Use of immunodotting to select the desorption agent for immunochromatography
In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen – antibody recognition. To reus...
| Autores: | , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión aceptada para publicación |
| Fecha de publicación: | 2004 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/41474 |
| Acceso en línea: | http://hdl.handle.net/10261/41474 |
| Access Level: | acceso abierto |
| Palabra clave: | Dotting Immunodotting Immunoaffinity Immunochromatography Lactoglobulin Desorption |
| Sumario: | In immunochromatography, a technique of increasing use, the sample containing the antigen (Ag) to be purified or determined is introduced into a chromatographic column containing an antibody (Ab) bound to the packing material. The antigen is retained based on antigen – antibody recognition. To reuse the immunocolumn for subsequent assays, the antigen has to be eluted without causing irreversible damage of antibodies. Selection of conditions for performing immunochromatography is usually made by trial and error. This way of working is time consuming and it may ruin the column. In this article, the feasibility of using immunodotting to select the conditions to be employed in one immunochromato- graphic assay is shown. An immunodotting method is developed to select the best desorption agent for an enzyme-linked immunoaffinity chromatography (ELIAC) assay to determine h-lactoglobulin (h-LG). The effect of several factors on the immunodotting performance is studied. The way of performing solvent exchange to treat the antibody with different solutions considered as potential desorption agents to check their effect is shown. Effectiveness of the solution chosen by immunodotting (4 M MgCl2 in 20 mM Tris, pH 5.9) as desorption agent is demonstrated by immunochromatographic assays. The immunodotting and solvent exchange methods developed should be useful to choose solvents and conditions for any other kind of assay. |
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