Name: Geliştirme ve doğrulama vericiyi bir tek molekül dijital enzim bağlı Immunosorbent Assay insan Interferon-α için dizi (Video) | JoVE Journal
Uploaded: 2018-06-14T14:00:00.000+00:00
Duration: 8 min 26 s
Description: Keşfedin tek molekül dijital ELISA yöntemiyle insan IFN-α alt tiplerini ultra hassas şekilde tespit etmeyi. Bu yöntem otoimmün hastalıklarda interferon yanıtlarını anlamak ve biyobelirteç keşfi için güçlü bir araç sunar.

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DD and YJC acknowledge support from the ANR (Project IFNX, no. CE17001002) and ImmunoQure AG for providing monoclonal antibodies. We thank Brigitte Bader-Meunier, Nathalia Bellon, Christine Bodemer, Alex Belot, Isabelle Melki, and Pierre Quartier for providing clinical samples. YJC acknowledges the European Research Council (GA 309449: Fellowship to YJC), and a state subsidy managed by the National Research Agency (France) under the &#34;Investments for the Future&#34; program bearing the reference ANR-10-IAHU-01.

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<li>Decalf, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+DPP4+activity+in+humans+establishes+its+in+vivo+role+in+CXCL10+post-translational+modification%3A+prospective+placebo-controlled+clinical+studies%5BTitle%5D)+AND+%22EMBO+Mol.+Med%22%5BJournal%5D)">Inhibition of DPP4 activity in humans establishes its in vivo role in CXCL10 post-translational modification: prospective placebo-controlled clinical studies.</a> EMBO Mol. Med. 8, 679-683 (2016).</li>
<li>Rabing Brix Petersen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rhodopsin+in+plasma+from+patients+with+diabetic+retinopathy+-+development+and+validation+of+digital+ELISA+by+Single+Molecule+Array+%28Simoa%29+technology%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">Rhodopsin in plasma from patients with diabetic retinopathy - development and validation of digital ELISA by Single Molecule Array (Simoa) technology.</a> J. Immunol. Methods. 446, 60-69 (2017).</li>
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<li>Meyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AIRE-Deficient+Patients+Harbor+Unique+High-Affinity+Disease-Ameliorating+Autoantibodies%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.</a> Cell. 166, 582-595 (2016).</li>
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<li>Pleil, J., Angrish, M., Madden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunochemistry+for+high-throughput+screening+of+human+exhaled+breath+condensate+%28EBC%29+media+implementation+of+automated+Quanterix+SIMOA+instrumentation%5BTitle%5D)+AND+%22J.+Breath+Res%22%5BJournal%5D)">Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media implementation of automated Quanterix SIMOA instrumentation.</a> J. Breath Res. 9, 047108(2015).</li>
<li>Schiess, R., Wollscheid, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+Proteomic+Strategy+for+Clinical+Biomarker+Discovery%5BTitle%5D)+AND+%22Mol.+Oncol%22%5BJournal%5D)">Targeted Proteomic Strategy for Clinical Biomarker Discovery.</a> Mol. Oncol. 3, 33-44 (2009).</li>
<li>Wilson, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Simoa+HD-1+Analyzer%3A+A+Novel+Fully+Automated+Digital+Immunoassay+Analyzer+with+Single-Molecule+Sensitivity+and+Multiplexing%5BTitle%5D)+AND+%22J.+Lab.+Autom%22%5BJournal%5D)">The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing.</a> J. Lab. Autom. 21, 533-547 (2016).</li>
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</ol>

The authors have nothing to disclose.

Herein, we described the development and validation of a highly reproducible, ultrasensitive single molecule array digital ELISA for direct quantification of IFN-&#945; protein in human samples (steps summarized in Table 1). One of the most critical steps in the development of the assay is the choice of antibody pair16, with the characteristics in terms of kinetics and epitope binding being key to a successful assay. It is important to avoid the use of paired monoclonal antibodies that target the same epitope or that cause steric impediment. Polyclonal antibodies could be used as detection antibodies to overcome such limitations. If at any given step the desired sensitivity is not achieved, further optimization possibilities should be considered. This may include the use of alternative antibody pairs, changes in parameters such as protein type (e.g. BSA &#60; casein), pH (6.0 - 8.5), ionic strength, buffering capacity (e.g. NaCl and phosphate concentrations), carrier beads, and/or presence of surfactants. A wide range of parameters con be optimized when developing a single molecule array assay. However, overall, conditions that give high signal:background ratios and minimal LODs are the ones preferred (see Figure 1, Figure 2,&#160;and Figure 3).
Regarding specificity, the IFN-&#945; assay showed no cross reactivity for any other IFNs tested (&#946;, &#947;, &#955;1, &#955;2, &#969;) (Figure 4a) and was capable of detecting all 13 IFN-&#945; subtypes (Figure 4b). However, the assay showed a lower affinity for the IFN-&#945;2 subtype, (Figure 4b and Supplementary Table 4). Interestingly, different sensitivities for the different classes of IFN-&#945;2 (a, b, c) were also observed, which may be due to distinct manufacturing procedures or different amino acid sequences, as the IFN-&#945;2 subtypes were obtained from different commercial suppliers. With this sole exception, all the IFN-&#945; species gave very similar responses. The specificity of the assay was further demonstrated by pre-treatment of the samples with the anti-IFN-&#945; A clone, which abrogated the signal (Figure 6 and Supplementary Table 6).
One of the main advantages of this technology is that any analyte of interest can potentially be targeted16. Furthermore, different biological specimens can be tested, such as serum, plasma, cerebrospinal fluid, cellular lysates, culture supernatants31 and even breath32. Usually, a 1:3 dilution of plasma is performed to avoid potential clogging of the single molecule array analyzer. However, the analyte of interest could be present at very low concentrations in the relevant biological sample and higher concentrations of sample might be required (depending on assay sensitivity)33. Although there is potential for multiplexing while maintaining good sensitivity34, it is a more challenging process and assays for measuring greater than 6 proteins within the same experiments have yet to be developed35.
The ability to detect and quantify cytokines and other biological relevant proteins at such low concentrations opens up a whole new range of applications33,36. It is well known that numerous proteins exert their effects even at very low concentrations, which up until now were below the limit of detection of the best ELISAs37. While other immunoassay technologies offer advantages over conventional ELISA19, we demonstrate here that single molecule array digital ELISA is a reproducible and robust platform for the ultrasensitive detection of low concentration cytokines in human samples. As such, this technology offers enormous potential for biomarker discovery and improved patient management of a wide range of diseases.

Type I IFNs are a family of cytokines which play a central role in orchestrating antiviral immune responses. They were first discovered by Isaacs and Lindenmann 60 years ago1,2 and it is now known that this heterogeneous family of polypeptides comprises 14 different subclasses (13 IFN-&#945; subtypes and 1 IFN-&#946;). Type I IFNs are essential to the clearance of viral infections, but have also been implicated in the pathology of a variety of human disease states, including the autoimmune disorders systemic lupus erythematosus (SLE), juvenile dermatomyositis (JDM), and the type I interferonopathies in which constitutive type I IFN-induced signaling results in pathology3,4,5,6,7.
Studying type I IFN protein levels in biological samples has been challenging since its initial identification as an &#34;interfering substance&#34;1,2. Currently, sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) is the most widely used method for detection of IFN-&#945; protein. Despite being specific, simple, and rapid, type I IFN ELISAs present important limitations, such as limited sensitivity. In addition, the measurement of all IFN-&#945; subtypes requires the use of multiple assays each with their own detection capacity and sensitivity. While there are commercial ELISAs that detect different subtypes of IFN-&#945;, their sensitivity is limited (1.95 pg/mL, 12.5 pg/mL, and 12.5 pg/mL, respectively) which is often insufficient to detect IFN-&#945; protein in biological samples. To overcome this limitation, several biological proxy assays have been developed to quantify type I IFN by measuring induced gene expression or functional activity8,9,10,11,12,13,14. Nonetheless, these assays do not provide a direct measurement of the IFN-&#945; protein.
In this study, single molecule array digital ELISA technology was used to develop an assay for the detection of single IFN-&#945; protein molecules. Digital ELISA utilizes the same basic chemistry as conventional ELISA, however, the reaction takes place in arrays comprising 50,000 individual 46 femtoliter sized wells15,16. Single protein molecules are captured by antibody-coated paramagnetic beads and labeled with a biotinylated detection antibody, followed by binding of an enzyme conjugate, streptavidin-&#946;-galactosidase (SBG). Subsequently, the beads are suspended with a fluorogenic enzyme substrate, resorufin-&#946;-D-galactopyranoside (RGP), into single-molecule arrays. By decreasing the volume reaction 2 billion times17, a high local concentration of fluorescent signal is achieved and single molecule counts become feasible, as each molecule generates a signal that can now be reliably measured18. In essence single molecule arrays are capable of counting single immunocomplexes and determining an average number of enzymes per bead (AEB). Counting the microwells in which a signal is detected permits quantification/digitalization of protein molecules, as there is a direct correlation between protein concentration and the ratio between immunocomplexed-beads and a total number of beads present in the femtoliter-sized chambers.
Yeung et al. performed an extensive cross-platform evaluation study using nine different technologies and four cytokine immunoassays with the aim of comparing assay precision, sensitivity, and data correlation across the different platforms19. One of the key findings of the study was that single molecule arrays and single molecule counting immunoassay presented the highest sensitivity for detection of cytokines in human serum within the sub-pg/mL concentration range. Single molecule array digital ELISA cytokine assays have been used to study the role of TNF-&#945; and IL-6 in Crohn&#39;s disease20, IFN-&#945; in interferonopathy and auto-immune patients 7, and the different post-translationally modified forms of C-X-C motif chemokine 10 (CXCL10) in chronic hepatitis and healthy donors receiving sitagliptin21,22. Other applications include measurement of rhodopsin in patients with diabetic retinopathy23; the study of brain pathologies through serum/plasma measurements of neurofilament light24&#160;and amyloid-&#946; 1-42 peptide25, in the context of multiple sclerosis and Alzheimer&#39;s disease, respectively. Single molecule array assays can also be used for improved pathogen detection such as characterization of the HIV viral reservoir26, and also for detection of DNA27 and micro RNAs28. A major advantage of the single molecule array technology is this high versatility, as an assay against any analyte of interest can be developed if a specific antibody pair is available. In addition, homebrew assay kits are commercially available, allowing the development of new assays, the protocol of which is detailed in a modified form below.
Here, a detailed description of the development and validation steps for a single molecule array assay is presented that results in enhanced sensitivity for IFN-&#945; protein detection. Antibodies for single molecule array assays should be highly specific, avoiding cross reactivity with related proteins (with species specificity considered if relevant). Ideally, antibodies with a KD smaller than 10-9 M should be chosen; high affinity ensures strong binding, with the production of a higher signal. The kinetics of the antibody-antigen binding are also important, and fast Kon and slow Koff will favor antigen-antibody complex assembly. Starting with an antibody pair that has a good performance in classical ELISA, with a limit of detection (LOD) of 1 - 100 pg/mL, increases the chances of obtaining a highly sensitive single molecule array assay. Chang and colleagues performed detailed kinetic studies of the biomolecular interactions occurring at every single step, proposing a set of equations to predict analytical sensitivity18. They showed that single molecule array digital ELISA appears to be effective within a broad range of antibody affinities (KD~10&#8722;11&#160;- 10&#8722;9 M), as well as that signal generation is more dependent on the on-rate of such antibodies.
To utilize high affinity antibodies we took advantage of antibodies isolated from patients with the autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED)29. For reasons that are not yet understood, the large majority of AIRE-deficient individuals develop a core set of high-avidity antibodies against all IFN-&#945; subtypes29, and we selected two anti-IFN-&#945; antibody clones of complementary binding affinities for the different IFN-&#945; subtypes, as estimated by IC50 values. The combination of these unique high-affinity antibodies with single molecule array technology enabled the direct quantification of IFN-&#945; at attomolar (fg/mL) concentrations. Such ultrasensitive IFN-&#945; protein detection will contribute to a better understanding of the nature, regulation and biological impact of the IFN-induced response in different disease settings.

<table><tbody><tr><td>Anti-IFN-&amp;alpha; antibody A (8H1 clone)</td><td>ImmunoQure AG</td><td>NA</td><td>Not commercially available</td></tr><tr><td>Anti-IFN-&amp;alpha; antibody B (12H5 clone)</td><td>ImmunoQure AG</td><td>NA</td><td>Not commercially available</td></tr><tr><td>Anti-IFN-&amp;alpha; antibody EBI-1 clone</td><td>eBioscience</td><td>BMS216C</td><td></td></tr><tr><td>Anti-IFN-&amp;alpha; antibody BMS216BK clone</td><td>eBioscience</td><td>BMS216BK</td><td>Not an ELISA kit but bulk abs</td></tr><tr><td>IFN &amp;alpha;2c</td><td>eBioscience</td><td>BMS305</td><td>OK</td></tr><tr><td>IFN &amp;alpha;I (17)</td><td>PBL</td><td>11150-1</td><td></td></tr><tr><td>IFN &amp;alpha;2a</td><td>PBL</td><td>11100-1</td><td></td></tr><tr><td>IFN &amp;alpha;2a</td><td>PeproTech</td><td>No longer available</td><td></td></tr><tr><td>IFN &amp;alpha;2b</td><td>PBL</td><td>11105-1</td><td></td></tr><tr><td>IFN &amp;alpha;1</td><td>PBL</td><td>11175-1</td><td></td></tr><tr><td>IFN &amp;alpha;4a (M1)</td><td>PBL</td><td>11177-1</td><td></td></tr><tr><td>IFN &amp;alpha;4b (a4)</td><td>PBL</td><td>11180-1</td><td></td></tr><tr><td>IFN &amp;alpha;B2 (a8)</td><td>PBL</td><td>11115-1</td><td></td></tr><tr><td>IFN &amp;alpha;C (a10)</td><td>PBL</td><td>11120-1</td><td></td></tr><tr><td>IFN &amp;alpha;D (a1)</td><td>PBL</td><td>11125-1</td><td></td></tr><tr><td>IFN &amp;alpha;G (a5)</td><td>PBL</td><td>11135-1</td><td></td></tr><tr><td>IFN &amp;alpha;F (a21)</td><td>PBL</td><td>11130-1</td><td></td></tr><tr><td>IFN &amp;alpha;H2 (a14)</td><td>PBL</td><td>11145-1</td><td></td></tr><tr><td>IFN &amp;alpha;J1 (a7)</td><td>PBL</td><td>11160-1</td><td></td></tr><tr><td>IFN &amp;alpha;K (a6)</td><td>PBL</td><td>11165-1</td><td></td></tr><tr><td>IFN &amp;alpha;WA (a16)</td><td>PBL</td><td>11190-1</td><td></td></tr><tr><td>IFN &amp;lambda;1</td><td>PeproTech</td><td>300-02L</td><td></td></tr><tr><td>IFN &amp;lambda;2</td><td>PeproTech</td><td>300-02K</td><td></td></tr><tr><td>IFN &amp;omega;</td><td>PeproTech</td><td>300-02J</td><td></td></tr><tr><td>IFN &amp;beta;</td><td>PeproTech</td><td>300-02BC</td><td></td></tr><tr><td>IFN &amp;gamma;</td><td>PeproTech</td><td>300-02</td><td></td></tr><tr><td>Anti-IFN-&amp;alpha; ELISA</td><td>PBL</td><td>41115.1</td><td></td></tr><tr><td>96-well plates</td><td>Corning</td><td>3904</td><td></td></tr><tr><td>ISRE-Reporter</td><td>Qiagen</td><td>CCS-008L</td><td></td></tr><tr><td>Fu-GENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Mediuem</td><td>ThermoFischer Scientific</td><td>31985062</td><td></td></tr><tr><td>Dual-Luciferase Reporter assay</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Nonidet P40 Substitute</td><td>Sigma-Aldrich</td><td>74385</td><td></td></tr><tr><td>Spectrophotometer NanoDrop 1000</td><td>Thermo Scientific</td><td>No longer available</td><td></td></tr><tr><td>Amicon micocentrifuge tubes - 0.5mL filtres</td><td>Merck Millipore</td><td>UFC505096</td><td></td></tr><tr><td>Bead Conjugation Buffer</td><td>Quanterix</td><td>102040</td><td>Can not be ordered separately</td></tr><tr><td>Non-Encoded Paramagnetic Beads</td><td>Quanterix</td><td>101360</td><td></td></tr><tr><td>Bead Wash Buffer</td><td>Quanterix</td><td>102040</td><td>Can not be ordered separately</td></tr><tr><td>EDC</td><td>Fisher Scientific</td><td>11844071</td><td></td></tr><tr><td>Bead Blocking Buffer</td><td>Quanterix</td><td>102040</td><td>Can not be ordered separately</td></tr><tr><td>Bead Diluent Buffer</td><td>Quanterix</td><td>101362</td><td></td></tr><tr><td>Detector and Sample Diluent</td><td>Quanterix</td><td>101359</td><td></td></tr><tr><td>Biotinylation Reaction Buffer</td><td>Quanterix</td><td>102040</td><td>Can not be ordered separately</td></tr><tr><td>NHS-PEG4-Biotin</td><td>Fisher Scientific</td><td>11891195</td><td></td></tr><tr><td>diH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Centrifuge for 1.7mL microcentrifuge tubes (Heraens Fresco 21 Centrifuge)</td><td>Thermo Scientific</td><td>75002478</td><td></td></tr><tr><td>Standard laboratory vortex mixer (MS2 Minishaker)</td><td>IKA</td><td>MS2</td><td></td></tr><tr><td>Pipettes (10, 20, 200 and 1000 &amp;mu;l)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Tips (10, 20, 200 and 1000 &amp;mu;l)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>1.7mL microcentrifuge tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Magnetic separator that accomodates 1.7mL microcentrifuge tubes (Life Technologies DynaMag-2)</td><td>ThermoFisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Mini benchtop centrifuge (Galaxy MinisStar)</td><td>VWR</td><td>521-2844</td><td></td></tr><tr><td>Mixer/Shaker (Eppendorf Thermomixer Comfort)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Single molecule array (Simoa) reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SBG, Bead and Detector Barcode Labels</td><td>Quanterix</td><td>101652</td><td></td></tr><tr><td>15 mL Reagent Bottles</td><td>Quanterix</td><td>102411</td><td></td></tr><tr><td>Simoa specimen 96-well plates</td><td>Quanterix</td><td>101457</td><td></td></tr><tr><td>Simoa Discs</td><td>Quanterix</td><td>100001</td><td></td></tr><tr><td>Simoa 2.0 conductive Tips</td><td>Quanterix</td><td>101726</td><td></td></tr><tr><td>Simoa Cuvettes</td><td>Quanterix</td><td>100803</td><td></td></tr><tr><td>Simoa SBG Reagent</td><td>Quanterix</td><td>102295</td><td></td></tr><tr><td>Simoa RGP Reagent</td><td>Quanterix</td><td>101736</td><td></td></tr><tr><td>Simoa System Buffer 1</td><td>Quanterix</td><td>100486</td><td></td></tr><tr><td>Simoa System Buffer 2</td><td>Quanterix</td><td>100487</td><td></td></tr><tr><td>Sealing Oil</td><td>Quanterix</td><td>100206</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Simoa HD-1 Analyzer</td><td>Quanterix</td><td>100032</td><td></td></tr><tr><td>&lt;u&gt;Technologies&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Single molecule array (Simoa)</td><td>Quanterix</td><td></td><td></td></tr><tr><td>Single molecule counting Erenna Immunoassay Systems</td><td>Singluex</td><td></td><td></td></tr></tbody></table>

development and validation of an ultrasensitive single molecule array digital enzyme-linked immunosorbent assay for human interferon-&#945;

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

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

In summary, a pan-IFN-α single molecule array assay with a limit of detection of 0.69 fg/mL, using a 2-step configuration with capture beads coated with 0.3 mg/mL of anti-IFN-α A antibody and anti-IFN-α B detection antibody biotinylated at a biotin:antibody ratio of 30 was developed. The concentrations used for biotinylated detection antibody and SBG were 0.3 µg/mL and 150 pM, respectively. This highly specific assay is capable of detecting all 13 IFN-α subtypes and does not cross react with other type of IFNs. Thus, while it is not possible to identify and quantify each individual species of IFN-α, all of them can be detected and measured together giving a total concentration value for IFN-α, that comprises the 13 different subclasses.
To explore the potential diagnostic value of this newly developed assay, IFN-α protein in plasma and serum from healthy individuals were measured and compared with samples from patients suffering from SLE and JDM. As illustrated in Figure 7, high levels of IFN-α protein were detected in both disease cohorts as compared to healthy controls. The dotted line indicates the LOD of a conventional commercially available ELISA, illustrating how this approach would not detect IFN-α protein in these patient groups despite the known associations of this cytokine with these phenotypes. Furthermore, IFN-α could be quantified over 5 logs of magnitude, illustrating the wide dynamic range of the assay, with detectable levels between 1 - 10 fg/mL confirming the highly sensitive nature of the assay.
<img alt="IFNα concentration graph; scatter plot comparing Healthy, SLE, JDM with data distribution analysis." class="xfigimg" src="/files/ftp_upload/57421/57421fig7.jpg" data-altupdatedat="1788250015000" data-alt="Figure 7"> 
Figure 7: Quantification of IFN-α protein in plasma and serum from patient cohorts. This figure has been modified from Rodero et al. 2017. Plasma from healthy controls (n = 20) and patients suffering from SLE (n = 72) and JDM (n = 43) were tested with the pan-IFNα single molecule array assay. Analysis was performed using one-way ANOVA test (Kruskal-Wallis) and Dunn's multiple comparison testing between groups. Median is depicted. Dotted line indicates LOD of a reference human anti-IFN-α ELISA (1.95 pg/mL = 1950 fg/mL). <a href="/files/ftp_upload/57421/57421fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Considerations</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td rowspan="3">1. Antibody pair choice</td>
			<td>Target different epitopes</td>
			<td rowspan="3">Table 2</td>
		</tr>
		<tr>
			<td>High affinity</td>
		</tr>
		<tr>
			<td>Fast Kon / Slow Koff</td>
		</tr>
		<tr>
			<td>2. Antibody pair orientation</td>
			<td rowspan="3">Choose conditions with the lowest Limit of Detection</td>
			<td rowspan="3">Figure 1</td>
		</tr>
		<tr>
			<td>3. Capture antibody concentration</td>
		</tr>
		<tr>
			<td>4. Biotin:Detector antibody ratio</td>
		</tr>
		<tr>
			<td>5. 2 vs. 3-step configuration</td>
			<td>Choose condition with the highest Signal:Background ratio</td>
			<td>Figure 2</td>
		</tr>
		<tr>
			<td>6. Detector antibody concentration</td>
			<td rowspan="2">Choose conditions with the lowest Limit of Detection</td>
			<td rowspan="2">Figure 3</td>
		</tr>
		<tr>
			<td>7. SBG concentration</td>
		</tr>
		<tr>
			<td>8. Specificity</td>
			<td>Assess cross-reactivity</td>
			<td rowspan="2">Figure 4</td>
		</tr>
		<tr>
			<td>9. Sensitivity</td>
			<td>Assess recognition of subtypes (if applicable)</td>
		</tr>
		<tr>
			<td>10. Reproducibility</td>
			<td>Assess assay variability</td>
			<td>Figure 5</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of steps for development and optimization of a single molecule array assay. This table summarizes the different steps required for the development and optimization of a single molecule array assay, from the initial choice of antibody pair until the assessment of reproducibility.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>8H1 (A)</td>
			<td>12H5 (B)</td>
			<td>Sifalimumab</td>
			<td>Rontalizumab</td>
		</tr>
		<tr>
			<td>IFNα1</td>
			<td>28.3</td>
			<td>51.3</td>
			<td>460</td>
			<td>22.63</td>
		</tr>
		<tr>
			<td>IFNα2</td>
			<td>2563</td>
			<td>10.7</td>
			<td>9.02</td>
			<td>2.15</td>
		</tr>
		<tr>
			<td>IFNα4</td>
			<td>5.11</td>
			<td>2.99</td>
			<td>35.35</td>
			<td>325.3</td>
		</tr>
		<tr>
			<td>IFNα5</td>
			<td>2.01</td>
			<td>19.6</td>
			<td>93.29</td>
			<td>2.49</td>
		</tr>
		<tr>
			<td>IFNα6</td>
			<td>64.7</td>
			<td>3.03</td>
			<td>4.97</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFNα7</td>
			<td>0.9</td>
			<td>0.63</td>
			<td>233.1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFNα8</td>
			<td>302</td>
			<td>0.83</td>
			<td>691</td>
			<td>10.86</td>
		</tr>
		<tr>
			<td>IFNα10</td>
			<td>2.54</td>
			<td>0.71</td>
			<td>43.2</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFNα14</td>
			<td>3224</td>
			<td>2.1</td>
			<td>14.52</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>IFNα16</td>
			<td>1.83</td>
			<td>32.99</td>
			<td>59.18</td>
			<td>28.65</td>
		</tr>
		<tr>
			<td>IFNα17</td>
			<td>2.21</td>
			<td>0.77</td>
			<td>890.9</td>
			<td>23.86</td>
		</tr>
		<tr>
			<td>IFNα21</td>
			<td>2.78</td>
			<td>12.87</td>
			<td>1769</td>
			<td>5.8</td>
		</tr>
	</tbody>
</table>
Table 2: Pan-alpha antibody selection. IC50 (ng/mL) determined using the interferon-stimulated response element (ISRE)-Luciferase neutralization assay. Table shows IC50 values of mAbs used in single molecule array assay for all IFN-α subtypes. 10,000 HEK 293 MSR cells were seeded in white half-area 96-well plates and reverse-transfected with 50 ng premixed ISRE-Firefly luciferase reporter and Renilla luciferase constructs using transfection reagents according to the manufacturer's instructions. The luciferase-expressing construct served as an internal normalization control. Cells were incubated overnight in Reduced Serum Medium supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.5% fetal bovine serum at 37 °C, 5% CO2 in a humidified atmosphere. After overnight incubation, cells were stimulated for 24 h with medium containing mixtures of recombinant human IFN-α with or without anti-IFN-α mAbs or control IgG that had been preincubated for 1 h at 37 °C. After 24 h of stimulation, dual luciferase reporter assays were performed according to the manufacturer's instructions. « - » Not determined. Sifalimumab is a fully human, immunoglobulin G1 κ monoclonal antibody that binds to and neutralizes the majority of IFN-α subtypes; Rontalizumab is a humanized monoclonal antibody against IFN-α. Adapted from Meyer et al. 2016 and Rodero et al. 2017.
Supplementary Table 1: Selection of antibody orientation, capture antibody concentration on beads and biotinylation ratio. The two possible different configurations were tested, using the anti-IFN-α A as capture antibody and anti-IFN-α B as detection antibody (A), and vice versa (B). IFNα-2c was used as antigen. Different capture antibody concentrations as well as biotin:antibody (B:A) ratios were also tested for both configurations. Saturation (Sat). Orange: AEB values that were used for LOD calculation; these concentrations were considered blank as the AEB values remained steady. LOD was calculated as Mean Blank + 3SD. <a href="/files/ftp_upload/57421/Supplementary_Table_1.xlsx">Please click here to download this file.</a>
Supplementary Table 2: Test of 2 vs 3-step configuration. The 2 and 3 -step configurations were assessed using IFNα-2c as antigen. AEB values as well as signal/background rations (S/B) are shown for both conditions. <a href="/files/ftp_upload/57421/Supplementary_Table_2.xlsx">Please click here to download this file.</a>
Supplementary Table 3: Detector and SBG concentration optimization. Three different concentrations of biotinylated detector antibody (0.1, 0.3, and 0.6 µg/mL) together with three different concentrations of SBG (50, 150, and 250 pM) were assessed. AEB values are shown for the nine tested conditions. Orange: AEB values that were used for LOD calculation; these concentrations were considered blank as the AEB values remained steady. LOD was calculated as Mean Blank + 3SD. <a href="/files/ftp_upload/57421/Supplementary_Table_3.xlsx">Please click here to download this file.</a>
Supplementary Table 4: Specificity and sensitivity of the optimized IFNα single molecule array assay. Average enzyme per beads values are shown when IFN-β, IFN-λ1, IFN-λ2, IFN-ω, and IFN-γ recombinant proteins were tested (A), together with the 16 subtypes of IFN-α (B). « - » Not determined. <a href="/files/ftp_upload/57421/Supplementary_Table_4.xlsx">Please click here to download this file.</a>
Supplementary Table 5: Reproducibility of the optimized IFNα single molecule array assay. The mean AEB values for all three independent runs are shown up to a concentration of 10.000 fg/mL. « - » Not determined. Saturation (Sat). <a href="/files/ftp_upload/57421/Supplementary_Table_5.xlsx">Please click here to download this file.</a>
Supplementary Table 6: Protein competition assay. Average enzyme per beads values are shown for all conditions tested. Measurements were done in duplicate. <a href="/files/ftp_upload/57421/Supplementary_Table_6.xlsx">Please click here to download this file.</a>

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

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

This method can help answer key questions on the nature, regulation, and biological impact of the interferon induced responses in different disease settings, including auto-immunity and infection. The main advantage of this technique is its own presidented sensitivity. This technique has great potential for biomarker discovery to improve patient management in many diseases as it can quantify cytokines with unprecedented sensitivity.A major step with this approach was to neutralize activities as related to patients with autoimmune polyindocrine system type one. To begin, load 280 million paramagnetic beads into a microfuge tube, taking note of the volume. Then pulse spin the beads and put the tube on a magnetic separator for a minute.Remove and discard the diluant solution thus isolating the beads. Next, wash the beads with BWB twice and BCB twice. For each wash, add 200 microliters and vortex the tube for five seconds.Then separate the beads from the solution using the magnetic separator. After one minute of separation, discard the diluant. Next, add 190 microliters of BCB per bead-volume.Then vortex the tube briefly, pulse spin the tube, and put the washed beads on ice. To activate the beads, combine one milliliter of cold BCB with 10 milligrams of EDC, and vortex until the solution is homogeneous. Work quickly and safely when using EDC, due to it's inherent instability and equinus solution.Then add 10 microliters of cold, diluted EDC per bead-volume to the bead suspension and vortex the beads briefly. Next, put the beads on a shaker at room temperature for 30 minutes. To conjugate antibodies to the beads, first vortex and puls spin the activated beads.Then put the beads in a magnetic separator for a minute and discard the BCB supernatant. Next, remove the tube from the magnet and wash the beads twice with 200 microliters of BCB. Then vortex, pulse spin, and magnetically separate the beads to remove the buffer.Then, quickly add 200 microliters of cold, buffer-exchanged antibody to the activated beads. After vortexing the beads into solution, put the suspension on the room temperature shaker for two hours at 1, 000 RPM. Begin with giving the antibody-coated bead suspension a pulse spin and then use the magnetic column to remove the buffer.Next, check the concentration of the protein in the buffer. Use a low volume spectraphotometer. At this point, the antibodies are coupled to the beads and any value above zero means that the beads are saturated with antibodies.Now, wash the beads with 200 microliters of BWB, as performed in all the previous washes. Then, transfer the supernatant to a new tube and check the protein concentration. Quantification of proteins being released from the beads allows measurement of antibody coupling.Then, wash the beads with 200 microliters of BWB again. Next, add 200 microliters of bead-blocking buffer, vortex the tube for five seconds and transfer it to the room temperature shaker for 30 minutes. After 30 minutes the beads will be blocked.Then, wash them once using 200 microliters of BWB, followed by washing them twice with 200 microliters of bead diluent buffer for each wash. Then, store the coated and blocked beads in 200 microliters of bead diluent buffer at four degrees Celsius. This section of the video suggests strategies for how to tweak the assay conditions using the single molecule array analyzer software in home brew configuration.Begin with testing different combinations of capture antibody conjugated beads and biotinylation detection antibody in both antibody combinations. Next, test combinations of the three different capture antibody concentrations with two different biotinylation ratios for detection and capture antibodies and vice versa. Then choose the combination that offers the lowest level of detection and use it for further steps.In this case, a 30 to one ration of biotin to antibody gives the best limit of detection. Next, compare a two-step configuration with a three-step configuration. Three-step configurations have three different incubation steps.One with the capture antibody, one with the detection antibody, and a final, third one for the SBG enzyme labeling. Two-step configurations combine the capture and the detection incubations. Run the single-molecule array analyzer using the selected capture and detector antibody concentrations and rations in the two and three-step configurations, pre-configured in the analyzer, following the manufacturer's instructions.Then choose the configuration that allows for the highest sensitivity and keep it for future steps. In this case, the two-step configuration gives slightly higher sensitivity at every test point. Next, optimize the concentrations of the detector antibody and the SBG.Test three different concentrations of detector antibody with three different concentrations of SBG. Choose the concentrations that give the highest sensitivity and keep them for future steps. In this case, 0.3 micrograms per milliliter of antibody and 150 pica molar SBG have the optimal level of detection with low background levels with fortuitous medium amplitude.For additional optimization steps, consult the text protocol. Therein are procedures for optimizing assay specificity and reproducibility and an assay for protein competition. Using an assay optimized to detect 13 sub-classes of interferon-alpha, plasma and serum were analyzed in SLE patients, JDM patients, and healthy controls.High levels of interferon-alpha protein were detected in both disease cohorts. The dotted line indicates the level of detection of a conventional, commercially available Eliza which would not detect the interferon-alpha protein in these patient groups, despite the known role of this cytokine in these diseases. After watching this video, you should have a good understanding on how to develop and validate a single molecule array assay for your analyze of choice.While attempting this procedure, it's important to remember that during the cell choice of antibodies is. Since its development, this assay has allowed a better characterization of the role of interferon-alpha in a diversity of auto-immune diseases and infections. This assay has also served to monitor responses to new therapies and to identify the cellular actors responsible for certain auto-immune disorders.

development-validation-an-ultrasensitive-single-molecule-array

Research

JoVE Journal

Immunology and Infection

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays.  Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes.  Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera.  The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 μl volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Detection Method for Influenza Virus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

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

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

This video demonstrates an imaging method to visualize in situ interferon-gamma (IFN-γ) secretion in the mouse spleen. Following bacterial infection and the induction of IFN-γ accumulation inside T cells and natural killer cells, the spleen is harvested from the mouse. Upon obtaining tissue sections, immunostaining is performed to visualize IFN-γ inside the cells.

Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

This video demonstrates secreted embryonic alkaline phosphatase assay using HEK-Blue interferon-α/β reporter cells to quantify the type-I interferons (IFNs) released following stimulation of plasmacytoid dendritic cells by HIV-1 Infected CD4+ T cells.

Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay

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.

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

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

This report describes an automated protocol for multiplex immunofluorescence (mIF) assays on an automated slide stainer. It demonstrates the high reproducibility of spatial proteomics on standard high-throughput tissue autostainers and whole-slide fluorescence imagers, which is ideal for custom mIF assays across a large number of tissue slides in translational research.

High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

Keşfedin tek molekül dijital ELISA yöntemiyle insan IFN-α alt tiplerini ultra hassas şekilde tespit etmeyi. Bu yöntem otoimmün hastalıklarda interferon yanıtlarını anlamak ve biyobelirteç keşfi için güçlü bir araç sunar.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

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