Related Experiment Videos
Solid-phase immunoassay using a flow cytometer: quantitative and qualitative determination of protein antigens and a
1Laboratory of Cell Biology, Genetic Engineering Research Institute, Korea Institute of Science and Technology, Daejon.
Insights
A novel flow cytometric immunoassay offers a reliable alternative for fluorescence detection. This method provides reproducible, quantitative results for various analytes, including immunoglobulin (Ig) and human chorionic gonadotropin (hCG).
Area of Science:
- Immunology
- Analytical Chemistry
- Biotechnology
Background:
- Traditional immunoassays often face limitations in sensitivity, reproducibility, and dynamic range.
- Flow cytometry offers high-throughput particle analysis, making it a potential tool for sensitive immunoassays.
Purpose of the Study:
- To develop and validate a fluoroimmunoassay utilizing a flow cytometer as the detection system.
- To assess the performance of this assay for both qualitative and quantitative analyses of various analytes.
Main Methods:
- Development of a fluoroimmunoassay using fluorescein-labeled antibodies.
- Utilized cyanogen bromide-activated agarose beads as solid-phase supports.
- Employed a flow cytometer for fluorescence detection and data acquisition.
Main Results:
- Successfully applied the flow cytometric immunoassay for qualitative determination of murine immunoglobulin (Ig) isotypes.
- Achieved quantitative determination of Ig, human chorionic gonadotropin (hCG), and progesterone with high reproducibility.
- Demonstrated a broader working range compared to traditional enzyme immunoassays.
Conclusions:
- Flow cytometry provides a reliable and sensitive platform for fluorescence detection in immunoassays.
- Agarose beads are effective solid-phase supports for microparticle-based flow cytometric immunoassays.
- This approach offers a robust alternative for quantitative and qualitative analysis of various biological analytes.
Abstract:
A fluoroimmunoassay employing a flow cytometer as the fluorescence-detecting device is described. Three kinds of antigens, murine immunoglobulin (Ig), human chorionic gonadotropin (hCG) and progesterone were chosen as examples of the assay using fluorescein-labeled antibodies. Cyanogen bromide-activated agarose beads were used as solid-phase supporters. The flow cytometric immunoassay was applied to both qualitative and quantitative analyses; determination of murine Ig isotypes, quantitative determination of Ig, hCG and a hapten, progesterone. This assay produced very reproducible and less-fluctuating data since thousands of particles in the assay were collected and processed to produce a single value for fluorescence intensities. Furthermore, the working range of the assay in terms of antigen concentration was much broader than that of enzyme immunoassay. Therefore, we believe that microparticles like agarose beads could be useful solid-phase supporters in immunoassay, and the flow cytometer could provide a reliable alternative to the fluorescence-detecting device in immunoassay.