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>
	<li>Pr&#252;ss-&#220;st&#252;n, A., Gore, F., Bartram, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safer water, better health: costs, benefits and sustainability of interventions to protect and promote health. , (2008).</li><li>Augustine, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+approaches+to+developing+a+multiplex+immunoassay+for+determining+human+exposure+to+environmental+pathogens.">Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.</a> J Immunol Methods. 425, 1-9 (2015).</li><li>Griffin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+salivary+antibody+immunoassays+for+the+detection+of+incident+infections+with+Norwalk+virus+in+a+group+of+volunteers.">Application of salivary antibody immunoassays for the detection of incident infections with Norwalk virus in a group of volunteers.</a> J Immunol Methods. 424, 53-63 (2015).</li><li>Griffin, S., Chen, I., Fout, G., Wade, T., Egorov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+multiplex+microsphere+immunoassay+for+the+quantitation+of+salivary+antibody+responses+to+selected+waterborne+pathogens.">Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens.</a> J Immunol Methods. 364 (1-2), 83-93 (2011).</li><li>Ferguson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+diagnostic+uses+of+saliva.">Current diagnostic uses of saliva.</a> J Dent Res. 66, 420-424 (1987).</li><li>Mandel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+uses+of+saliva.">The diagnostic uses of saliva.</a> J Oral Pathol Med. 19, 119-125 (1990).</li><li>Malamud, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva+as+a+Diagnostic+Fluid.">Saliva as a Diagnostic Fluid.</a> Brit Med J. 305, 207-208 (1992).</li><li>Ballam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+is+useful+in+the+salivary+diagnosis+of+Helicobacter+pylori+infection.">Western blotting is useful in the salivary diagnosis of Helicobacter pylori infection.</a> J Clin Pathol. 53, 314-317 (2000).</li><li>Estevez, P., Satoguina, J., Nwakanma, D., West, S., Conway, D., Drakeley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva+as+a+source+of+anti-malarial+antibodies+to+examine+population+exposure+to+Plasmodium+falciparum.">Human saliva as a source of anti-malarial antibodies to examine population exposure to Plasmodium falciparum.</a> Malar J. 10, 104 (2011).</li><li>Abd-Alla, M., Jackson, T., Reddy, S., Ravdin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+invasive+amebiasis+by+enzyme-linked+immunosorbent+assay+of+saliva+to+detect+amebic+lectin+antigen+and+anti-lectin+immunoglobulin+G+antibodies.">Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies.</a> J Clin Microbiol. 38 (6), 2344-2347 (2000).</li><li>Toyoguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reactivity+to+Cryptosporidium+parvum+in+saliva+of+calves+after+experimental+infection.">Antibody reactivity to Cryptosporidium parvum in saliva of calves after experimental infection.</a> J Vet Med Sci. 62 (11), 1231-1234 (2000).</li><li>Choo, S., Zhang, Q., Seymour, L., Akhtar, S., Finn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+and+booster+salivary+antibody+responses+to+a+7-valent+pneumococcal+conjugate+vaccine+in+infants.">Primary and booster salivary antibody responses to a 7-valent pneumococcal conjugate vaccine in infants.</a> J Infect Dis. 182 (4), 1260-1263 (2000).</li><li>Tourinho, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+cutoff+ratio+for+detecting+antibodies+against+hepatitis+A+virus+in+oral+fluids+by+enzyme+immunoassay.">Importance of the cutoff ratio for detecting antibodies against hepatitis A virus in oral fluids by enzyme immunoassay.</a> J Virol Methods. 173 (2), 169-174 (2011).</li><li>Moorthy, M., Daniel, H., Kurian, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+saliva+as+an+alternative+to+plasma+for+the+detection+of+hepatitis+C+virus+antibodies.">An evaluation of saliva as an alternative to plasma for the detection of hepatitis C virus antibodies.</a> Indian J Med Microbiol. 26 (4), 327-332 (2008).</li><li>Moe, C., Sair, A., Lindesmith, L., Estes, M., Jaykus, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+norwalk+virus+infection+by+indirect+enzyme+immunoassay+detection+of+salivary+antibodies+to+recombinant+norwalk+virus+antigen.">Diagnosis of norwalk virus infection by indirect enzyme immunoassay detection of salivary antibodies to recombinant norwalk virus antigen.</a> Clin Diagn Lab Immunol. 11 (6), 1028-1034 (2004).</li><li>Yap, G., Sil, B., Ng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+saliva+for+early+dengue+diagnosis.">Use of saliva for early dengue diagnosis.</a> PLoS Negl Trop Dis. 5 (5), e1046 (2011).</li><li>Schramm, W., Angulo, G., Torres, P., Burgess-Cassler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+saliva-based+test+for+detecting+antibodies+to+human+immunodeficiency+virus.">A simple saliva-based test for detecting antibodies to human immunodeficiency virus.</a> Clin Diagn Lab Immunol. 6 (4), 577-580 (1999).</li><li>Chart, H., Perry, N., Willshaw, G., Cheasty, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+saliva+for+antibodies+to+the+LPS+of+Escherichia+coli+O157+in+patients+with+serum+antibodies+to+E.+coli+O157+LPS.">Analysis of saliva for antibodies to the LPS of Escherichia coli O157 in patients with serum antibodies to E. coli O157 LPS.</a> J Med Microbiol. 52 (Pt 7), 569-572 (2003).</li><li>Ashbolt, N., Bruno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+and+refinement+of+the+WHO+risk+framework+for+recreational+waters+in+Sydney,+Australia.">Application and refinement of the WHO risk framework for recreational waters in Sydney, Australia.</a> J Water Health. 1 (3), 125-131 (2003).</li><li>Westrell, T., Schonning, C., Stenstrom, T., Ashbolt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QMRA+(quantitative+microbial+risk+assessment)+and+HACCP+(hazard+analysis+and+critical+control+points)+for+management+of+pathogens+in+wastewater+and+sewage+sludge+treatment+and+reuse.">QMRA (quantitative microbial risk assessment) and HACCP (hazard analysis and critical control points) for management of pathogens in wastewater and sewage sludge treatment and reuse.</a> Water Sci Technol. 50 (2), 23-30 (2004).</li><li>Ashbolt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+contamination+of+drinking+water+and+disease+outcomes+in+developing+regions.">Microbial contamination of drinking water and disease outcomes in developing regions.</a> Toxicology. 198 (1-3), 229-238 (2004).</li><li>Eisenberg, J., Soller, J., Scott, J., Eisenberg, D., Colford, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+model+to+assess+microbial+health+risks+associated+with+beneficial+uses+of+biosolids.">A dynamic model to assess microbial health risks associated with beneficial uses of biosolids.</a> Risk Anal. 24, 221-236 (2004).</li><li>Wade, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Report on 2009 National Epidemiologic and Environmental Assessment of Recreational Water Epidemiology Studies, US EPA. US EPA Report Number: EPA/600/R-10/168: US EPA2011. , (2011).</li><li>Fujii, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+surveillance+development+for+tropical+infectious+diseases+using+simultaneous+microsphere-based+multiplex+assays+and+finite+mixture+models.">Serological surveillance development for tropical infectious diseases using simultaneous microsphere-based multiplex assays and finite mixture models.</a> PLoS Negl Trop Dis. 8, e3040 (2014).</li><li>Rota, M., Massari, M., Gabutti, G., Guido, M., De Donno, A., Ciofi degli Atti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measles+serological+survey+in+the+Italian+population:+interpretation+of+results+using+mixture+model.">Measles serological survey in the Italian population: interpretation of results using mixture model.</a> Vaccine. 26 (34), 4403-4409 (2008).</li><li>Vyse, A., Gay, N., Hesketh, L., Pebody, R., Morgan-Capner, P., Miller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+serological+surveys+using+mixture+models:+the+seroepidemiology+of+measles,+mumps+and+rubella+in+England+and+Wales+at+the+beginning+of+the+21st+century.">Interpreting serological surveys using mixture models: the seroepidemiology of measles, mumps and rubella in England and Wales at the beginning of the 21st century.</a> Epidemiol Infect. 134 (6), 1303-1312 (2006).</li><li>Baughman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+diagnostic+cutoff+points+for+levels+of+serum+antibodies+to+pertussis+toxin,+filamentous+hemagglutinin,+and+fimbriae+in+adolescents+and+adults+in+the+United+States.">Establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the United States.</a> Clin Diagn Lab Immunol. 11 (6), 1045-1053 (2004).</li><li>Clotilde, L., Bernard, C., Hartman, G., Lau, D., Carter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbead-based+immunoassay+for+simultaneous+detection+of+Shiga+toxins+and+isolation+of+Escherichia+coli+O157+in+foods.">Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.</a> J Food Prot. 74 (3), 373-379 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Luminex User Software Manual (RUO) xPONENT 3.1 Rev. 2. 3, 6-102 (2014).</li><li>Waterboer, T., Sehr, P., Pawlita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+non-specific+binding+in+serological+Luminex+assays.">Suppression of non-specific binding in serological Luminex assays.</a> J Immunol Methods. 309 (1-2), 200-204 (2006).</li></ol>

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 ...

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...