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Flow Cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Updated: Jul 2, 2026

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Published on: December 12, 2011

Multiplexed microsphere-based flow cytometric immunoassays.

Kathryn L Kellar¹, Aida J Mahmutovic, Kakali Bandyopadhyay

¹National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

Current Protocols in Cytometry

|September 5, 2008

Summary

This summary is machine-generated.

Multiplexed microsphere immunoassays offer a cost-effective and sensitive alternative to traditional ELISAs for measuring multiple analytes. Rigorous validation is crucial to eliminate cross-reactivities in these advanced diagnostic tools.

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Area of Science:

Biotechnology
Immunology
Analytical Chemistry

Background:

Multiplexed microsphere-based immunoassays enable simultaneous measurement of multiple analytes using flow cytometry.
These assays adapt the ELISA format to microspheres for quantifying multiple antigens, offering advantages over traditional methods.
Potential cross-reactivities necessitate systematic elimination during validation.

Purpose of the Study:

To describe the development and validation of multiplexed microsphere immunoassays.
To highlight the advantages of these assays, including smaller sample volumes and reduced costs.
To detail protocols for coupling analytes and biotinylating antibodies for assay development.

Main Methods:

Utilizing spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes.
Adapting ELISA format to microspheres for sandwich immunoassays to quantitate antigens.
Employing antibody-capture immunoassays for detecting multiple antibodies in specimens.

Main Results:

Demonstrated that multiplexed immunoassays are as reproducible, reliable, and sensitive as ELISAs.
Showcased the ability to combine sandwich and competitive immunoassays in a single array.
Provided protocols for analyte coupling and antibody biotinylation.

Conclusions:

Multiplexed microsphere immunoassays provide a sensitive, cost-effective, and efficient platform for multiplexed analyte detection.
The described methods facilitate the development of robust assays for both antigen and antibody quantification.
Systematic validation is essential to ensure assay specificity and reliability in complex biological systems.