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.

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

Research

JoVE Journal

Immunology and Infection

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 characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

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

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

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

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 cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

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 and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

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 methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

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 allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

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 mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using 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 epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

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 ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, including&#160;the cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public ...