Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

To detect islet autoantibodies — a biomarker of type-1 diabetes — take human serum samples and add acetic acid to inhibit interfering components.

Incubate and then add an alkaline buffer to neutralize the acid.

Transfer the treated serum to a multi-well plate containing a mixture of human islet autoantigens, including biotinylated capture and ruthenium-conjugated antigens, each at an optimal concentration for the assay.

The islet autoantibody in the positive sample bridges the ruthenium-conjugated antigen to the biotinylated capture antigen, forming an antigen-antibody complex.

Transfer the mixture into a streptavidin-coated electrochemiluminescence plate pre-treated with a blocking agent and equipped with working and counter electrodes.

Incubate to allow biotinylated complex interactions with the bound streptavidin and then wash.

Add a buffer containing tripropylamine or TPA.

In an electrochemiluminescence instrument, the voltage application between the electrodes initiates the oxidation of ruthenium and TPA, generating TPA radicals.

The TPA radicals excite ruthenium complexes, which emit photons while returning to their ground state.

A high chemiluminescence in the positive sample indicates the presence of islet autoantibodies.

Mix the human islet autoantigen with biotin or sulfo-tag in 1:5 molar ratio in a tube, and cover the tube with aluminum foil since both the tags are light-sensitive. Then, incubate the tube for an hour at room temperature. In the meanwhile, when the incubation is going on, prime the spin column with twofold phosphate-buffered saline.

Then, centrifuge the column at 1,000 times g for two minutes each time. Next, centrifuge the autoantigen tag mixture in the spin column at 1,000 times g for 2 minutes. Then, aliquot and store that labeled antigen at negative 80 degrees Celsius.

Next, use the rational concentration of the biotin sulfo-tag-labeled antigen based on the checkerboard assay for the antigen buffer to prepare 3 milliliters of antigen solution per 96-well plate.

Then in each well, add 4 microliters of serum and adjust the final volume to 20 microliters with 1-fold phosphate-buffered saline. Then, add 20 microliters of labeled antigen solution in each well, and cover the plate with sealing foil to prevent light. Then, transfer the plate on a shaker at room temperature for 2 hours.

Once the shaker stops, leave the plate at 4 degrees Celsius in the refrigerator for 18 to 24 hours. Now, mix 15 microliters of serum with 18 microliters of 0.5 molar acetic acid for each sample, and incubate the same at room temperature for 45 minutes.

On the basis of the checkerboard assay, use the rational concentration of the biotin sulfo-tag-labeled antigen to prepare the antigen solution.

Then, aliquot 35 microliters of the antigen buffer in each well of a new PCR plate. Just before the chorus of incubation of the serum mixture is completed, add 3.8 microliters of 1 molar Tris buffer at pH nine along the side of each well on the antigen plate.

Once the incubation is over, quickly transfer 25 microliters of the acetic acid-treated serum in each well of the antigen plate, and agitate the solution. Then, cover the PCR plate with sealing foil to avoid light, and place on a shaker at room temperature for 2 hours. Once done, transfer the plate at 4 degrees Celsius in the refrigerator, and incubate for 18 to 24 hours.

Remove the streptavidin plate from the refrigerator at 4 degrees Celsius, and let the plate reach room temperature. After the streptavidin plate attains room temperature, add 150 microliters of 3% blocker A in each well, and cover the PCR plate with sealing foil, and incubate in the refrigerator overnight.

The next day, remove the streptavidin plate from the refrigerator and expel the buffer from the wells. Then, dry the plate by setting it upside down on some dry paper towels for the remaining buffer to soak. Then, add 150 microliters of 1-fold PBST buffer to wash the well for three consecutive washes.

Next, transfer 30 microliters of serum antigen incubate in each well of the streptavidin plate, and cover the plate with foil to avoid light. Then, transfer the plate on a shaker at room temperature for an hour. After one hour, discard the serum antigen incubate from the plate, and add 150 microliters of 1-fold PBST buffer to wash the well three times. Once the washing is over, add 150 microliters of reading buffer to each well to read on a plate reader.