISA) [3]. Although ELISA is an accepted assay in clinical practice, the relatively poor limit of detection with spectrophotometry detection is one cause of its lack of sensitivity. In all instances, the immobilized antigens are discarded after only one use. In this work we develop a CE sensitive method as an alternative to ELISA; we proposed an on line IA-CE with modify magnetic microbeads. Immunoaffinity capillary electrophoresis (IA-CE) is a hybrid technique that combines immunocapture and CE separation [4]. The aim in the present work was developed an IA-CE using antigens immobilized into the surface of magnetic microbeads. The running buffer was phosphate 40 mM. Purified antigens of H. pylori were immobilized on magnetic microbeads modified with amino groups. The sensitizations of the magnetic microbeads were achieved on line. The serum samples used for diagnosis were diluted 100 folds and injected during 10 min. The anti-human IgG fluorescein conjugate was injected during 5 min. The release and separation buffer was phosphate 40 mM with 10%v/v of acetic acid. This buffer was used to separate all bindings antigen-antibody. The only remaining bondings were the covalent ones (magnetic microbeads-antigen). A laser-induced fluorescence detector was used to quantify the labeled antibody released. The integration of a CE methodology based on the use of modified paramagnetic microbeads with a laser-induced fluorescence detector increased the capability to determinate the low levels of IgG antibodies specific to H. pylori with high sensibility. Other advantages of this assay were the short time of analysis (20 min) and a less sample consumed than conventional immunoassay techniques. Moreover, this method allowed us to use the immobilized microbeads several time before they were discarded. [1] A.F. Cutler, F. Havstad, C. Ma, M. Blaser, G.I. Perez-Perez, T. Schubert. Gastroenterology 109,1995, 136. [2] A.A. van Zwet, J.C. Thijs, R. Roosendaal, E.J. Kuipers, S. Pena, J. de Graaff. Eur. J. Gastroenterol. Hepatol. 8, 1996, 501. [3] A.R. Stacy, G.D. Bell, D.G. Newell, in: G. Gasbarrini, S. Petrolani (Eds.), Basic and Clinical Aspects of H. pylori Infection, Springer, Berlin, 1998, p. 159. [4] L. K. Amundsen, H. Sirén, Electrophoresis 28, 2007, 99.