Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

Summary

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

Abstract

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

Introduction

Eighty-eight percent of diarrhea-related illness worldwide is associated with human exposure to contaminated water, unsafe food, and poor sanitation/hygiene, causing approximately 1.5 million deaths, the majority of whom are children¹. This is a major cause of concern for public health officials and policy makers. In an effort to investigate exposures and illnesses associated with waterborne and other environmental pathogens, we developed a multiplex immunoassay to measure antibodies in human samples^2-4. This method can be applied to epidemiological studies to determine human exposure to these pathogens and to better define immunoprevalence and incident infections.

Saliva holds considerable promise as an alternative to serum for human biomarker research. Among the advantages of using saliva are the non-invasiveness and ease of sample collection, low cost, and samples can easily be collected from children^5-7. Serum and saliva samples have been studied extensively for antibodies against H. pylori^2,3,8, Plasmodium falciparum⁹, Entamoeba histolytica¹⁰, Cryptosporidium parvum^3,11, Streptococcus pneumonia¹², hepatitis viruses A and C^13-14, noroviruses^2-4,15, T. gondii^2-4, dengue virus¹⁶, human immunodeficiency virus (HIV)¹⁷, and Escherichia coli O157:H7¹⁸.

A multiplex immunoassay allows for the analysis of multiple analytes simultaneously within a single sample volume and within a single cycle or run. Multiplexed antigens from C. jejuni, T. gondii, H. pylori, hepatitis A virus, and two noroviruses were used to measure human salivary IgG^2-4 and IgA^3,4 and plasma IgG^2,3 antibody responses to these pathogens using a bead-based multiplexing immunoassay. When used in conjunction with epidemiological studies of exposure to microbes in water, soil and food, the type of assay described in this study may provide valuable information to enhance the understanding of infections caused by environmental pathogens. Moreover, salivary antibody data obtained from such studies can be used to improve risk assessment models^19-22.

Protocol

Approval was obtained from the Institutional Review Board (IRB # 08-1844, University of North Carolina, Chapel Hill, NC, USA) for the collection of stimulated crevicular saliva samples from beachgoers at Boquerón Beach, Puerto Rico, as part of the United States Environmental Protection Agency (USEPA) National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study²³ to assess swimming associated exposures and illnesses. Study subjects provided informed consent and were instructed on the use of the saliva collection device by trained USEPA contractors. The saliva samples were shipped on ice and, upon receipt, they were centrifuged and stored at -80 °C as described⁴.

1. Bead Activation

1. Resuspend bead set stocks by vortexing and sonicating for 20 sec and transfer approximately 5.0 x 10⁶ of the stock beads (400 µl) to microcentrifuge tubes.
   Note: The beads are supplied at a concentration of 12.5 x 10⁶ beads/ml.
2. Pellet the stock beads by centrifuging at 10,000 x g for 2 min.
3. Remove the supernatant and resuspend the pelleted beads in 100 µl distilled water (dH₂O) by vortex and sonication for 20 sec. Repeat step 1.2.
4. Remove the supernatant and resuspend the washed beads in 80 µl of 100 mM sodium phosphate monobasic, pH 6.2 by vortex and sonication for 20 sec.
5. Immediately before use, make a 50 mg/ml N-hydroxysulfosuccinimide (Sulfo-NHS) solution by adding 200 µl dH₂O to the 10 mg Sulfo-NHS aliquot. Mix by vortex.
6. Add 10 µl of the 50 mg/ml Sulfo-NHS to the beads. Mix by vortex.
7. Immediately before use, make a 50 mg/ml 1-ethyl-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) solution by adding 200 µl distilled water (dH₂O) to the 10 mg EDC aliquot. Mix by vortex.
8. Add 10 µl of 50 mg/ml EDC solution to the beads. Mix by vortex. Incubate the beads for 20 min at room temperature, in the dark, with mixing by vortex at 10 min intervals. Pellet the activated beads by microcentrifugation at 10,000 x g for 2 min.
9. Remove the supernatant and resuspend the beads in 250 µl of 50 mM 2-[N-Morpholino]ethanesulfonic acid (MES), pH 5.0 by vortex and sonication for 20 sec.
10. Pellet the activated beads by microcentrifugation at 10,000 x g for 2 min.
11. Repeat steps 1.9 and 1.10.
    Note: This provides a total of two washes with 50 mM MES, pH 5.0.
12. Resuspend the beads in 100 µl of 50 mM MES, pH 5.0 by vortex and sonication for 20 sec.

2. 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)-polyoxyethylenesorbitan 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 skin. Do not breathe dust/fume/gas/mist/vapors or spray. Wear appropriate personal protective equipment (PPE's) when handling and dispose of 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 2.5 and 2.6.
   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.

3. Bead Count

1. Prepare a 1:10 dilution of the coupled beads in water or PBS buffer.
2. Load 10 µl of the bead dilution onto a hemocytometer at the sample introduction point.
3. Count the beads seen in one of the 4 x 4 corner grids. Calculate the total number of coupled beads using the following formula: Count (1 corner of 4 x 4 grid) x (1 x 10⁴) x (dilution factor) x resuspension volume in ml.

4. Confirmation of Antigen Coupling

1. Resuspend the stock mixture of beads coupled to antigens of interest by vortex and sonication for 20 sec.
2. 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 (PBS-1% BSA, pH 7.4).
3. Prepare at least 7 two-fold serial dilutions of anti-species IgG primary antibody according to manufacturer's recommendations in 96-well round bottom plate with PBS-1% BSA buffer.
4. Pre-wet a separate 8-well column (8 rows) of a 96-well filter bottom plate for each antigen coupling confirmation test with 100 µl of wash buffer and remove supernatant by vacuum. Add 50 µl of working bead mixture (antigen-coupled beads) to the pre-wet wells.
5. Add 50 µl of antibody dilutions to rows 1-7 of each column of the 96-well filter plate and 50 µl of PBS-1% BSA buffer to row 8 in lieu of diluted antibody to serve as background wells. Mix with a multi-channel pipettor by pipetting up and down 5 times. Perform the same procedure for every antigen-coupled bead set to be confirmed.
6. 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.
7. Wash wells with 100 µl of wash buffer and remove supernatant by vacuum. Repeat 1x. Resuspend the beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
8. Dilute biotinylated anti-species specific IgG secondary detection antibody to 16 µg/ml in PBS-1% BSA.
9. Add 50 µl of diluted secondary antibody to each well by pipetting up and down 5 times.
10. Cover filter plate and allow 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.
11. Resuspend the beads in 50 µl of PBS-1% BSA with a multi-channel pipettor.
12. Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 µg/ml in PBS-1% BSA.
13. Add 50 µl of reporter to each well and mix by pipetting up and down 5 times.
14. Cover filter plate and allow 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.
15. Resuspend beads in 100 µl of PBS-1% BSA and analyze 50 µl using the analyzer²⁹.
    Note: Results of the bead-based multiplex immunoassay are measured in Median Fluorescence Intensity (MFI). Always refer to the latest version of the software manual, if available to avoid errors.

5. Salivary Multiplex Immunoassay

1. Remove saliva from the -80 °C freezer and allow 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 filter plate with 100 µl of wash buffer and remove 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 filter plate and allow 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 filter plate and allow 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 analyzer²⁹.
    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.

Results

One unique bead set was used as a control to measure non-specific binding and sample to sample variability. These beads were treated identically to the antigen coupled beads with the exception that they were not incubated with any antigen in the coupling step. MFI values >500 obtained from the control beads incubated with all saliva samples were removed from further analyses due to suspected contamination from serum and the remaining responses were log distributed. The saliva can be c...

Discussion

These results indicate that the multiplex immunoassay method is useful for discriminating between saliva samples that are immunopositive or immunonegative. To determine immunopositivity, a single cut-off point was developed by calculating the mean plus three standard deviations of the log transformed MFI responses of the control uncoupled beads tested with all of the saliva samples. The cut-off point afforded the ability to assess exposure and immunoprevalence to either a single or multiple pathogens. This discriminative...

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
Hemacytometer (Bright Line)	Housser Scientific	3200	Used to count coupled beads
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