A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva
Published: January 31, 2024
Abstract
Source: Augustine, S. A. J., et al. Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens. J. Vis. Exp. (2016).
This video demonstrates a bead-based multiplex immunoassay technique to simultaneously detect antibodies for multiple environmental pathogens in human saliva. Fluorescent beads were coupled to antigens from different pathogens, which were captured using antibodies in the human saliva sample. Upon labeling the antibodies using a fluorescent reporter, the beads were analyzed using flow cytometry to assess the presence of different pathogen-specific antibodies in the saliva.
Protocol
1. Bead Coupling
- Couple the antigens to the bead sets using the concentrations shown in Table 1.
- Add each antigen to the activated beads and bring the total volume to 500 µl in 50 mM MES, pH 5.0. Mix the antigens and beads by vortex.
- Incubate the antigens and beads for 2 hr with mixing by rotation (~15 rpm) at room temperature in the dark. Pellet the coupled beads by microcentrifugation at 10,000 x g for 2 min.
- Remove the supernatant and resuspend the beads in 500 µl of phosphate-buffered saline (PBS)-bovine serum albumin (BSA)-polyoxyethylene sorbitan monolaurate (Tween-20)-sodium azide (PBS-TBN) pH 7.4 by vortex and sonication. Pellet the beads by microcentrifugation at 10,000 x g for 2 min and remove the supernatant.
Caution: Sodium azide is an acutely toxic chemical. It is fatal if swallowed or gets in contact with the skin. Do not breathe dust/fumes/gas/mist/vapors or spray. Wear appropriate personal protective equipment (PPEs) when handling and disposing of it in accordance with appropriate laws. - Resuspend the beads in 1 ml of PBS-TBN by vortex and sonication for 20 sec.
- Pellet the beads by microcentrifugation at 10,000 x g for 2 min.
- Repeat steps 1.5 and 1.6.
Note: This provides a total of two washes with PBS-TBN. - Resuspend the coupled and washed beads in 1 ml of PBS, 1% BSA, 0.05% Azide, pH 7.4. Store the coupled beads in a 2-8 °C refrigerator in the dark.
2. Salivary Multiplex Immunoassay
- Remove saliva from the -80 °C freezer and allow it to thaw at room temperature.
- Resuspend antigen-coupled bead stocks by vortex and sonication for 20 sec.
- Prepare a working bead mixture by diluting the coupled bead stocks to a final concentration of 100 beads/µl of each unique bead set in PBS-1% BSA buffer.
- Prepare a 1:4 dilution of saliva with PBS-1% BSA buffer in a 96-well, deep well plate.
- Pre-wet the filter plate with 100 µl of wash buffer and remove the supernatant by vacuum.
- Add 50 µl of a working bead mixture and an equal volume of diluted saliva to 95 wells of the 96-well filter plates for a 1:8 final dilution. Mix reactions with a multi-channel pipettor. To the one control well, add 50 µl antigen-coupled beads plus 50 µl of PBS-1% BSA buffer (as a replacement for saliva).
- Cover and allow to incubate in the dark at room temperature for 1 hr on a microplate shaker at 500 rpm. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
- Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
- Dilute biotinylated goat anti-human IgG secondary detection antibody to 16 µg/ml in PBS-1% BSA.
- Add 50 µl diluted secondary antibody to each well and mix contents with a multi-channel pipettor.
- Cover the filter plate and allow it to incubate in the dark at room temperature for 30 min on a plate shaker. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
- Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
- Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 µg/ml in PBS-1% BSA.
- Add 50 µl reporter to each well and mix with a multi-channel pipettor.
- Cover the filter plate and allow it to incubate in the dark at room temperature for 30 min on a plate shaker. Remove supernatant by vacuum. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat wash 1x.
- Resuspend beads in 100 µl of PBS-1% BSA and analyze 50 µl using the analyzer.
Note: Results of the multiplex immunoassay are measured in Median Fluorescence Intensity (MFI) units. Always refer to the latest version of the software manual, if available to avoid errors.
Table 1: Working bead mix for the multiplex immunoassay. After coupling and confirmation were completed, a master mix consisting of each bead set was prepared as shown. The master mix was distributed among all of the wells in the 96-well plate. All of the wells were incubated with saliva with the exception of one background control well which contained only beads and PBS-1% BSA buffer.
| Antigen | Bead Set | Coupling buffer | Ag Concentration (µg) | # of beads in 1 corner | # of beads/µl | Vol. for 100 B/uL for 4 ml (µl) |
| C. jejuni | 8 | MES 5.0 | 50 | 64 | 6,400 | 94 |
| H. pylori | 33 | MES 5.0 | 25 | 71 | 7,100 | 85 |
| Hepatitis A | 42 | MES 5.0 | 100 | 91 | 9,100 | 66 |
| T. gondii | 30 | MES 5.0 | 25 | 85 | 8,500 | 71 |
| Norovirus GII.4 | 55 | MES 5.0 | 5 | 72 | 7,200 | 83 |
| Norovirus GI.1 | 67 | MES 5.0 | 5 | 63 | 6,300 | 95 |
| Uncoupled | 80 | MES 5.0 | 60 | 6,000 | 100 | |
| Total volume of beads (µl) | 594 | |||||
| Total volume of buffer needed (µl) | 3,406 | |||||
Disclosures
The authors have nothing to disclose.
Materials
| Equipment and Software | |||
| Microcentrifuge | Thermo Electron Corporation | 75002446 | Used to centrifuge samples |
| Vortex Mixer | VWR | G560 | Used to mix samples |
| Sonicator (mini) | Fisher Scientific | 15-337-22 | Used to separate beads |
| Pipettors P10, P20, P100, P1000, 8 ch. | Capp | Various | |
| Multiscreen Vacuum Manifold | Millipore | MSVMHTS00 | Used in washing steps to remove supernatant |
| MicroShaker | VWR | 12620-926 | Used to agitate beads during incubations |
| Tube rack (1.5mL and 0.5mL) (assorted) | VWR | 30128-346 | |
| Weighing Scale | Mettler or other | Used to measure wash reagents for making buffers | |
| Dynabead Sample Mixer | Invitrogen | 947-01 | Used during coupling incubation step |
| MatLab (R2014b) | The MathWorks, Inc. | Used to analyze antibody response data | |
| Microsoft Excel 2014 | Microsoft Corporation | Used to analyze antibody response data | |
| Luminex Analyzer with xPonent 3.1 software | Luminex Corporation | LX200-XPON3.1 | Instrument and software used to run assay |
| Antigens | |||
| GI.1 Norwalk Virus : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| GII.4 Norovirus VA387 : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| Hepatitis A Virus : grade II concentrate from cell culture | Meridian Life Sciences | 8505 | Antigen coupled at 100 µg |
| Helicobacter pylori : lysate | Meridian Life Sciences | R14101 | Antigen coupled at 25 µg |
| Toxoplasma gondii : recombinant p30 (SAG1) | Meridian Life Sciences | R18426 | Antigen coupled at 25 µg |
| Campylobacter jejuni : heat killed whole cells | KPL | 50-92-93 | Antigen coupled at 50 µg |
| Primary Antibodies | |||
| Guinea pig anti-Norovirus | (CCHMC)* | NA | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| BacTraceAffinity Purified Antibody to Helicobacter pylori | KPL | 01-93-94 | Used for coupling confirmation |
| Goat pAb to Toxoplasma gondii | Abcam | Ab23507 | Used for coupling confirmation |
| BacTrace Goat anti-Campylobacter species | KPL | 01-92-93 | Used for coupling confirmation |
| Secondary Antibodies | |||
| Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L) | Jackson | 705-065-149 | Used for coupling confirmation |
| Biotinylated Rabbit anti-Goat IgG (H+L) | KPL | 16-13-06 | Used for coupling confirmation |
| Biotinylated Goat anti-Mouse IgG (H+L) | KPL | 16-18-06 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG(H+L) | KPL | 176-1506 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(γ) | KPL | 16-10-02 | Used for Salivary Immunoassay |
| Consumables | |||
| 1.5 mL copolymer microcentrifuge tubes | USA Scientific | 1415-2500 | Used as low binding microcentrifuge tubes |
| 10 µL pipette tip refills | BioVentures | 5030050C | |
| 200 µL pipette tip refills | BioVentures | 5030080C | |
| 1000 µL pipette tip refills | BioVentures | 5130140C | |
| Aluminum foil | Various Vendors | Used keep beads in the dark during incubations | |
| Deep Well plates | VWR | 40002-009 | Used for diluting saliva samples |
| Multiscreen Filter Plates | Millipore | MABVN1250 | Used to run assays |
| Oracol saliva collection system | Malvern Medical Developments Limited | Used for saliva collection | |
| Reagents | |||
| Carboxylated microspheres (beads) | Luminex Corporation | Dependent on bead set | Antigens are coupled to the microspheres |
| EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) | Pierce | 77149 or 22980 | Used in bead activation |
| Sulfo-NHS (N-hydroxysulfosuccinimide) | Pierce | 24510 | Used in bead activation |
| Steptavidin-R-phycoerythrin (1mg/mL) | Molecular Probes | S-866 | Used as reporter |
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | M-2933 | Used for coupling |
| Tween-20 (Polyoxyethylenesorbitan monolaurate) | Sigma | P-9416 | Used in wash buffer to remove non-specific binding |
| Protein Buffers | |||
| PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)** | Filter Sterilize and store at 4°C | ||
| PBS, pH 7.4 | Sigma | P-3813 | 138 mM NaCl, 2.7 mM KCl |
| BSA | Sigma | A-7888 | 0.1% (w/v) |
| Tween-20 | Sigma | P-9416 | 0.2% (v/v) |
| Sodium Azide (0.05% azide)** | Sigma | S-8032 | **Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE's. Dispose of according to applicable laws. |
| MES/ Coupling Buffer (0.05 M MES, pH 5.0) | |||
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | S-3139 | |
| 5 N NaOH | Fisher | SS256-500 | |
| Assay Buffer (PBS, 1% BSA, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 1% BSA, pH 7.4 | Sigma | P-3688 | 138 mM NaCl, 2.7 mM KCl, 1% BSA |
| Activation Buffer (0.1 M NaH2PO4, pH 6.2) | Filter Sterilize and store at 4°C | ||
| NaH2PO4 (Sodium phosphate, monobasic anhydrous) | Sigma | S-3139 | 0.1M NaH2PO4 |
| 5 N NaOH | Fisher | SS256-500 | |
| Wash Buffer (PBS, 0.05% Tween-20, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 0.05% Tween-20, pH 7.4 | Sigma | P-3563 | 138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN |
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Cite This Article
A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva. J. Vis. Exp. (Pending Publication), e21892, doi: (2024).