A Homogeneous and Multiplexed Immunoassay for High-Throughput Screening Using Fluorometric Microvolume Assay Technology
| Autor: | Julia Mellentin-Michelotti, Sheri Miraglia, Lolita Evangelista, Pau-Miau Yuan, Elana Swartzman |
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| Rok vydání: | 1999 |
| Předmět: |
Fluorophore
High-throughput screening Biophysics Enzyme-Linked Immunosorbent Assay Biochemistry chemistry.chemical_compound medicine Humans Fluorometry Multiplex HUVEC Cells Fluorescent Antibody Technique Indirect Molecular Biology Cells Cultured Immunoassay Multiplexed immunoassay Chromatography medicine.diagnostic_test Interleukin-6 Microchemistry Interleukin-8 Cell Biology Fluorescence chemistry Fluorescent Antibody Technique Direct Homogeneous Endothelium Vascular |
| Zdroj: | Analytical Biochemistry. 271:143-151 |
| ISSN: | 0003-2697 |
| Popis: | We have developed a simple, homogeneous bead-based immunoassay for use with fluorometric microvolume assay technology (FMAT). The FLISA (fluorescence-linked immunosorbent assay) can be easily adapted from existing immunoassays, is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity, and can be readily performed in 96- and 384-well plates. Additionally, the FLISA utilizes 100-fold less primary antibody than the conventional immunoassay. The scanner uses a helium/neon laser to image and measure bead-bound fluorescence while the background fluorescence is ignored. Consequently, no wash steps are required to remove unbound antibody, ligand, and fluorophore. Furthermore, the instrument is capable of detecting two different fluorescent dyes, allowing for multiplexed assays based on color. Fluorescent bead-based immunoassays were developed for the cytokines IL-6 and IL-8, and their use in both one-color and two-color FLISAs is demonstrated. Although no wash steps were employed, the FLISA was able to accurately measure the concentrations of IL-6 and IL-8 in the growth media of cytokine-stimulated HUVEC cells. In addition, a simulated high-throughput two-color FLISA positively identified those wells in a 384-well plate that contained different amounts of IL-6 and/or IL-8 peptide. The homogeneous, multiplex and multiplate format of the FLISA reduces hands-on time and reagent usage, and is therefore ideally suited for high-throughput screening. |
| Databáze: | OpenAIRE |
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