Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

Overview

This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.

Protocol

1. Covalent Coupling of Capture Antibodies to MagPlex Microspheres

Each capture antibody is conjugated to a specific carboxylated magnetic microsphere set using a two-step procedure as described below.

NOTE: A coupling kit including all of the reagents, buffers, and materials required for microsphere coupling is also available (see TABLE OF MATERIALS). 

1. Resuspend the stock uncoupled microsphere suspension according to the instructions described in the product information sheet provided with your microspheres.
   NOTE: Resuspension instructions will depend on the size and volume of your stock microsphere vials.
2. Transfer 2.5 x 10⁶ of the stock microspheres to a recommended microcentrifuge tube (see TABLE OF MATERIALS).
3. Place the tube into a magnetic separator and allow separation to occur for 60 seconds.
4. With the tube still positioned in the magnetic separator, remove the supernatant. 
   NOTE: Take care not to disturb the microspheres.
5. Remove the tube from the magnetic separator and resuspend the microspheres in 100 µL of dH₂O by vortex and sonication for 20 seconds.
6. Repeat steps 3 and 4.
7. Remove the tube from the magnetic separator and resuspend the washed microspheres 80 µL of 0.1 M sodium phosphate monobasic, pH 6.2 by vortex and sonication for 20 seconds.
8. Add 10 µL of 50 mg/mL N-hydroxysulfosuccinimide (sulfo-NHS) (diluted in 0.1 M sodium phosphate monobasic,          pH 6.2) to the microspheres and mix gently by a vortex.
9. Add 10 μL of 50 mg/mL 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (diluted in 0.1 M sodium phosphate monobasic, pH       6.2) to the microspheres and mix gently by a vortex.
10. Incubate for 20 minutes at room temperature with gentle mixing by vortex at 10-minute intervals.
11. Place the tube into a magnetic separator and allow separation to occur for 60 seconds.
12. With the tube still positioned in the magnetic separator, remove the supernatant. 
    NOTE: Take care not to disturb the microspheres.
13. Remove the tube from the magnetic separator and resuspend the microspheres in 250 µL phosphate-buffered saline (PBS), pH 7.4 by vortex and sonication for approximately 20 seconds.
14. Place the tube into a magnetic separator and allow separation to occur for 60 seconds.
15. With the tube still positioned in the magnetic separator, remove the supernatant. 
    NOTE: Take care not to disturb the microspheres.
16. Repeat steps 13-15 for a total of two washes.
17. Remove the tube from the magnetic separator and resuspend the activated and washed microspheres in 100 µL of PBS, pH 7.4 by vortex and sonication for approximately 20 seconds.
18. Add 12.5 μg of capture antibody to the resuspended microspheres (i.e., 5 μg/1 million microspheres).
    NOTE: Monoclonal antibodies are preferred for capture but polyclonal antibodies may also be used.
    NOTE: 5 μg protein per 1 million beads typically performs well but we recommend titrating the amount to achieve optimal assay performance.
19. Bring total volume to 500 μL with PBS, pH 7.4.
20. Mix coupling reaction by a vortex.
21. Incubate for 2 hours with mixing by rotation at room temperature in the dark.
22. Place the tube into a magnetic separator and allow separation to occur for 60 seconds.
23. With the tube still positioned in the magnetic separator, remove the supernatant.
    NOTE: Take care not to disturb the microspheres.
24. Remove the tube from the magnetic separator and resuspend the coupled microspheres in 1 ml of PBS, pH 7.4 containing 0.02% Tween-20, 0.1% bovine serum albumin (BSA), 0.05% sodium azide (PBS-TBN) by vortex and sonication for 20 seconds.
25. Place the tube into a magnetic separator and allow separation to occur for 60 seconds.
26. With the tube still positioned in the magnetic separator, remove the supernatant. 
    NOTE: Take care not to disturb the microspheres.
27. Repeat steps 24-26 for a total of two washes.
28. Remove the tube from the magnetic separator and resuspend the coupled microspheres in 500 µL of PBS-TBN.
    NOTE: Determine the number of microspheres recovered using a cell counter or hemocytometer.
29. Store coupled microspheres refrigerated at 2-8°C in the dark.
    NOTE: A protocol to confirm coupling efficiency is also available.

2. Biotin Labeling of Detection Antibodies

1. Detection antibodies were biotin-labeled using EZ-Link sulfosuccinimidyl-6-(biotinamido) hexanoate according to the manufacturer's instructions with a 20-fold molar excess of biotin reagent to achieve 4-6 biotin groups per antibody molecule.
   NOTE: Polyclonal antibodies are typically used for detection, but monoclonal antibodies may also be used if specific for a different epitope than the capture antibody.
   NOTE: Alternatively, commercially available biotinylated antibodies may be used for detection.  

3. Detection of Organisms by Multiplexed Capture Sandwich Immunoassay

The coupled microsphere sets and labeled detection antibodies generated in 1 and 2 above are used in a multiplexed capture sandwich immunoassay to detect and quantify the bacteria present in a sample. An overview of the assay workflow is shown in Figure 1.

1. Select the stock vials of antibody-coupled MagPlex bead sets from 2-8ºC storage.
2. Vortex and sonicate for 20 seconds.
3. Prepare a working multiplexed bead mix so that each bead set is at a final concentration of 2500 microspheres per 50 µL for each reaction well (50,000 beads/set/ml) in PBS-TBN.
   NOTE: Use a spreadsheet to determine preparation parameters for the working bead mix.
   NOTE: Internal control beads (RP1 Monitor Microspheres) may also be included to monitor assay and instrument performance.
4. Pipette 50 µL of working bead mix into the appropriate wells of a 96-well plate.
5. Pipette 50 µL of standard or sample to appropriate wells.
6. Pipette 50 µL of PBS-TBN to each background well.
7. Cover the plate with a foil seal to protect the beads from light and incubate on a plate shaker at room temperature for 1 hour with shaking at 800 rpm.
8. Place the plate on a magnetic plate separator for 1 minute.
9. Keeping the plate on the magnetic plate separator, carefully remove the foil and invert the plate to empty the wells, then tap to dry on a thick layer of laboratory paper towels 3-4 times.
   NOTE: Alternatively, manually aspirate the wells using a multi-channel pipettor or use an automated plate washer.
10. Use a multi-channel pipettor to add 200 µL of PBS-TBN to each well and shake for 1 minute on a plate shaker at 800 rpm.
11. Place the plate on a magnetic plate separator for 1 minute.
12. Keeping the plate on the magnetic plate separator, invert the plate to empty the wells, then tap to dry on a thick layer of laboratory paper towels 3-4 times.
    NOTE: Alternatively, manually aspirate the wells using a multi-channel pipettor or use an automated plate washer.
13. Repeat steps 10-12 for a total of two washes.
    NOTE: Remove as much buffer as possible before proceeding to the next step to avoid potential dilution of the reaction.
14. Use a multi-channel pipettor to add 50 µL of assay buffer to each well and shake for 1 minute on a plate shaker at 800 rpm.
15. Dilute the biotinylated detection antibody to 4 µg/ml in PBS-TBN.
16. Add 50 µL of the diluted detection antibody to each well.
17. Cover the plate with a foil seal to protect the beads from light and incubate on a plate shaker at room temperature for 1 hour with shaking at 800 rpm.
18. Place the plate on a magnetic plate separator for 1 minute.
19. Keeping the plate on the magnetic plate separator, invert the plate to empty the wells, then tap to dry on a thick layer of laboratory paper towels 3-4 times.
    NOTE: Alternatively, manually aspirate the wells using a multi-channel pipettor or use an automated plate washer.
20. Wash each well two times following the procedure described in steps 10-12.
    NOTE: Remove as much buffer as possible before proceeding to the next step to avoid potential dilution of the reaction.
21. Use a multi-channel pipettor to add 50 µL of assay buffer to each well and shake for 1 minute on a plate shaker at 800 rpm. Streptavidin, R-Phycoerythrin Conjugate (SAPE).
22. Dilute the SAPE reporter to 4 µg/mL in assay buffer.
23. Add 50 µL of the diluted SAPE to each well.
24. Cover the plate with a foil seal to protect the beads and SAPE from light and incubate on a plate shaker at room temperature for 30 minutes with shaking at 800 rpm.
25. Place the plate on a magnetic plate separator for 1 minute.
26. Keeping the plate on the magnetic plate separator, invert the plate to empty the wells, then tap to dry on a thick layer of laboratory paper towels 3-4 times.
    ​NOTE: Alternatively, manually aspirate the wells using a multi-channel pipettor or use an automated plate washer.
27. Wash each well two times following the procedure described in steps 10-12.
28. Add 100 µL of PBS-TBN to each well and shake for 1 minute on a plate shaker at 800 rpm.
29. Analyze 50 µL of each reaction on the Luminex analyzer (according to the User Manual for the analyzer used), collecting a minimum of 50 beads per bead set for analysis. A brief description of the xMAP INTELLIFLEX® is provided below.
    1. Select the drop-down menu in the upper left corner of the screen and navigate to Plate Configuration.
    2. Select Run Plate.
    3. Select the Eject icon to eject the plate carrier.
    4. Load the plate onto the plate carrier and select the Retract icon to retract the plate carrier.
    5. Select the Run icon to run the plate.

Representative Results

Figure 1: Multiplexed capture sandwich immunoassay workflow. A multiplexed mixture of antibody-coupled microspheres is dispensed into each well, and the sample is added. After incubation for 1 hour, the reactions are washed, and the biotinylated detection antibodies are added. Following a 1 hour incubation, the reactions are washed again, and the SAPE reporter is added. Reactions are incubated for 30 minutes, washed, and the results are read on an xMAP flow analyzer.

Materials

Name	Company	Catalog Number	Comments
MagPlex® Microspheres	Luminex®	MC100xx-YY	See website for individual catalog numbers (https://www.luminexcorp.com/magplex-microspheres/#order)
1.5 mL microcentrifuge tubes	Eppendorf™	22431081	Protein LoBind®
0.1 M monobasic sodium phosphate (NaH2PO4), pH 6.2	MilliporeSigma	S3139	Activation buffer
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)	Thermo Scientific™	Pierce™ 77149
N-hydroxysulfosuccinimide (Sulfo-NHS)	Thermo Scientific ™	Pierce™ 24510
Phosphate-buffered saline (PBS), pH 7.4	MilliporeSigma	P3813	Coupling buffer
xMAP® Antibody Coupling Kit	Luminex®	40-50016	Optional kit available for microsphere coupling.
Anti-Escherichia coli O157:H7 antibody	SeraCare	5310-0326	Polyclonal, used for both capture and detection
Anti-Salmonella CSA-Plus antibody	SeraCare	5310-0321	Polyclonal, used for both capture and detection
Anti-Campylobacter spp. antibody	SeraCare	5310-0324	Polyclonal, used for both capture and detection
Anti-Listeria antibody	SeraCare	5310-0319	Polyclonal, genus-specific, used for both capture and detection
PBS, pH 7.4 containing 0.02% Tween-20, 0.1% BSA, 0.05% sodium azide (PBS–TBN)	MilliporeSigma	P3563 A7888 S8032	Up to 1% BSA may be used
Anti-Escherichia coli O157:H7 Antibody	SeraCare	5310-0326	Polyclonal
(EZ-Link Sulfo-NHS-LC-Biotin) (Pierce).	Thermo Scientific™	A39257
E. coli O157:H7	SeraCare	5370-0013	Heat-killed
Salmonella serotype Typhimurium	SeraCare	5370-0002	Heat-killed
Campylobacter jejuni	SeraCare	5370-0011	Heat-killed
Listeria monocytogenes	SeraCare	5370-0010	Heat-killed
Streptavidin, R-Phycoerythrin Conjugate (SAPE)	Thermo Scientific™	S866