Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Multianalyte immunobead assay uses multiple antigen-coated microspheres to recognize their corresponding target antibodies simultaneously.

To begin this assay, take a mixture of microbeads in a tube containing the appropriate buffer. Each bead is coated with a specific antigen on its surface, allowing easy differentiation of beads from one another.

Pipette the coated immunobead mixture into the wells of a microtiter assay plate. Supplement the wells with the serum sample containing different isotypes of target antibodies. These variants have subtle differences in their structures, allowing each well to have multiple primary antibodies.

Incubate, to allow sample antibody isotypes to attach to specific antigens captured on respective immunobeads. Wash with an assay buffer to remove unbound primary antibodies. Add fluorescent dye tagged-secondary antibodies, corresponding to each variant of primary antibody, into the well.

Each labeled secondary antibody binds to the Fc site of the respective primary antibody. Multiple bead-captured antigen-primary antibody complexes are fluorescently labeled. Wash the plate to remove unbound secondary antibodies.

Read the plate using a fluorescent analyzer capable of concurrently detecting different fluorophores at their respective wavelengths. Depending upon the signal detection, various isotypes of serum antibodies and their binding to different antigens can be assessed.

For assay performance, resuspend the coupled microspheres by vortex for 30 seconds, and sonicate for 60 to 90 seconds. Then, remove the required quantity of each bead colloid from the respective tube, and combine the bead colloids in a new 1.5-millimeter microcentrifuge tube.

Next, insert the tube into a magnetic separator, and allow separation for 60 seconds. With the tube still in the magnetic separator, remove the supernatant, without disturbing the bead pellet.

After removing the beads from the separator, resuspend the beads in 100 microliters of assay buffer. Vortex for 30 seconds before placing the tube into a magnetic separator for 60 seconds, to separate the beads. Repeat the washing twice.

Next, adjust the concentration of the three-plex working microsphere mixture by adding an appropriate volume of assay buffer to generate a final concentration of 100 microspheres per 1 µL, for each target.

Aliquot 25 microliters of the microsphere mixture into each well of a 96-well plate. Dilute plasma or serum specimens 500-fold in assay buffer, and prepare standard specimens according to titration desired.

Add 25 microliters of assay buffer as the blank sample, and add each of the diluted specimens or standard, into each designated well of a 96-well specimen plate. When done, cover the plate with an aluminum seal or foil to incubate for 1 hour at room temperature on a plate shaker set to 700 rotations per minute.

Prepare a solution of anti-human detection antibodies, or secondary antibody solutions, at 4 µg/mL with assay buffer, as described in the manuscript. Place the plate on a magnetic separator to wash rapidly, and then, forcefully invert the plate over a biohazard container to remove liquid from the wells. With the plate still inverted, forcefully tap the plate against a thick wad of paper.

Then, wash each well with 100 microliters of assay buffer, and remove the liquid by forceful inversion over a biohazard container. Repeat the wash twice. After the last wash, discard all used wads of paper into a biohazard container.

Next, add 25 microliters of secondary antibody working solution to each well of a 96-well plate, and cover the plate with aluminum foil to incubate on a plate shaker at 700 rotations per minute.

After 30 minutes of incubation, wash the wells of the plate twice with assay buffer, as demonstrated earlier. Then, add 100 microliters of assay buffer into each well of the 96-well plate. Cover the plate with aluminum foil, and incubate for five minutes on a plate shaker at room temperature.

Analyze a 60-microliter sample via the instrument analyzer according to the system manual.