Name: bead based multiplex assay for analysis of tear cytokine profiles
Uploaded: 2017-10-13T13:00:00
Description: Watch this Scientific Journal Video about Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles at JoVE.com

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 an...

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&#160;</td>
			<td>Luminex Corp, Austin, TX, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xPonent software&#160;</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 ...

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