The research work was supported by the Centre Grant from Tan Tock Seng Hospital Personalised Seed Funding Program 2015; Singapore Eye Research Institute Pilot Grant and Tan Tock Seng Hospital Pitch for Fund grant.

The protocols used in this study were approved by the Institutional review board of Tan Tock Seng Hospital, Singapore.
1. Tear Cytokine Analysis
<ol>
	<li>Collection of tears:
	<ol>
		<li>Ask the subject to sit comfortably on an examination chair and place his/her head against the headrest.</li>
		<li>Ask the subject to look up, carefully open the Schirmer strips (made of Whatman filter paper no. 41) and place the rounded end of the strip on the inferior fornix of the eye and instruct the subject to close the eyes for 5 min. While collecting the tears take great care to minimize ocular surface contact.30</li>
		<li>Ask the subject to open his/her eyes to remove the strip and place it in a sterile 1.5 mL microcentrifuge tube. Immediately transfer the tube to the laboratory or store at -80o C until testing for cytokine profiling.</li>
	</ol>
	</li>
	<li>Elution of tear sample from tear flow strip:
	<ol>
		<li>Cut the tear flow strip to a length of 0.5 cm (to standardize the quantity of tears to be tested) and place it in a sterile 1.5 mL microcentrifuge tube.</li>
		<li>Add 30 &#181;L of assay buffer and incubate at room temperature for 5 min followed by centrifuging the tube at 14,000 x g for 1 min.</li>
		<li>Transfer the supernatant into another 1.5 mL microcentrifuge tube and discard the strip. Place the sample containing the tube on ice and use the eluted tear sample immediately for cytokine profiling.</li>
	</ol>
	</li>
	<li>Preparation of reagents: 
	NOTE: The forty one analytes to be tested in each sample by bead based multiplex assay are interleukin (IL)-1&#945;, IL-1&#946;, IL-R&#945;, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-15, IL-17A, interferon-alpha (IFN- &#945;) 2, IFN-&#947;, IFN-gamma-inducible protein 10 (IP-10, CXCL10), macrophage-derived chemokine (MDC), macrophage inflammatory protein (MIP)-1&#945; and MIP-1&#946;, monocyte chemotactic protein (MCP)-1, MCP-3, tumor necrosis factor-alpha (TNF-&#945;), TNF-&#946;, growth-regulated oncogene (GRO), tumor growth factor alpha (TGF-&#945;), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, platelet derived growth factor (PDGF)-AA, PDGF-AB/BB, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), eotaxin, fractalkine, soluble CD40 ligand (sCD40L), Fms-like tyrosine kinase 3 ligand (Flt-3L) and regulated upon activation, normal T-cell expressed and secreted protein (RANTES).
	<ol>
		<li>Bring the antibody bead solution (50X) vials to room temperature (20&#160;- 25 oC) and vortex the vials for 1 min.</li>
		<li>Count the number of wells required for the assay, and calculate the quantity of total antibody bead cocktail solution (25 &#181;L per well) and each antibody bead solution (50X) required for the assay.</li>
		<li>Prepare the cocktail solution by adding the calculated amount of each antibody bead solution and fill to the desired final volume by adding the remaining amount of bead diluent. Ensure that the final concentration of each antibody in the cocktail is 1X. Always prepare at least 20% extra volume of cocktail solution in case of pipetting errors. 
		NOTE: As an example, for a 96-well assay prepare 3000 &#181;L of antibody bead cocktail solution. The required amount of each antibody bead solution = 3000/stock concentration of each antibody bead solution; 3000/50 = 60 &#181;L of each antibody bead solution
		<ol>
			<li>Add 60 &#181;L of each of the 41 antibody bead solutions (60 X 41 = 2460 &#181;L) into a cocktail bottle and add 540 &#181;L of bead diluent solution to the antibody bead mixture to make 3000 &#181;L of final working solution (1X).</li>
			<li>Prior to use, mix the bead cocktail solution properly. After the assay store the remaining volume at 2-8 oC for up to 30 days.</li>
		</ol>
		</li>
		<li>Prepare the quality controls (QC) by adding 250 &#181;L deionized water to QC 1 and QC 2 stocks.</li>
		<li>Mix properly by inverting the bottles several times and vortex for 10 s. Transfer the solution to properly labeled polypropylene microcentrifuge tubes and store the remaining solution at -20 &#176;C which can be used for up to 30 days.</li>
		<li>Prepare the wash buffer by adding 270 mL deionized water to 30 mL of 10X wash buffer solution and mix well (before dilution, bring the 10X buffer to room temperature and dissolve all salt precipitates by mixing). Store the unused wash buffer (1X) at 2-8 &#176;C which can be used for up to 30 days.</li>
		<li>Prepare the human cytokine standard stock (10,000 pg/mL of all analytes) by adding 250 &#181;L deionized water to the stock vial. Mix well by inverting several times and vortex the vial for 10 s. Allow it to stand for 10 min and transfer the stock solution to properly labeled polypropylene microcentrifuge tubes. After the assay, store the remaining solution at -20 &#176;C, which can be used for up to 30 days.</li>
		<li>Prepare the working human cytokine standards by 5-fold serial dilutions (50 &#181;L -&#62; 200 &#181;L) with assay buffer to get 2000, 400, 80, 16, and 3.2 pg/mL.</li>
		<li>After standard preparation, use the working standards within 60 min and use the assay buffer as blank/background (-pg/mL).</li>
	</ol>
	</li>
	<li>Cytokine profiling by bead based multiplex assay:
	<ol>
		<li>Bring all reagents to room temperature and vortex for 5-10 seconds before adding them to the 96-well microtiter plate. If eluted tear samples are stored at -80oC prior to the assay, thaw the frozen tear extracts on ice and centrifuge at 1000 X g for 5 min.</li>
		<li>Prepare an assay work sheet in a vertical configuration for working human cytokine standards [0 (Blank), 3.2, 16, 80, 400, 2,000, and 10,000 pg/mL], QC1, QC2 and samples.</li>
		<li>Add 200 &#181;L of 1X wash buffer to each well of the plate, seal it with plate sealer and keep it on a plate shaker at room temperature (20-25 oC) for 10 min.</li>
		<li>Decant the 1X wash buffer by inverting the plate and tap it onto absorbent towels several times to remove the any residual amount of wash buffer in the wells.</li>
		<li>Add 25 &#181;L of each working human cytokine standard, QC1, QC2, blank (assay buffer) and samples into the appropriate wells.</li>
		<li>Add 25 &#181;L of assay buffer into each well.</li>
		<li>Add 25 &#181;L of 1X antibody bead cocktail solution into each well. As the antibody bead solution is light sensitive, seal the plate with plate sealer and cover it with aluminum foil to protect it from light during the assay.</li>
		<li>Incubate the plate at 4 oC overnight on a shaker.</li>
		<li>Place the plate on the plate rack of an automatic magnetic plate washer. Let it sit for 1 min to settle the magnetic beads at the bottom of the well and aspirate the well contents. Add 200 &#181;L of wash buffer per well and let it sit for 1 min and then aspirate the well contents. Repeat the wash once again (for plate washing, follow the kit manufacturer&#39;s instructions).</li>
		<li>Add 25 &#181;L of detection antibodies solution into each well, seal the plate, cover it with aluminum foil, and incubate at room temperature for 60 min on a shaker.</li>
		<li>Add 25 &#181;L of Streptavidin-Phycoerythrin solution into each well, seal the plate, cover it with aluminum foil, and incubate at room temperature for 30 min on a shaker.</li>
		<li>Place the plate on a magnetic plate washer. Let it sit for 1 min and then aspirate the well contents. Add 200 &#181;L of wash buffer per well. Let it sit for 1 min and then aspirate the well contents. Repeat the wash once again.</li>
		<li>Add 150 &#181;L of sheath fluid to each well and place the plate on a shaker for 5 min at room temperature to resuspend the antibody beads.</li>
		<li>Read the plate immediately using the bead based multiplex assay plate reader and analyze the cytokine concentrations using a 5-parameter curve-fitting algorithm. A schematic flow diagram of tear cytokine profiling is shown in Figure 1.</li>
	</ol>
	</li>
	<li>Reading the assay plate (instrument set up) and data analysis:
	<ol>
		<li>Switch on the bead based multiplex assay reader and pre-warm the laser for 30 min.</li>
		<li>Launch the bead based multiplex assay software. Under the &#39;Automated Maintenance&#39; tab, select the &#39;Calibration-verification&#39; option. Vortex each reagent vial of the calibration and verification beads for 30 s. Place 5 drops of each reagent into the designated wells. Fill the designated reservoirs with deionized water and 70% ethanol.</li>
		<li>Create a new protocol
		<ol>
			<li>Open the &#39;Protocols&#39; page and then the &#39;Protocols&#39; tab. Click &#34;Create New Protocol&#34;; the &#39;settings&#39; tab will open.</li>
			<li>In the &#39;Name&#39; box, type the name of the protocol.</li>
			<li>Type a description in the box to the right of the &#39;Name&#39; box.</li>
			<li>In the &#39;Version&#39; box, type the version of the protocol.</li>
			<li>In the &#39;Manufacturer&#39; box, type the manufacturer information for the protocol.</li>
			<li>Define settings in the &#39;Acquisition Settings&#39; section as follows. Set &#39;Volume&#39;: 100 &#181;L, &#39;Timeout&#39;: 60 s; &#39;DD Gating&#39;: 8,000 to 15,000; &#39;Reporter Gain&#39;: Standard PMT; &#39;Bead type&#39;: Magnetic bead.</li>
			<li>Define settings in the &#39;Analysis Settings&#39; section, selecting &#39;Quantitative&#39; as the analysis type. Set &#39;Number of Standards&#39;: 6; &#39;Number of Controls&#39;: 0; Tick the &#39;Mean of Replicates&#39; box; 
			Tick the &#39;Analyze results while acquiring samples&#39; box.</li>
			<li>Click &#34;Next&#34;; the &#39;Analytes&#39; tab opens, click on the desired analytes (bead ID) in the numbered analyte grid.</li>
			<li>Click and type the corresponding analyte name in the &#39;Name&#39; column to the right of the analyte grid (analytes&#39; names and their corresponding bead region details were mentioned in Table 1).</li>
			<li>Click and type the desired unit of measurement (i.e. pg/mL) in the &#39;Units&#39; box to the left of the &#39;Apply All&#39; button. Then click &#34;Apply All&#34;.</li>
			<li>Click and type the desired bead count for each analyte (i.e. 50) in the &#39;Count&#39; box. Click &#34;Apply All&#34;.</li>
			<li>Click &#34;Next&#34;; the Plate Layout tab opens. Highlight the wells for the standards and select &#34;2&#34; under replicate count and click on the &#34;S&#34; Standard button. Repeat this step for the background &#34;B&#34; and samples &#34;U&#34; wells.</li>
			<li>Click &#34;Save&#34;.</li>
		</ol>
		</li>
		<li>Create New Standards/Control Lots
		<ol>
			<li>Open the &#39;Protocols&#39; page, and then the &#39;Standards &#38; Controls&#39; tab. Click &#34;Create New Standard/Control Lots&#34;.</li>
			<li>Open the &#39;Select Protocol&#39; box. Select the protocol that was created in step 1.5.3.</li>
			<li>Key in the information of the standards accordingly: Std/Ctrl kit number, Std/Ctrl kit name, expiration, and manufacturer.</li>
			<li>Key in the value of the highest standard for each analyte (i.e. 10000 pg/mL). Key in 5 under dilution and click on &#34;Apply All&#34; to automatically generate the expected concentrations for the rest of the standards.</li>
			<li>Click &#34;Save&#34;.</li>
		</ol>
		</li>
		<li>Create a New Batch from an existing protocol
		<ol>
			<li>Open the &#39;Batches&#39; page and click &#34;Create a New Batch tab from an existing Protocol&#34;.</li>
			<li>Type the batch name in the &#39;Batch Name&#39; box and type a description about the batch in the &#39;Enter Optional Description&#39; box.</li>
			<li>Click on the protocol generated previously (in step 1.5.3).</li>
			<li>Click &#34;Next&#34;; the next tab that opens is the &#39;Stds &#38; Ctrls&#39; tab. View the details of assay standards and select &#34;Next&#34;.</li>
			<li>On the &#39;Plate Layout&#39; tab, assign well commands for this batch and click &#34;Save&#34; to save batch information to the &#39;Pending Batch&#39; list.</li>
			<li>Load the plate into the bead based multiplex assay plate reader, shake it for 5 min and run the batch from the pending batch list. Data acquired will be saved in the .csv file format.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Convert the .csv file generated from the bead based multiplex assay software to the .rbx file (results data file) format using the rbx conversion software. Launch the conversion software, select the .csv file under select xPONENT file(s) and click on the &#34;generate&#34; button; the .rbx file will be saved in the selected output folder.</li>
		<li>Launch the analysis software and open the .rbx file.</li>
		<li>Optimize the standard curves using the 4PL/5PL curve fit.
		<ol>
			<li>Select the following in the &#39;Standard Curve&#39; tab: &#39;Regression Type&#39;: &#39;Logistic-5PL&#39;; &#39;Axis Transformation&#39;: &#39;Log(x) - Linear(y)&#39;; tick the &#39;Show Conc Range Lines&#39; box; tick the &#39;Show Unknown Samples&#39; box; Tick the &#39;Show Control Samples&#39; box; Tick the &#39;Apply across all analytes&#39; box; tick the &#39;Show report after optimization&#39; box.</li>
			<li>Click the &#34;Optimize&#34; button for auto-optimization.</li>
		</ol>
		</li>
		<li>Label the sample identities under &#39;Enter Sample Info&#39; tab.</li>
		<li>Obtain the observed concentrations in the &#39;Report Table&#39; tab and click on &#34;export report table&#34; to generate the data in a spreadsheet file for further analysis.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose. No financial interest or conflict of interest.

Cytokines are small cellular secreted proteins and potent immune modulators regulating immune responses.32 The expression profiles of various cytokines in tear samples are related to various pathological conditions of the eye and studies on cytokine profiles help to understand the mechanisms of disease pathogenesis, determine the state of ocular health, disease severity, diagnosis and progression.2,5 Lower protein concentrations and small ...

Tears are produced by the lacrimal gland and accessory glands and coat the outer surface of the eye. Tear film consists of an outer lipid layer and an inner aqueous layer that includes soluble proteins, mucins and membrane bound mucins. Tears prevent microbial invasion, supply nutrients, and provide lubrication to the ocular surface. Tears act as an interface between the air and tissue for oxygen transport to the cornea.1 Tear film is made up of proteins, carbohydrates, lipids and electrolytes. The association between tear proteins with ocular and systemic diseases like glaucoma, dry eye disease, vernal conjunctivitis, diabetes mellitus, thyroid-associated orbitopathy and cancer has been identified in several studies.2,3,4 Tear samples can be collected by microcapillary tubes or tear flow strips (Schirmer strips). Additionally, the Schirmer&#39;s test is a standard procedure performed in the cornea and refractive surgery clinics, the results of which may be used for cytokine analysis assays. Non-invasive sample collection, accessibility of the bio-specimen, and the association of tear composition with various physiological and pathological conditions make tear film a potential source of biomarkers for several ocular and systemic diseases.4,5,6
Tear cytokines plays an important role in studying the ocular surface health and inflammatory conditions of various ocular diseases.7 Abnormal concentrations of several cytokines in tear samples were reported to be associated with dry eye disease, vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), seasonal allergic conjunctivitis, and uveitis.8,9,10,11,12,13 Tear proteins can be analyzed by traditional methods like mass spectrometry, western blotting and enzyme-linked immunosorbent assays (ELISA).14,15 However, the limitations of these methods are poor sensitivity and a larger volume of sample required for the analysis of multiple tear cytokines in each patient.16,17 Bead based multiplex assays have been developed to analyze multiple analytes in complex mixture samples and successfully applied on tear samples to analyze multiple cytokines in different diseases.6,18 A combination of sandwich ELISA and flow cytometry techniques enable these assays to become more sensitive than ELISA for the quantification of multiple analytes in a single sample.19 This method can be applied on a variety of clinical samples and cell culture supernatants and helps in the study of the immune responses in several pathophysiological conditions.20,21,22,23,24
There are several studies comparing bead based multiplex assays with ELISA and have reported a correlation between the methods. Loo et al. compared bead based multiplex assay with conventional ELISA for the detection of adiponectin, resistin, leptin and ghrelin in human serum or plasma samples and reported a strong correlation (r&#62;0.9) between the assays.25 Dupont et al. reported a strong correlation between bead based multiplex assays and ELISA for the detection of IL-1&#946;, IL-4, IL-5, IL-6, IL-10, IFN-&#978;, and TNF-&#945; in phytohemagglutinin and lipopolysaccharide stimulated whole blood collected from pregnant women.26 Pickering et al. reported another strong correlation between bead based multiplex assay and ELISA for the detection of serum antibodies to Haemophilus influenza type b polysaccharide (r=0.96), toxoids of Clostridium tetani (r=0.96), and Corynebacterium diphtheriae (r=0.91).27 Biagini et al. reported a high positive correlation (r=0.852) between bead based multiplex assay and ELISA for detection of Bacillus anthracis anti-PA IgG in serum samples.28 Wang et al. reported a correlation between bead based multiplex assay and ELISA for the detection of Alzheimer&#39;s disease biomarkers amyloid-&#946; 42 (r=0.77), total tau (r=0.94), and tau phosphorylated on amino acid 181 (r=0.82) in cerebrospinal fluid samples.29 These studies have demonstrated the applicability of bead based multiplex assays on variety of clinical samples, smaller sample volume requirements and a correlation with standard ELISA, which make bead based multiplex assays a promising alternative to traditional ELISA methods for the detection of multiple analytes in different type of samples in different disease phenotypes. Here we describe a standardized protocol for bead based multiplex assay for cytokine profiling for 41 analytes in tear samples collected from healthy subjects using Schirmer strips.

<table><tbody><tr><td>Milliplex MAP human cytokine / chemokine magnetic bead panel -1 kit</td><td>Merck, USA</td><td>HCYTOMAG-60K-41</td><td></td></tr><tr><td>Flexmap 3D luminex instrument&amp;nbsp;</td><td>Luminex Corp, Austin, TX, USA</td><td></td><td></td></tr><tr><td>xPonent software&amp;nbsp;</td><td>Luminex Corp, Austin, TX, USA</td><td></td><td></td></tr><tr><td>RBXGenerator software</td><td>BIO-RAD, France</td><td></td><td></td></tr><tr><td>Bio-Plex Manager 6.1</td><td>BIO-RAD, France</td><td></td><td></td></tr><tr><td>Plate shaker</td><td>Corning, USA</td><td></td><td></td></tr><tr><td>TECAN Microplate Washer</td><td>Tecan, Switzerland</td><td>HydroSpeed</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Scientific Industries inc, USA</td><td>G560E</td><td></td></tr><tr><td>Pipettes</td><td>Mettler Toledo, CA, USA</td><td></td><td></td></tr><tr><td>1.5ml microcentrifuge tube</td><td>Axygen, USA</td><td>MCT-150-C</td><td></td></tr><tr><td>Schirmer tear flow test strip</td><td>Eye Care and Cure, USA</td><td>101657</td><td></td></tr><tr><td>Flexmap 3D Calibration Kit</td><td>Luminex Corp, Austin, TX, USA</td><td>40-028</td><td></td></tr><tr><td>Flexmap 3D Verfication Kit</td><td>Luminex Corp, Austin, TX, USA</td><td>40-029</td><td></td></tr><tr><td>Ocular examination chair</td><td></td><td></td><td></td></tr></tbody></table>

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.

Tear samples were collected from 8 eyes of 4 healthy subjects using tear flow strips and cytokine levels were analyzed using the above mentioned protocol. All subjects were male and the age of the four subjects was 36, 42, 44 and 52 years respectively. Ocular surface health and dry eye disease was evaluated by clinical tests (tear film break up time, Schirmer&#39;s test, corneal staining, conjunctival staining, lid/meibomian gland examination, corneal tear signs and visual signs and sympt...

Watch this Scientific Journal Video about Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles at JoVE.com

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

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 ...

bead-based-multiplex-assay-for-analysis-of-tear-cytokine-profiles

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.7K Views.  Tan Tock Seng Hospital. 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.

Video: Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

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

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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 ...

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or activating an endogenous regulator of the immune system have the potential to cause serious immune-related adverse events such as cytokine release syndrome (CRS). It is, therefore crucial to assess the safety profile of such drugs with a combination of in vivo and in vitro experiments before first-in-human dose administration. Cytokine release assays (CRAs), where the proposed antibody therapeutic is co-cultured with human immune cells (such as peripheral blood mononuclear cells (PBMCs), whole blood, or otherwise) and the amount of inflammatory cytokine produced is measured, are critical for hazard identification. However, different labs using different control antibodies can threaten the harmonization of CRAs, and clinically relevant controls (such as TGN1412) can be difficult to source, which can lead to less accurate or reliable results or data which are difficult to compare between laboratories. The inclusion of positive and negative controls in a CRA can ensure the accuracy and reliability of the results. The National Institute for Biological Standards and Control (NIBSC) has produced a panel of lyophilized antibody controls intended for use in various CRA platforms to harmonize results across various laboratories and assay methods. A set of three different positive control antibodies include anti-CD52, anti-CD3, and anti-CD28 superagonist (SA), which are known to induce dose-dependent CRS in patients. Each antibody is provided with an isotype-matched negative control antibody. This panel of reference reagents has previously been shown to have good inter-lab reproducibility and are suitable controls to increase the confidence and robustness of safety data from a variety of CRA platforms.

The use of cytokine release assay reference reagents allows for more reproducible and standardized in vitro safety profiles of immunotherapeutic monoclonal antibodies. Here we describe how cytokine release assays can be used alongside a reference reagent panel to predict the safety of some therapeutic monoclonal antibodies.

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or ...

Non-Invasive Biomarker Discovery for Tear Proteomics Using LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...

Medicine

Protocol for Tear Protein Extraction in Experimental Animal Models

Optimizing Tear Collection in Mice for mRNA and Protein Analysis

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other tissues and organs. Molecular analysis of this biofluid offers a non-invasive way to diagnose or monitor diseases, assess medical treatment efficacy, and identify possible biomarkers. Due to the limited sample volume, collecting tear samples requires specific skills and appropriate tools to ensure high quality and maximum efficiency. Various tear sampling methodologies have been described in human studies. In this article, a comprehensive description of an optimized protocol is presented, specifically tailored for extracting tear-related protein information from experimental animal models, especially mice. This method includes the pharmacological stimulation of tear production in 2-month-old mice, followed by sample collection using Schirmer strips and the evaluation of the efficacy and efficiency of the protocol through standard procedures, SDS-PAGE, qPCR, and digital PCR (dPCR). This protocol can be easily adapted for the investigation of the tear protein signature in a variety of experimental paradigms. By establishing an affordable, standardized, and optimized tear sampling protocol for animal models, the aim was to bridge the gap between human and animal research, facilitating translational studies and accelerating advancements in the field of ocular and systemic disease research.

Author Spotlight: Isolating Biomolecules from Mouse Tears &#8212; A Methodology for Molecular Analysis and Biomarker Research

The identification of RNA and protein biomarkers from tears in mouse models holds great promise for early diagnostics in various diseases. This manuscript provides a comprehensive protocol for optimizing the efficacy and efficiency of mRNA and protein isolation from mouse tears.

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other ...

Biology

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Source: Lehmann, J. S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56440">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> J. Vis. Exp. (2017).
This video demonstrates a multiplex bead-based immunoassay for quantifying multiple cytokine targets in a sample. A mixture of distinct microspheres conjugated to cytokine-specific antibodies is used to bind the cytokines present in the sample. Streptavidin-tagged reporters are immobilized on the bound-cytokines using biotinylated detection antibodies. Flow cytometry identifies the bound cytokines based on the unique sizes and internal fluorescence intensities of the microspheres, while the fluorescence intensity of the reporters indicates the number of cytokines.

1. Biological Sample Preparation
NOTE: The chosen anatomical site and volume of blood collection are left to the discretion of each individual ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

A Multiplex Bead-Based Assay for Analyzing Cytokines in Human Tear Samples

Source: Balne, P. K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55993">Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles.</a> J. Vis. Exp. (2017)
This video illustrates a bead-based multiplex assay for analyzing cytokines in human tears. The method allows for the simultaneous analysis of multiple cytokines from a single sample, providing enhanced sensitivity for studying immune responses in various pathophysiological conditions.

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Summary

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

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Optimizing Tear Collection in Mice for mRNA and Protein Analysis

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

A Multiplex Bead-Based Assay for Analyzing Cytokines in Human Tear Samples