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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of antigen-conjugated microspheres

1. Select three different vials of magnetic microspheres with unique bead regions, recording bead ID and lot information for each vial used.
   NOTE: The following steps will be followed for each distinct microsphere region. Microspheres are light-sensitive and should be protected from prolonged exposure to light. During wash steps, be careful not to disturb the microspheres. If disturbed, allow a second 60 s separation.
2. Vortex the microsphere stock for 60 s and sonicate for 5 min prior to use to dissociate aggregated beads.
3. Transfer 1.0 x 10⁶ beads to a 1.5 mL low-protein binding microcentrifuge tubes.
4. Insert the tube into a magnetic separator and allow separation to occur for 60 s. With the tube still in the magnetic separator, carefully remove the supernatant without disturbing the bead pellet.
5. Remove the tube from the magnetic separator, resuspend the beads with 100 µL of HPLC-grade water, and vortex for 30 s. Place the tube back into the magnetic separator for 60 s, and subsequently remove the supernatant. Repeat this wash protocol twice.
6. Remove the tube from the magnetic separator and resuspend the washed microspheres in 90 µL of 100 mM Monobasic Sodium Phosphate, pH 6.2 (Activation Buffer) by vortex for 30 s.
7. Add 10 µL of 50 mg/mL Sulfo-NHS (diluted with Activation Buffer) to the microspheres and vortex gently for 10 s. Add 10 µL of 50 mg/mL EDC solution (diluted with Activation Buffer) and vortex gently for 10 s. Incubate microspheres for 20 min at room temperature (RT) with a gentle vortex every 10 min.
8. Repeat wash steps 1.4-1.5 with 50 mM MES, pH 5.0 (Coupling Buffer) in lieu of LC/MS-grade water for a total of two washes.
9. Remove the tube from the magnetic separator and resuspend the beads with 100 µL of Coupling Buffer by vortex for 30 s followed immediately by the addition of the desired quantity of protein.
10. Bring the total volume to 150 µL with Coupling Buffer. Mix coupling reaction by vortex for 30 s and incubate for 2 h by rotation at RT.
    NOTE: For assays defined herein, conjugations were performed with the following protein concentrations: Spike S1: 5 µg; Nucleocapsid: 5 µg; Membrane: 12.5 µg.
11. Repeat wash step 1.4 with Phosphate Buffered Saline (PBS)-1% Goat Serum Albumin, 0.01% Polysorbate-20 (Quench Buffer) in lieu of LC/MS-grade water for a total of two washes. Resuspend the washed microspheres in 100 µL of Quench Buffer containing 0.05% sodium azide by vortex for 30 s.
    NOTE: Permit the beads to quench fully for at least 6 h prior to proceeding to any other procedure.
12. Count the number of recovered microspheres using an automated cell counter or a hemocytometer. Record the observed bead concentration.
13. Refrigerate the coupled microspheres at 4 °C in the dark.

2. Procedures

1. Assay performance (Base protocol)
   1. Resuspend the coupled microspheres by vortex for 30 s and sonicate for ~60-90 s.
   2. Remove the required quantity of each bead colloid from the respective tube and combine the bead colloids in a new 1.5 mL microcentrifuge tube.
   3. Insert the tube into a magnetic separator and allow separation to occur for 60 sec. With the tube still in the magnetic separator, carefully remove the supernatant without disturbing the bead pellet.
   4. Remove the beads from the separator, resuspend the beads with 100 µL of PBS-1% bovine serum albumin, 0.01% Polysorbate-20 (Assay Buffer), and vortex for 30 s. Place the tube into a magnetic separator and allow separation to occur for 60 s. Repeat this washing protocol (i.e., steps 2.1.3-2.1.4) twice.
   5. Adjust the concentration of the 3-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.
   6. Aliquot 12.5 µL of the microsphere mixture prepared in step 2.1.5 into each well of a 384-well plate or 25 µL into each well of a 96-well plate.
   7. Dilute the plasma/serum specimens 500-fold in Assay Buffer. Prepare standard specimens according to titration desired.
   8. Add 12.5 µL of Assay Buffer as the blank sample and add each of the diluted specimen or standard into each designated well of a 384-well specimen plate or 25 µL of the blank, diluted specimen or standard into each well of a 96-well specimen plate.
   9. Cover the plate with an aluminum seal or foil and incubate for 1 h at RT on a plate shaker set to 700 rpm.
      NOTE: Schematic for the dilution curves is provided in Table 1.
   10. Prepare a solution of anti-human detection antibodies (secondary antibody solutions) at 4 µg/ mL with Assay Buffer as specified in step 2.1.11.
   11. Prepare Goat-anti-human IgM, conjugated with Super Bright 436 (SB)/Goat-anti-human IgA, Phycoerythrin (PE) Conjugate detections antibodies at 4 µg/mL; or Goat-anti-human IgM, SB Conjugate/ Goat-anti-human IgG, PE Conjugate detections antibodies at 4 µg/mL.
       NOTE: For a 384-well plate format, 12.5 µL/well of the prepared secondary antibody solution is required, and for a 96-well plate format, 25 µL/well is required.
   12. Place the plate on a magnetic separator, wash rapidly, and forcefully invert 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.
   13. Wash each well with 100 µL of Assay Buffer and remove the liquid by forceful inversion over a biohazard container, as previously described. Repeat these steps (2.1.12-2.1.13) for a total of two washes. Discard all used wads of paper into a biohazard container.
   14. Add 12.5 µL of the secondary antibody working solution to each well of a 384-well plate or 25 µL to each well of a 96-well plate. Cover the plate with an aluminum seal or foil and incubate for 30 min at RT on a plate shaker set to 700 rpm.
   15. Repeat wash steps 2.1.12-2.1.13.
   16. Add 75 µL of Assay Buffer into each well of a 384- well plate or 100 µL into each well of a 96-well plate. Cover the plate with an aluminum seal or foil and incubate for 5 min at RT on a plate shaker set to 700 rpm.
   17. Analyze 60 µL via the instrument analyzer according to the system manual.

Table 1: Dilution series for standard curves: Table of dilution factors used for the standard curves for the IgG serotype; presented as a 7-point, 1:5 serial dilution curve starting at 1 µg/mL for α-Spike S1 and α-Nucleocapsid and 5 µg/mL for α-Membrane antibodies.