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Immunology and Infection

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

Published: September 12th, 2016



1National Exposure Research Laboratory, U.S. Environmental Protection Agency, 2National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education, 4Department of Biological Sciences, McMicken College of Arts and Sciences, University of Cincinnati

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.

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.

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 samples2-4. This method can be applied to epidemiological studies to determine human exposure to these pathogens and to better define immunoprevalence and i....

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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 .......

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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.......

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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.......

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Clarissa Curioso was supported through an appointment to the Research Participation Program at the U.S. Environmental Protection Agency administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA.


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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
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
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
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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