Name: developing a salivary antibody multiplex immunoassay to measure human exposure to environmental pathogens
Uploaded: 2016-09-12T15:00:00.000+00:00
Duration: 9 min 8 s
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 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 &#176;C as described4.
1. Bead Activation
<ol>
	<li>Resuspend bead set stocks by vortexing and sonicating for 20 sec and transfer approximately 5.0 x 106 of the stock beads (400 &#181;l) to microcentrifuge tubes. 
	Note: The beads are supplied at a concentration of 12.5 x 106 beads/ml.</li>
	<li>Pellet the stock beads by centrifuging at 10,000 x g for 2 min.</li>
	<li>Remove the supernatant and resuspend the pelleted beads in 100 &#181;l distilled water (dH2O) by vortex and sonication for 20 sec. Repeat step 1.2.</li>
	<li>Remove the supernatant and resuspend the washed beads in 80 &#181;l of 100 mM sodium phosphate monobasic, pH 6.2 by vortex and sonication for 20 sec.</li>
	<li>Immediately before use, make a 50 mg/ml N-hydroxysulfosuccinimide (Sulfo-NHS) solution by adding 200 &#181;l dH2O to the 10 mg Sulfo-NHS aliquot. Mix by vortex.</li>
	<li>Add 10 &#181;l of the 50 mg/ml Sulfo-NHS to the beads. Mix by vortex.</li>
	<li>Immediately before use, make a 50 mg/ml 1-ethyl-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) solution by adding 200 &#181;l distilled water (dH2O) to the 10 mg EDC aliquot. Mix by vortex.</li>
	<li>Add 10 &#181;l 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.</li>
	<li>Remove the supernatant and resuspend the beads in 250 &#181;l of 50 mM 2-[N-Morpholino]ethanesulfonic acid (MES), pH 5.0 by vortex and sonication for 20 sec.</li>
	<li>Pellet the activated beads by microcentrifugation at 10,000 x g for 2 min.</li>
	<li>Repeat steps 1.9 and 1.10. 
	Note: This provides a total of two washes with 50 mM MES, pH 5.0.</li>
	<li>Resuspend the beads in 100 &#181;l of 50 mM MES, pH 5.0 by vortex and sonication for 20 sec.</li>
</ol>
2. Bead Coupling
<ol>
	<li>Couple the antigens to the bead sets using the concentrations shown in Table 1.</li>
	<li>Add each antigen to the activated beads and bring the total volume to 500 &#181;l in 50 mM MES, pH 5.0. Mix the antigens and beads by vortex.</li>
	<li>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.</li>
	<li>Remove the supernatant and resuspend the beads in 500 &#181;l 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&#39;s) when handling and dispose of in accordance with appropriate laws.</li>
	<li>Resuspend the beads in 1 ml of PBS-TBN by vortex and sonication for 20 sec.</li>
	<li>Pellet the beads by microcentrifugation at 10,000 x g for 2 min.</li>
	<li>Repeat steps 2.5 and 2.6. 
	Note: This provides a total of two washes with PBS-TBN.</li>
	<li>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 &#176;C refrigerator in the dark.</li>
</ol>
3. Bead Count
<ol>
	<li>Prepare a 1:10 dilution of the coupled beads in water or PBS buffer.</li>
	<li>Load 10 &#181;l of the bead dilution onto a hemocytometer at the sample introduction point.</li>
	<li>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.</li>
</ol>
4. Confirmation of Antigen Coupling
<ol>
	<li>Resuspend the stock mixture of beads coupled to antigens of interest by vortex and sonication for 20 sec.</li>
	<li>Prepare a working bead mixture by diluting the coupled bead stocks to a final concentration of 100 beads /&#181;l of each unique bead set in PBS-1% BSA buffer (PBS-1% BSA, pH 7.4).</li>
	<li>Prepare at least 7 two-fold serial dilutions of anti-species IgG primary antibody according to manufacturer&#39;s recommendations in 96-well round bottom plate with PBS-1% BSA buffer.</li>
	<li>Pre-wet a separate 8-well column (8 rows) of a 96-well filter bottom plate for each antigen coupling confirmation test with 100 &#181;l of wash buffer and remove supernatant by vacuum. Add 50 &#181;l of working bead mixture (antigen-coupled beads) to the pre-wet wells.</li>
	<li>Add 50 &#181;l of antibody dilutions to rows 1-7 of each column of the 96-well filter plate and 50 &#181;l 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.</li>
	<li>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.</li>
	<li>Wash wells with 100 &#181;l of wash buffer and remove supernatant by vacuum. Repeat 1x. Resuspend the beads in 50 &#181;l of PBS-1% BSA with a multi-channel pipettor.</li>
	<li>Dilute biotinylated anti-species specific IgG secondary detection antibody to 16 &#181;g/ml in PBS-1% BSA.</li>
	<li>Add 50 &#181;l of diluted secondary antibody to each well by pipetting up and down 5 times.</li>
	<li>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 &#181;l of wash buffer and remove supernatant by vacuum. Repeat wash 1x.</li>
	<li>Resuspend the beads in 50 &#181;l of PBS-1% BSA with a multi-channel pipettor.</li>
	<li>Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 &#181;g/ml in PBS-1% BSA.</li>
	<li>Add 50 &#181;l of reporter to each well and mix by pipetting up and down 5 times.</li>
	<li>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 &#181;l of wash buffer and remove supernatant by vacuum. Repeat wash 1x.</li>
	<li>Resuspend beads in 100 &#181;l of PBS-1% BSA and analyze 50 &#181;l 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.</li>
</ol>
5. Salivary Multiplex Immunoassay
<ol>
	<li>Remove saliva from the -80 &#176;C freezer and allow to thaw at room temperature.</li>
	<li>Resuspend antigen coupled bead stocks by vortex and sonication for 20 sec.</li>
	<li>Prepare a working bead mixture by diluting the coupled bead stocks to a final concentration of 100 beads/&#181;l of each unique bead set in PBS-1% BSA buffer.</li>
	<li>Prepare a 1:4 dilution of saliva with PBS-1% BSA buffer in a 96 well, deep well plate.</li>
	<li>Pre-wet filter plate with 100 &#181;l of wash buffer and remove supernatant by vacuum.</li>
	<li>Add 50 &#181;l 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 &#181;l antigen-coupled beads plus 50 &#181;l of PBS-1% BSA buffer (as a replacement for saliva).</li>
	<li>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 &#181;l of wash buffer and remove supernatant by vacuum. Repeat wash 1x.</li>
	<li>Resuspend beads in 50 &#181;l of PBS-1% BSA with a multi-channel pipettor.</li>
	<li>Dilute biotinylated goat anti-human IgG secondary detection antibody to 16 &#181;g/ml in PBS-1% BSA.</li>
	<li>Add 50 &#181;l diluted secondary antibody to each well and mix contents with a multi-channel pipettor.</li>
	<li>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 &#181;l of wash buffer and remove supernatant by vacuum. Repeat wash 1x.</li>
	<li>Resuspend beads in 50 &#181;l of PBS-1% BSA with a multi-channel pipettor.</li>
	<li>Dilute streptavidin-R-phycoerythrin reporter (SAPE) to 24 &#181;g/ml in PBS-1% BSA.</li>
	<li>Add 50 &#181;l reporter to each well and mix with a multi-channel pipettor.</li>
	<li>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 &#181;l of wash buffer and remove supernatant by vacuum. Repeat wash 1x.</li>
	<li>Resuspend beads in 100 &#181;l of PBS-1% BSA and analyze 50 &#181;l 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.</li>
</ol>

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The United States Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to Agency&#39;s administrative review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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 power may be useful in population studies to investigate health risks associated with exposure to environmental and other pathogens.
There are several other methods that can be used to determine a cut-off point depending on the distribution of the MFIs, uncoupled controls and what question the study is designed to answer. Some examples for determining cut-offs include finite mixed modeling24-26, mean plus 3 standard deviations of responses to controls, mean plus 2 standard deviations of responses to controls, and three times the mean of responses to controls27,28. For simple screenings, less stringent criteria may be used but for epidemiological studies, it may be more appropriate to employ a more stringent definition to reduce the probability of reporting false positives. Our initial application is to look at immunoconversion and immunoprevalence between swimmers and non-swimmers. In this study, the uncoupled control MFIs were not normally distributed and the results were log transformed.
Advantages to performing a bead-based multiplex immunoassay include the use of very low sample volumes, reduction in time and labor and the ability to measure responses for up to 500 analytes simultaneously while requiring a minimal amount of reagents. By contrast, traditional methods of assessing antibody levels indicative of exposure and/or infection (e.g., ELISA testing) can take several hours to complete, are labor intensive, and can only be used to measure one analyte at a time. These traditional methods also require larger volumes of patient/participant samples. For example, most ELISA&#39;s require at least 100 &#181;l of sample while a multiplex immunoassay may require 50 &#181;l or less.
Limitations of a multiplex immunoassay include the need for rigorous optimization to determine the optimal concentrations of primary capture and secondary detection antibodies, antigens and reporter. Cross-reactivity is also a major concern in immunoassays as antibodies against a particular antigen may cross-react with antigens from other organisms and thereby lead to false positive readings. Optimization, cross-reactivity and non-specific binding were addressed previously2-4. The success of any such assay is largely dependent on the ability to obtain highly immunogenic antigens as well as specific antibodies to these antigens for use in the bead coupling and coupling confirmation steps. Another limitation is the limited availability of characterized (diagnostically positive and negative) samples to validate the assays.
There are a number of critical steps within the protocol. These include: ensuring the antigens that will be coupled to the beads do not contain foreign proteins, azide, glycine, Tris or any primary amines. If any of these agents exist in the antigen preparation, they should be removed by dialysis or chromatography. It is recommended that one should start with 5 &#181;g of antigen per 5 million beads and titrate to find the optimal concentration. Choose the optimal detection antibody concentration that gives the best sensitivity and dynamic range. Bear in mind that signals tend to decrease as antigen and detection antibody concentrations increase (hook effect). For example, if the signal decreases as antigen concentration increases then detection antibody may be limiting. Conversely, if signals decrease as detection antibody increases then reporter (SAPE) may be limiting. Check the multiplex for cross- reactivity by combining the optimal coupling for each protein (5,000 of each bead set per well). Test the multiplexed bead sets by combining with the optimal detection antibody amount. Optimize the reporter and make a standard curve of the antigen by testing each antigen individually.
Troubleshooting a multiplex immunoassay to improve sensitivity and to increase median fluorescent signal are critically important. To improve sensitivity, decrease either the amount of antigen coupled to the beads or the detection antibody concentration. The use of a higher affinity antibody for capture and/or detection may also improve sensitivity. Increasing the amount of antigen coupled to the beads may increase the median fluorescent signal. One study demonstrated that pre-incubating serum samples with polyvinylalcohol plus polyvinylpyrrolidone (PVX) buffer is able to suppress non-specific binding in serological assays using the bead-based multiplex assay30 such as the one that we are describing here. However, another study by our collaborators found no significant effect between PBS-BSA and PVX buffers. They concluded further that because of the higher viscosity of the PVX buffer, it created problems with bead acquisition in the analyzer and formed foam in the microplate wells3.
In conclusion, we have presented a method that is capable of measuring the presence of human salivary IgG antibodies to multiple pathogens simultaneously in a very small sample volume. In the future, this assay will be used to detect the presence of salivary antibodies associated with swimming related exposures or infections and to help inform risk assessment models. Additionally, the assay can be used to measure antibodies associated with airborne and foodborne exposures, determine incident infections or immunoconversions and provide critical immunological information to epidemiologists conducting questionnaire-type population studies.

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><tbody><tr><td>&lt;strong&gt;Equipment and Software&lt;/strong&gt;</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&amp;nbsp;</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 (1.5 ml and 0.5 ml) (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>&lt;strong&gt;Antigens&lt;/strong&gt;</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>*Cincinnati Childrens&#039; Hospital. Final conc. 5 &amp;micro;g.</td></tr><tr><td>GII.4 Norovirus VA387: p-particle</td><td>Xi Jiang (CCHMC)*</td><td>NA</td><td>*Cincinnati Childrens&#039; Hospital. Final conc. 5 &amp;micro;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 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Helicobacter pylori&lt;/em&gt;: lysate</td><td>Meridian Life Sciences</td><td>R14101</td><td>Antigen coupled at 25 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Toxoplasma gondii&lt;/em&gt;: recombinant p30 (SAG1)</td><td>Meridian Life Sciences</td><td>R18426</td><td>Antigen coupled at 25 &amp;micro;g</td></tr><tr><td>&lt;em&gt;Campylobacter jejuni&lt;/em&gt;: heat killed whole cells</td><td>KPL</td><td>50-92-93</td><td>Antigen coupled at 50 &amp;micro;g</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</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 &lt;em&gt;Helicobacter pylori&lt;/em&gt;</td><td>KPL</td><td>01-93-94</td><td>Used for coupling confirmation</td></tr><tr><td>Goat pAb to&lt;em&gt; Toxoplasma gondii&lt;/em&gt;</td><td>Abcam</td><td>Ab23507</td><td>Used for coupling confirmation</td></tr><tr><td>BacTrace Goat anti-&lt;em&gt;Campylobacter&lt;/em&gt; species</td><td>KPL</td><td>01-92-93</td><td>Used for coupling confirmation</td></tr><tr><td>&lt;strong&gt;Secondary&amp;nbsp; Antibodies&lt;/strong&gt;</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>&lt;strong&gt;Consumables&lt;/strong&gt;</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 &amp;micro;l pipette tip refills</td><td>BioVentures</td><td>5030050C</td><td></td></tr><tr><td>200 &amp;micro;l pipette tip refills</td><td>BioVentures</td><td>5030080C</td><td></td></tr><tr><td>1,000 &amp;micro;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>&lt;strong&gt;Reagents&lt;/strong&gt;</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-[3-dimethylaminopropyl] carbodiimide hydrochloride)</td><td>Pierce</td><td>77149 or 22980</td><td>Used in bead activation</td></tr><tr><td>Sulfo-NHS (&lt;em&gt;N&lt;/em&gt;-hydroxysulfosuccinimide)</td><td>Pierce</td><td>24510</td><td>Used in bead activation</td></tr><tr><td>Steptavidin-R-phycoerythrin (1 mg/ml)</td><td>Molecular Probes</td><td>S-866</td><td>Used as reporter</td></tr><tr><td>MES (2-[&lt;em&gt;N&lt;/em&gt;-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>&lt;strong&gt;Protein Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)&lt;/strong&gt;**</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;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>**&lt;strong&gt;Caution&lt;/strong&gt;: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE&#039;s. Dispose of according to applicable laws.</td></tr><tr><td>&lt;strong&gt;MES/Coupling Buffer (0.05 M MES, pH 5.0)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MES (2-[&lt;em&gt;N&lt;/em&gt;-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>&lt;strong&gt;Assay Buffer (PBS, 1% BSA, pH 7.4)&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;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>&lt;strong&gt;Activation Buffer (0.1 M NaH&lt;/strong&gt;&lt;sub&gt;&lt;strong&gt;2&lt;/strong&gt;&lt;/sub&gt;&lt;strong&gt;PO&lt;/strong&gt;&lt;sub&gt;&lt;strong&gt;4&lt;/strong&gt;&lt;/sub&gt;&lt;strong&gt;, pH 6.2)&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;C</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Sodium phosphate, monobasic anhydrous)</td><td>Sigma</td><td>S-3139</td><td>0.1 M NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>5 N NaOH</td><td>Fisher</td><td>SS256-500</td><td></td></tr><tr><td>&lt;strong&gt;Wash Buffer (PBS, 0.05% Tween-20, pH 7.4)&lt;/strong&gt;</td><td></td><td></td><td>Filter Sterilize and store at 4 &amp;deg;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 contaminated with serum if the gums are rubbed too vigorously with the sponge attached to the collection device or if the study participant has periodontal disease. The log transformed MFI data were used to calculate an immunopositive cut-off point of 505 MFI based on the mean plus 3 standard deviations of the log MFI values. Additionally, antigen-coupled beads were added to specific wells that were treated as test wells in every manner except diluted saliva was replaced with PBS-1% BSA to evaluate background fluorescence and cross-reactivity. The MFI values obtained in these background wells were subtracted from the MFI values from each antigen-coupled bead set for each saliva sample.
Bead coupling was confirmed using animal derived species-specific primary detection antibodies specific for each antigen. To ensure that the antigen coupled beads were capable of approaching the dynamic range of the assay, we defined proper coupling as an MFI &#8805;18,000, observation of dose response and cross-reactivity between primary antibodies and non-targets of &#60;10% as described2. Representative coupling confirmations for C. jejuni and T. gondii of the antigens in the assay are shown in Figure 1.
Based on the cut-off point established (505 MFI), the immunoprevalence rates for the samples (n = 2,078) ranged from about 2% (n = 41) for T. gondii to nearly 50% (n = 1,009) for norovirus GI.1 (Figure 2). The data indicate that the immunoprevalence rate was highest for the noroviruses followed by hepatitis A virus and H. pylori.
While 32% (n = 672) of the samples were immunonegative for all of the pathogens in the assay, 68% (n = 1,406) were immunopositive to one or more pathogens. Figure 3 shows the breakdown of immunopositivity to one or more of the pathogens.
<img alt="Figure 1" src="/files/ftp_upload/54415/54415fig1.jpg" /> 
Figure 1: Coupling confirmation analyses for two of the organisms in the multiplex immunoassay. Coupling confirmation was performed for all of the antigens and the graphs for C. jejuni and T. gondii in this figure are representative of the coupling confirmations for the antigens in the multiplex immunoassay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54415/54415fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54415/54415fig2.jpg" /> 
Figure 2: Immunoprevalence to specific pathogens. Immunoprevalence to specific pathogens in the multiplex immunoassay ranged from about 2% for T. gondii to nearly 50% for norovirus GI.1. Antibodies against the norovirus antigens were more commonly observed followed by HAV (hepatitis A virus) and H. pylori. T. gondii and C. jejuni antibodies appeared less frequently in the saliva samples analyzed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54415/54415fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54415/54415fig3.jpg" /> 
Figure 3: Breakdown of immunopositivity to multiple pathogens simultaneously. The multiplex immunoassay allows for the analysis of immunopositivity to multiple pathogens simultaneously in a 50 &#181;l sample volume. Almost a third of the saliva samples assayed was immunonegative for antibodies against all of the antigens in the multiplex. Approximately 31% of the samples was immunopositive for one antigen while a quarter was positive to two antigens. 9.4% was immunopositive to three antigens. 3% was positive to four or more antigens simultaneously. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54415/54415fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Bead Set</td>
			<td>Coupling buffer</td>
			<td>Ag Concentration (&#181;g)</td>
			<td># of beads in 1 corner</td>
			<td># of beads/&#181;l</td>
			<td>Vol. for 100 B/uL for 4 ml (&#181;l)</td>
		</tr>
		<tr>
			<td>C. jejuni</td>
			<td>8</td>
			<td>MES 5.0</td>
			<td>50</td>
			<td>64</td>
			<td>6,400</td>
			<td>94</td>
		</tr>
		<tr>
			<td>H. pylori</td>
			<td>33</td>
			<td>MES 5.0</td>
			<td>25</td>
			<td>71</td>
			<td>7,100</td>
			<td>85</td>
		</tr>
		<tr>
			<td>Hepatitis A</td>
			<td>42</td>
			<td>MES 5.0</td>
			<td>100</td>
			<td>91</td>
			<td>9,100</td>
			<td>66</td>
		</tr>
		<tr>
			<td>T. gondii</td>
			<td>30</td>
			<td>MES 5.0</td>
			<td>25</td>
			<td>85</td>
			<td>8,500</td>
			<td>71</td>
		</tr>
		<tr>
			<td>Norovirus GII.4</td>
			<td>55</td>
			<td>MES 5.0</td>
			<td>5</td>
			<td>72</td>
			<td>7,200</td>
			<td>83</td>
		</tr>
		<tr>
			<td>Norovirus GI.1</td>
			<td>67</td>
			<td>MES 5.0</td>
			<td>5</td>
			<td>63</td>
			<td>6,300</td>
			<td>95</td>
		</tr>
		<tr>
			<td>Uncoupled</td>
			<td>80</td>
			<td>MES 5.0</td>
			<td></td>
			<td>60</td>
			<td>6,000</td>
			<td>100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="4">Total volume of beads (&#181;l)</td>
			<td>594</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="4">Total volume of buffer needed (&#181;l)</td>
			<td>3,406</td>
		</tr>
	</tbody>
</table>
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.

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 ...

developing-salivary-antibody-multiplex-immunoassay-to-measure-human

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  U.S. Environmental Protection Agency. 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.

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

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Chemistry

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengineering

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Biology

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Multiplex Immunoassay for Borrelia-Specific IgG and IgM in Serum

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

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

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