A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva

Overview

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

1. Couple the antigens to the bead sets using the concentrations shown in Table 1.
2. 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.
3. 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.
4. 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.
5. Resuspend the beads in 1 ml of PBS-TBN by vortex and sonication for 20 sec.
6. Pellet the beads by microcentrifugation at 10,000 x g for 2 min.
7. Repeat steps 1.5 and 1.6.
   Note: This provides a total of two washes with PBS-TBN.
8. 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

1. Remove saliva from the -80 °C freezer and allow it to thaw at room temperature.
2. Resuspend antigen-coupled bead stocks by vortex and sonication for 20 sec.
3. 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.
4. Prepare a 1:4 dilution of saliva with PBS-1% BSA buffer in a 96-well, deep well plate.
5. Pre-wet the filter plate with 100 µl of wash buffer and remove the supernatant by vacuum.
6. 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).
7. 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.
8. Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
9. Dilute biotinylated goat anti-human IgG secondary detection antibody to 16 µg/ml in PBS-1% BSA.
10. Add 50 µl diluted secondary antibody to each well and mix contents with a multi-channel pipettor.
11. 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.
12. Resuspend beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
13. Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 µg/ml in PBS-1% BSA.
14. Add 50 µl reporter to each well and mix with a multi-channel pipettor.
15. 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.
16. 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

Materials

Name	Company	Catalog Number	Comments
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