This article describes a detailed protocol for a microarray assay aimed at detecting multiple proteins in serum samples. The method involves the use of protein-specific capture antibodies and biotinylated detection antibodies to facilitate accurate quantification of protein concentrations.
Begin by assembling a pre-treated microarray assay slide with distinct spots containing protein-specific capture antibodies for multiple protein detection.
Load the dilutions of the protein standard and the pre-diluted serum samples into separate wells. These dilutions create a range of protein concentrations for accurate quantification.
Incubate to facilitate protein-antibody interactions. Wash to remove unbound proteins, disassemble, and dry the slide.
Invert the assay slide in a snap apparatus containing a rehydrated transfer slide spotted with protein-specific biotinylated detection antibodies in the same positions as the assay slide.
Close the apparatus and press to snap the slides over each other. This facilitates the transfer of the detection antibodies to corresponding spots on the assay slide, minimizing the cross-reactivity.
Separate the slides to allow the interaction of detection antibodies with bound proteins, forming immunocomplexes.
Reassemble the assay slide, rinse it, and introduce fluorophore-conjugated streptavidin, interacting with biotinylated antibodies.
Using a scanner, detect fluorescence spots at various locations, indicating multiple proteins in the serum sample. Compare the spot intensities with the standard for protein quantification.
To begin the immunoassay, remove the prepared assay slide from the freezer and allow it to warm to room temperature in its sealed bag for 30 minutes. Prepare seven serially diluted solutions of proteins in PBS with 0.05% polysorbate-20. Prepare appropriately diluted sample solutions in the same buffer.
Clamp a 24-well silicone gasket onto the room-temperature assay slide. Load the seven protein dilutions into seven of the eight wells in a column. Load protein-free PBS with 0.05% polysorbate-20 into the last well of that column. Load the sample solutions into the remaining wells.
Shake the slides at 450 RPM for 1 hour at room temperature or at 4 degrees Celsius overnight. Then, wash the slide three times. Remove the gasket and dry the slide under a stream of nitrogen gas.
Next, allow the detection antibody transfer slide to warm to room temperature for 30 minutes in its sealed bag. Then, incubate the slides for 20 minutes in a closed chamber with a relative humidity of 60% to rehydrate the arrays. Use the pogo pins to fix the detection transfer slide face-up in the snap apparatus.
Place the assay slide face-down on the pogo pins, and align the slide with the straight pins. Close the apparatus and snap-transfer the detection antibodies to the assay slide. Remove the slides from the apparatus and incubate the assay slide for one hour.
Clamp a 24-well silicone gasket to the slide, and wash the slide three times. Then, load into each well 80 microliters of a 2.5 microgram per milliliter solution of streptavidin fluorophore in PBS. Shake the slide at 450 RPM for 20 minutes.
Wash the slide three times with a 0.1% solution of polysorbate-20 in PBS, and once with distilled water. Then, remove the gasket and dry the slide. Scan the slide with a fluorescent scanner and measure the spot intensities. Determine the limits of detection and the protein concentrations.
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