This article describes a method for immobilizing antibodies on amine-functionalized filter-paper discs using glutaraldehyde. The process involves several steps including washing, blocking, and detection of immunoglobulin G (IgG) molecules.
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.
Add five milliliters of 0.05% glutaraldehyde solution to a 20-milliliter glass bottle. Immerse 15 amine-functionalized medium-flow filter paper discs in this solution, and keep for one hour with shaking at room temperature. Concurrently, repeat this step to prepare another 15 aldehyde-functionalized fast-flow filter paper discs.
To remove unreacted glutaraldehyde from the paper discs, first, place the medium-flow filter paper discs, and the fast-flow filter paper discs into separate 15-milliliter centrifuge tubes. Then, add five milliliters of deionized water to each tube, and shake the tubes for 10 seconds. Remove the water by aspirating with a pipette. Repeat two times to remove any unreacted glutaraldehyde.
Then, dry the paper discs in a 37 degrees Celsius oven. Once dry, add five microliters of 0.025 milligrams per milliliter mouse IgG-Fc fragment antibodies to the medium-flow filter paper discs, and eight microliters to the fast-flow filter paper discs.
After incubating for 20 minutes, add 10 microliters of 50 millimolar PBS pH 7.4 to each paper disc without removing the antibodies. Incubate for 40 minutes for the amine-aldehyde reaction. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.
After drying the paper discs in an oven at 37 degrees Celsius, block the paper discs with 15 microliters of blocking buffer for 10 minutes at room temperature. Then, wash each paper disc with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.
Next, run the IgG standards. Load 10 microliters of various IgG concentrations onto individual discs in triplicate. Incubate for one hour at room temperature. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.
Following the washes, load 10 microliters of HRP-conjugated mouse IgG-Fc fragment antibodies, and incubate for one hour at room temperature. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.
Finally, load a 10-microliter mixture of TMB and hydrogen peroxide onto each disc. Take images of all paper discs with a digital camera or smartphone after five minutes of incubation, and analyze as discussed in the text protocol.
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