Encyclopedia of Experiments: Biological Techniques
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Transcript
Begin the viral antigen microarray assay with a prepared viral antigen microarray slide.
The slide contains multiple pads, each with a single array containing hundreds of purified antigens of multiple viral strains printed onto spots arranged in a grid. Each spot contains a single antigen type adsorbed on the three-dimensional microporous nitrocellulose surface.
Secure the slide into a chamber for easy handling during subsequent steps. Add a blocking buffer containing proteins that bind to the slide's remaining surfaces to prevent the non-specific binding of antibodies to the surface.
Incubate the microarray slide with diluted human serum. The antibody isotypes in the serum, specific for the antigens on the slide, recognize and bind to the antigens' epitopes.
Remove the serum. Wash with a buffer to remove unbound antibodies and proteins on the slide.
Add quantum dot-conjugated secondary antibody mixtures, each with a different quantum dot. The isotype-specific secondary antibodies bind specifically to the respective serum antibody isotype bound to the antigens on the slide.
Use a microarray imager to quantify antibody binding to antigens within the microarray slide.
Upon excitation at appropriate wavelengths, the quantum dots bound to secondary antibodies return to the ground state and emit specific fluorescent signals that are detected by the photodetector.
The spot fluorescence intensity quantifies the serum antibody isotype within the serum bound to the individual antigen.
