One-step direct immunoassay with horseradish peroxidase as antigen for studying the functionality of antibody surfaces
| Autor: | A. Le Postollec, Gaëlle Coussot, Clément Faye, Michel Dobrijevic |
|---|---|
| Přispěvatelé: | ASP 2018, Laboratoire d'Astrophysique de Bordeaux [Pessac] (LAB), Université de Bordeaux (UB)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2018 |
| Předmět: |
Bioanalysis
Analytical chemistry One-Step 02 engineering and technology 01 natural sciences Horseradish peroxidase Antibodies Analytical Chemistry medicine Animals Antigens Binding site Horseradish Peroxidase ComputingMilieux_MISCELLANEOUS Immunoassay [SDU.ASTR]Sciences of the Universe [physics]/Astrophysics [astro-ph] biology medicine.diagnostic_test Chemistry 010401 analytical chemistry 021001 nanoscience & nanotechnology Combinatorial chemistry 0104 chemical sciences Covalent bond biology.protein 0210 nano-technology Biosensor Peroxidase |
| Zdroj: | Talanta Talanta, Elsevier, 2018, 178, pp.922-927. ⟨10.1016/j.talanta.2017.10.032⟩ |
| ISSN: | 0039-9140 |
| DOI: | 10.1016/j.talanta.2017.10.032 |
| Popis: | Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. "the active sites" that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD10%) are reported. |
| Databáze: | OpenAIRE |
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