Encyclopedia of Experiments: Immunology
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Transcript
Begin with the amine-functionalized filter-paper discs and immerse them in glutaraldehyde, a cross-linking reagent.
Glutaraldehyde reacts with the amine groups via an amine-aldehyde reaction.
Place the discs in a tube. Add water, shake, and aspirate to remove the residual glutaraldehyde.
Post-drying, introduce the immunoglobulin-G-Fc fragment-specific capture antibodies. Amine groups on these antibodies interact with aldehyde groups on paper discs, facilitating their covalent immobilization.
Wash to remove unbound antibodies and introduce a blocking buffer to prevent non-specific binding.
Next, add a test sample containing immunoglobulin G molecules onto the paper disc. The immunoglobulin G molecules interact with the capture antibodies.
Overlay with horseradish peroxidase-conjugated IgG-Fc fragment-specific detection antibodies that interact with the pre-bound immunoglobulin G.
Wash to remove unbound antibodies and add a mixture of chromogenic substrate and hydrogen peroxide.
In the presence of hydrogen peroxide, horseradish peroxidase catalyzes the oxidation of the chromogenic substrate, generating a blue color on the disc, indicating the presence of immunoglobulin G.
