Encyclopedia of Experiments: Immunology
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Transcript
Take an assay plate containing a mixture of magnetic polystyrene microspheres internally dyed with spectrally different fluorophores.
Each microsphere set is covalently coupled to distinct pathogenic bacteria-specific capture antibodies.
Add a heat-killed pathogenic bacterial mixture. Incubate.
Capture antibodies bind the target bacterial antigens, forming microsphere-bacteria complexes.
Use a magnetic separator to settle complexes. Discard unbound bacteria and wash with buffer.
Add buffer and biotinylated detection antibodies, which bind specifically to microsphere-bound bacteria.
Magnetically separate the complexes and wash with buffer.
Resuspend the complexes in buffer. Pipette reporter molecules comprising fluorophore-coupled streptavidin, which bind to the biotinylated detection antibodies.
Magnetically separate and wash the complexes. Resuspend in buffer.
During flow-based analysis, the first laser excites the microsphere's internal fluorophores, generating unique spectral signatures and identifying unique microsphere sets.
The second laser excites the fluorophore reporters bound to bacteria on microspheres, quantifying bacteria on various microsphere sets, enabling multiplexed quantitative bacterial detection.
