Name: developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens
Uploaded: 2016-09-12T15:00:00
Description: Watch this Scientific Journal Video about Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens at JoVE.com

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.

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 Boquer&#243;n 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 ...

<ol>
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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...

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 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 children5-7. Serum and saliva samples have been studied extensively for antibodies against H. pylori2,3,8, Plasmodium falciparum9, Entamoeba histolytica10, Cryptosporidium parvum3,11, Streptococcus pneumonia12, hepatitis viruses A and C13-14, noroviruses2-4,15, T. gondii2-4, 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 IgG2-4 and IgA3,4 and plasma IgG2,3 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 models19-22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Electron Corporation</td>
			<td>75002446</td>
			<td>Used to centrifuge samples</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>G560</td>
			<td>Used to mix samples</td>
		</tr>
		<tr>
			<td>Sonicator (mini)</td>
			<td>Fisher Scientific</td>
			<td>15-337-22</td>
			<td>Used to separate beads</td>
		</tr>
		<tr>
			<td>Pipettors P10, P20, P100, P1000, 8 ch.</td>
			<td>Capp</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer (Bright Line)</td>
			<td>Housser Scientific&#160;</td>
			<td>3200</td>
			<td>Used to count coupled beads</td>
		</tr>
		<tr>
			<td>Multiscreen Vacuum Manifold</td>
			<td>Millipore</td>
			<td>MSVMHTS00</td>
			<td>Used in washing steps to remove supernatant</td>
		</tr>
		<tr>
			<td>MicroShaker</td>
			<td>VWR</td>
			<td>12620-926</td>
			<td>Used to agitate beads during incubations</td>
		</tr>
		<tr>
			<td>Tube rack (1.5mL and 0.5mL) (assorted)</td>
			<td>VWR</td>
			<td>30128-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Scale</td>
			<td>Mettler or other</td>
			<td></td>
			<td>Used to measure wash reagents for making buffers</td>
		</tr>
		<tr>
			<td>Dynabead Sample Mixer</td>
			<td>Invitrogen</td>
			<td>947-01</td>
			<td>Used during coupling incubation step</td>
		</tr>
		<tr>
			<td>MatLab (R2014b)</td>
			<td>The MathWorks, Inc.</td>
			<td></td>
			<td>Used to analyze antibody response data</td>
		</tr>
		<tr>
			<td>Microsoft Excel 2014</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td>Used to analyze antibody response data</td>
		</tr>
		<tr>
			<td>Luminex Analyzer with xPonent 3.1 software</td>
			<td>Luminex Corporation</td>
			<td>LX200-XPON3.1</td>
			<td>Instrument and software used to run assay</td>
		</tr>
		<tr>
			<td>Antigens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GI.1 Norwalk Virus : p-particle</td>
			<td>Xi Jiang (CCHMC)*</td>
			<td>NA</td>
			<td>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;g.</td>
		</tr>
		<tr>
			<td>GII.4 Norovirus VA387 : p-particle</td>
			<td>Xi Jiang (CCHMC)*</td>
			<td>NA</td>
			<td>&#160;*Cincinnati Childrens&#39; Hospital. Final conc. 5 &#181;g.</td>
		</tr>
		<tr>
			<td>Hepatitis A Virus : grade II concentrate from cell culture</td>
			<td>Meridian Life Sciences</td>
			<td>8505</td>
			<td>Antigen coupled at 100 &#181;g</td>
		</tr>
		<tr>
			<td>Helicobacter pylori : lysate</td>
			<td>Meridian Life Sciences</td>
			<td>R14101</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Toxoplasma gondii : recombinant p30 (SAG1)</td>
			<td>Meridian Life Sciences</td>
			<td>R18426</td>
			<td>Antigen coupled at 25 &#181;g</td>
		</tr>
		<tr>
			<td>Campylobacter jejuni : heat killed whole cells</td>
			<td>KPL</td>
			<td>50-92-93</td>
			<td>Antigen coupled at 50 &#181;g</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-Norovirus</td>
			<td>(CCHMC)*</td>
			<td>NA</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Mouse anti-Hepatitis A IgG</td>
			<td>Meridian Life Sciences</td>
			<td>C65885M</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Mouse anti-Hepatitis A IgG</td>
			<td>Meridian Life Sciences</td>
			<td>C65885M</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>BacTraceAffinity Purified Antibody to Helicobacter pylori</td>
			<td>KPL</td>
			<td>01-93-94</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Goat pAb to Toxoplasma gondii</td>
			<td>Abcam</td>
			<td>Ab23507</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>BacTrace Goat anti-Campylobacter species</td>
			<td>KPL</td>
			<td>01-92-93</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Secondary&#160; Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L)</td>
			<td>Jackson</td>
			<td>705-065-149</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Biotinylated Rabbit anti-Goat IgG (H+L)</td>
			<td>KPL</td>
			<td>16-13-06</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Biotinylated Goat anti-Mouse IgG (H+L)</td>
			<td>KPL</td>
			<td>16-18-06</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG(H+L)</td>
			<td>KPL</td>
			<td>176-1506</td>
			<td>Used for coupling confirmation</td>
		</tr>
		<tr>
			<td>Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(&#7518;)&#160;</td>
			<td>KPL</td>
			<td>16-10-02</td>
			<td>Used for Salivary Immunoassay</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL copolymer microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td>Used as low binding microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030050C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5030080C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L pipette tip refills</td>
			<td>BioVentures</td>
			<td>5130140C</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Various Vendors</td>
			<td></td>
			<td>Used keep beads in the dark during incubations</td>
		</tr>
		<tr>
			<td>Deep Well plates</td>
			<td>VWR</td>
			<td>40002-009</td>
			<td>Used for diluting saliva samples</td>
		</tr>
		<tr>
			<td>Multiscreen Filter Plates</td>
			<td>Millipore</td>
			<td>MABVN1250</td>
			<td>Used to run assays</td>
		</tr>
		<tr>
			<td>Oracol saliva collection system</td>
			<td>Malvern Medical Developments Limited</td>
			<td></td>
			<td>Used for saliva collection</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated microspheres (beads)</td>
			<td>Luminex Corporation</td>
			<td>Dependent on bead set</td>
			<td>Antigens are coupled to the microspheres</td>
		</tr>
		<tr>
			<td>EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride)</td>
			<td>Pierce</td>
			<td>77149 or 22980</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Pierce</td>
			<td>24510</td>
			<td>Used in bead activation</td>
		</tr>
		<tr>
			<td>Steptavidin-R-phycoerythrin (1mg/mL)</td>
			<td>Molecular Probes</td>
			<td>S-866</td>
			<td>Used as reporter</td>
		</tr>
		<tr>
			<td>MES (2-[N-Morpholino]ethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>M-2933</td>
			<td>Used for coupling</td>
		</tr>
		<tr>
			<td>Tween-20 (Polyoxyethylenesorbitan monolaurate)</td>
			<td>Sigma</td>
			<td>P-9416</td>
			<td>Used in wash buffer to remove non-specific binding</td>
		</tr>
		<tr>
			<td>Protein Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)**</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3813</td>
			<td>138 mM NaCl, 2.7 mM KCl</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-7888</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P-9416</td>
			<td>0.2% (v/v)</td>
		</tr>
		<tr>
			<td>Sodium Azide (0.05% azide)**</td>
			<td>Sigma</td>
			<td>S-8032</td>
			<td>**Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE&#39;s. Dispose of according to applicable laws.</td>
		</tr>
		<tr>
			<td>MES/ Coupling Buffer (0.05 M MES, pH 5.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MES (2-[N-Morpholino]ethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td></td>
		</tr>
		<tr>
			<td>5 N NaOH</td>
			<td>Fisher</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer (PBS, 1% BSA, pH 7.4)&#160;</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, 1% BSA, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3688</td>
			<td>138 mM NaCl, 2.7 mM KCl, 1% BSA</td>
		</tr>
		<tr>
			<td>Activation Buffer (0.1 M NaH2PO4, pH 6.2)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>NaH2PO4 (Sodium phosphate, monobasic anhydrous)</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td>0.1M NaH2PO4</td>
		</tr>
		<tr>
			<td>5 N NaOH</td>
			<td>Fisher</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer (PBS, 0.05% Tween-20, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>Filter Sterilize and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>PBS, 0.05% Tween-20, pH 7.4</td>
			<td>Sigma</td>
			<td>P-3563</td>
			<td>138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN</td>
		</tr>
	</tbody>
</table>

developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens

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.

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 &#62;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...

Watch this Scientific Journal Video about Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens at JoVE.com

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

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 ...

The overall goal of this salivary antibody bead-based multiplex immunoassay is to identify the etiological agents responsible for environmentally associated symptomatic and asymptomatic illnesses by measuring the IGG antibody responses to multiple pathogens simultaneously. This method can help answer key questions in the field of exposure science. For example, which pathogens are responsible for causing illnesses in particular populations.The main advantage of this technique is that it can be used to quickly and cost-effectively measure antibodies to multiple analyze at one time using very small simple volumes. This method can provide insight to etiological agents and their sources of exposure, including foodborne, waterborne and airborne pathways. Official demonstration of this procedure is critical because if you don't set it up properly, the results can be unreliable.Begin by vortexing and sonicating the stock mixtures of beads coupled to the antigens of interest for 20 seconds at a time to generate homogeneous bead suspensions. Then dilute the coupled bead stocks to a final concentration of 100 beads per microliter of each unique bead set in PBS, supplemented with 1%BSA. Next, set up at least seven two-fold serial dilutions of anti-species IGG primary antibody, in PBS plus BSA in a 96 well round bottom plate according to the manufacturers recommendations.Pre-wet a separate eight 12 column of a 96 well filter bottom plate for each antigen coupling confirmation test with 100 microliters of wash buffer and remove the supernatant by vacuum. Then, add 50 microliters of the antigen coupled beads to the pre-wet wells at 50 microliters of the serially diluted antibody to rows one through seven of the 96 well filter plate, and add 50 microliters of PBS plus BSA to row eight as the diluted antibody background control. Use a multi-channel pipette to mix the contents of each of the wells up and down five times.Then cover the plate, and incubate the antibodies with the antigen coupled beads in the dark at room temperature on a microplate shaker at 500 rpm. After one hour, aspirate the supernatant and wash the wells with two 100 microliter aliquots of wash buffer. After the second wash, replace the buffer with 50 microliters of PBS plus BSA.Now dilute the biotinylated anti-species specific IGG secondary detection antibody, to 16 micrograms per milliliter in PBS plus BSA and add 50 microliters of diluted secondary antibody to each row. Incubate the beads and antibodies at room temperature in the dark for 30 minutes on a plate shaker. At the end of the incubation, aspirate the supernatant, and wash the beads with two 100 microliter aliquots of wash buffer.After the second wash re-suspend the beads in 50 microliters in PBS plus BSA and dilute the streptavidin r-phycoerythrin reporter to 24 micrograms per milliliter in PBS plus BSA. Add 50 microliters of the reporter to each well and mix by pipetting. Incubate the plate in the dark for 30 minutes at room temperature on the shaker, followed by two 100 microliter rinses in wash buffer as demonstrated.Then re-suspend the beads in 100 microliters of PBS plus BSA and evaluate the antigen coupled beads on the analyzer. After re-suspending the antigen coupled bead stocks by vortexing and sonication for 20 seconds, dilute the coupled bead stocks to a final concentration of 100 beads per microliter of each unique bead set in PBS plus BSA. Then prepare a one to four dilution of room temperature saliva in PBS plus BSA in a 96 well deep well plate.Pre-wet a filter plate with 100 microliters of wash buffer and aspirate the supernatant. Next, add 50 microliters of antigen coupled beads and an equal volume of the diluted saliva to 95 wells of the 96 well filter plate for one to eight final dilution. At the same time, add 50 microliters of antigen coupled beads, plus 50 microliters of PBS plus BSA alone to the control well.Incubate the beads in the dark at room temperature for one hour on the microplate shaker at 500 rpm. Then aspirate the supernatant and wash the wells two times with 100 microliters of wash buffer. After the second wash, re-suspend the beads in 50 microliters of PBS plus BSA.Dilute the secondary antibody, and add 50 microliters of it to each well. After mixing the well contents with a multi-channel pipette incubate the plate on the shaker in the dark for 30 minutes at room temperature, then wash the wells two times with 100 microliters of wash buffer and re-suspend the beads in 50 microliters of fresh PBS plus BSA. Now dilute the reporter and add 50 microliters to each well.Mix thoroughly with a multi-channel pipette. After 30 minutes at room temperature on the shaker in the dark, wash the wells two times as demonstrated, then re-suspend the beads in 100 microliters of PBS plus BSA and evaluate the samples on the analyzer. To ensure that the antigen coupled beads are capable of approaching the dynamic range of the assay, proper coupling is defined as a mean fluorescence intensity, greater than or equal to 18, 000.As demonstrated for these representative coupling confirmations of the antigens in the assay for C.jejuni and T.gondii. Based on the established cut-off point, the immunoprevalence rates for the samples ranged from about 2%for T.gondii to nearly 50%for Norovirus geno group 1.1 indicating that the immunoprevalence rate was highest for the Noroviruses, followed by Hepatitis A and H.Pylori. While 32%of the samples were immuno-negative for all of the pathogens in the assay, 68%were immuno-positive to one or more pathogen.Once mastered, this procedure can be completed in about four hours if performed properly. While attempting this procedure it's important to keep the beads in the dark to avoid photo-bleaching. Following this procedure, other methods like ELISAs and Western Blotting can be used to answer additional questions about sensitivity and specificity.As well as cross-reactivity of the antibodies. After its development, this technique paved the way for researchers in the fields of epidemiology, exposure science, and clinical microbiology, to explore human risks from environmental exposures. After watching this video you should have a very good understanding of how to develop, analyze, and interpret the results of a multiplex immunoassay, measuring antibodies to multiple pathogens simultaneously.Don't forget that working with bodily fluids and sodium azide can be extremely hazardous, and that precautions such as wearing appropriate personal protective equipment and proper reagent disposal should always be taken when performing this procedure.

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