Name: development and validation of an ultrasensitive single molecule array digital enzyme-linked immunosorbent assay for human interferon-α
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Description: Watch this Scientific Journal Video about Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-Î± at JoVE.com

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.

<ol>
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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;

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-&#945; 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-&#945; A antibody and anti-IFN-&#945; 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 &#181;g/mL and 150 pM, respectively. This highly specific assay is capable of detecting all 13 IFN-&#945; 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-&#945;, all of them can be detected and measured together giving a total concentration value for IFN-&#945;, that comprises the 13 different subclasses.
To explore the potential diagnostic value of this newly developed assay, IFN-&#945; 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-&#945; 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-&#945; protein in these patient groups despite the known associations of this cytokine with these phenotypes. Furthermore, IFN-&#945; 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="Figure 7" class="xfigimg" src="/files/ftp_upload/57421/57421fig7.jpg" /> 
Figure 7: Quantification of IFN-&#945; 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-IFN&#945; single molecule array assay. Analysis was performed using one-way ANOVA test (Kruskal-Wallis) and Dunn&#39;s multiple comparison testing between groups. Median is depicted. Dotted line indicates LOD of a reference human anti-IFN-&#945; 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>IFN&#945;1</td>
			<td>28.3</td>
			<td>51.3</td>
			<td>460</td>
			<td>22.63</td>
		</tr>
		<tr>
			<td>IFN&#945;2</td>
			<td>2563</td>
			<td>10.7</td>
			<td>9.02</td>
			<td>2.15</td>
		</tr>
		<tr>
			<td>IFN&#945;4</td>
			<td>5.11</td>
			<td>2.99</td>
			<td>35.35</td>
			<td>325.3</td>
		</tr>
		<tr>
			<td>IFN&#945;5</td>
			<td>2.01</td>
			<td>19.6</td>
			<td>93.29</td>
			<td>2.49</td>
		</tr>
		<tr>
			<td>IFN&#945;6</td>
			<td>64.7</td>
			<td>3.03</td>
			<td>4.97</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFN&#945;7</td>
			<td>0.9</td>
			<td>0.63</td>
			<td>233.1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFN&#945;8</td>
			<td>302</td>
			<td>0.83</td>
			<td>691</td>
			<td>10.86</td>
		</tr>
		<tr>
			<td>IFN&#945;10</td>
			<td>2.54</td>
			<td>0.71</td>
			<td>43.2</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IFN&#945;14</td>
			<td>3224</td>
			<td>2.1</td>
			<td>14.52</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>IFN&#945;16</td>
			<td>1.83</td>
			<td>32.99</td>
			<td>59.18</td>
			<td>28.65</td>
		</tr>
		<tr>
			<td>IFN&#945;17</td>
			<td>2.21</td>
			<td>0.77</td>
			<td>890.9</td>
			<td>23.86</td>
		</tr>
		<tr>
			<td>IFN&#945;21</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-&#945; 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&#39;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 &#176;C, 5% CO2 in a humidified atmosphere. After overnight incubation, cells were stimulated for 24 h with medium containing mixtures of recombinant human IFN-&#945; with or without anti-IFN-&#945; mAbs or control IgG that had been preincubated for 1 h at 37 &#176;C. After 24 h of stimulation, dual luciferase reporter assays were performed according to the manufacturer&#39;s instructions. &#171; - &#187; Not determined. Sifalimumab is a fully human, immunoglobulin G1 &#954; monoclonal antibody that binds to and neutralizes the majority of IFN-&#945; subtypes; Rontalizumab is a humanized monoclonal antibody against IFN-&#945;. 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-&#945; A as capture antibody and anti-IFN-&#945; B as detection antibody (A), and vice versa (B). IFN&#945;-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 IFN&#945;-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 &#181;g/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 IFN&#945; single molecule array assay. Average enzyme per beads values are shown when IFN-&#946;, IFN-&#955;1, IFN-&#955;2, IFN-&#969;, and IFN-&#947; recombinant proteins were tested (A), together with the 16 subtypes of IFN-&#945; (B). &#171; - &#187; 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 IFN&#945; single molecule array assay. The mean AEB values for all three independent runs are shown up to a concentration of 10.000 fg/mL. &#171; - &#187; 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>

Watch this Scientific Journal Video about Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-Î± at JoVE.com

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

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 ...

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

Research

JoVE Journal

Immunology and Infection

11.9K Views.  Institut Pasteur. 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-α subtypes in human samples.

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

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Chemistry

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

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 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Biochemistry

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;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.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Biology

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Medicine

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

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

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.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

ELISA Assays: Indirect, Sandwich, and Competitive

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4 
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Enzyme-linked immunosorbent assay (ELISA) is frequently used to measure the presence and/or concentration of an antigen, antibody, peptide, protein, hormone, or other biomolecule in a biological sample. It is extremely sensitive, capable of detecting low antigen concentrations. The sensitivity of ELISA is attributed to its ability to detect the interactions between a single antigen-antibody complex (1). Moreover, the inclusion of an enzyme-conjugated antigen-specific antibody permits the conversion of a colorless substrate into a chromogenic or fluorescent product that can be detected and easily quantitated by a plate reader. When compared to the values generated by titrated amounts of a known antigen of interest, the concentration of the same antigen in the experimental samples can be determined. Different ELISA protocols have been adapted to measure antigen concentrations in a variety of experimental samples, but they all have the same basic concept (2). Choosing the type of ELISA to perform, indirect, sandwich, or competitive, depends on a number of factors, including the complexity of the samples to be tested and the antigen-specific antibodies available to use. The indirect ELISA is frequently used to determine the outcome of an immunological response, such as measuring the concentration of an antibody in a sample. The sandwich ELISA is best suited for analyzing complex samples, such as tissue culture supernatants or tissue lysates, where the analyte, or antigen of interest, is part of a mixed sample. Finally, the competitive ELISA is most often used when there is only one antibody available to detect the antigen of interest. Competitive ELISAs are also useful for detecting a small antigen with only a single antibody epitope that cannot accommodate two different antibodies due to steric hinderance. The protocol will describe the basic procedures for the indirect, sandwich, and competitive ELISA assays.
The indirect ELISA assay is commonly used to measure the amount of antibodies in serum or in the supernatant of a hybridoma culture. The general procedure for the indirect ELISA assay is:
<ol>
	<li>Coat wells with antigens</li>
	<li>Add serum or hybridoma culture supernatant containing antibody (primary or 1&#176; antibody)</li>
	<li>Incubate and wash</li>
	<li>Add secondary (or 2&#176;) enzyme-conjugated antibody</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
The sandwich ELISA assay differs from the indirect ELISA assay in that the method does not involve coating the plates with a purified antigen. Instead, a &#34;capture&#34; antibody is used to coat the wells of the plate. The antigen is &#34;sandwiched&#34; between the capture antibody and a second &#34;detection&#34; enzyme-conjugated antibody - where both antibodies are specific for the same antigen, but at different epitopes (3). By binding to the capture antibody/antigen complex, the detection antibody remains in the plate. Either monoclonal antibodies or polyclonal antisera can be used as the capture and detection antibodies. The main advantage of the sandwich ELISA is that the sample does not have to be purified before analysis. Moreover, the assay can be quite sensitive (4). Many commercially available ELISA kits are of the sandwich variety and use tested, matched pairs of antibodies. The general procedure for the sandwich ELISA assay is:
<ol>
	<li>Coat wells with capture antibody</li>
	<li>Add test samples containing antigen</li>
	<li>Incubate and wash</li>
	<li>Add enzyme-conjugated detection antibody.</li>
	<li>Incubate and wash</li>
	<li>Add substrate</li>
</ol>
Most commercially available sandwich ELISA kits come with enzyme-conjugated detection antibodies. In cases where an enzyme-conjugated detection antibody is not available, a secondary enzyme-conjugated antibody specific for the detection antibody can be used. The enzyme on the secondary antibody performs the same role, which is to convert the colorless substrate to a chromogenic or fluorescent product. The above-mentioned secondary enzyme-conjugated antibody would more like to be used in a &#34;homemade&#34; sandwich ELISA developed by an investigator who has generated their own monoclonal antibodies, for example. One drawback to using a secondary enzyme-conjugated antibody is to be sure it only binds to the detection antibody, and not the capture antibody bound to the plate. This would result in a measurable product in all wells, regardless of the presence or absence of antigen or detection antibody.
Finally, the competitive ELISA assay is used to detect soluble antigens. It is simple to perform, but it is only suitable when the purified antigen is available in a relatively large amount. The general procedure for the competitive ELISA assay is:
<ol>
	<li>Coat wells with antigen</li>
	<li>Incubate and wash</li>
	<li>Preincubate test sample with enzyme-conjugated primary antibodies</li>
	<li>Add mixture to well</li>
	<li>Incubate and wash away any unbound enzyme-conjugated primary antibody</li>
	<li>Add substrate</li>
</ol>
The &#34;competition&#34; in this assay comes from the fact that more antigen in the test sample used in step 3 will result in less antibody available to bind to the antigen coating the well. Thus, the intensity of the chromogenic/fluorogenic product in the well at the end of the assay is inversely related to the amount of antigen present in the test sample.
A key component in any type of ELISA is the titrated standards of known concentrations that will allow the user to determine the antigen concentration present in the test samples. Typically, a series of wells are designated for creating a standard curve, where known amounts of a purified recombinant protein are added to the wells in decreasing amounts. When these wells are processed at the same time as the test samples, the user can then have a reference set of absorbance values obtained from a microplate reader for known protein concentrations to go along with the absorbance values for the test samples. The user can then calculate a standard curve to which the test samples can be compared for determining the amount of protein of interest present. The standard curve can also determine the degree of precision of the user&#39;s dilution making.
Finally, the last step in each of the ELISA types listed above calls for the addition of a substrate. The degree of conversion of the substrate to product is directly related to the amount of enzyme present in the well. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes found conjugated to antibodies. As expected, there are a number of substrates available specific for either enzyme that produce a chromogenic or fluorescent product. Moreover, substrates are available in a range of sensitivities that can increase the overall sensitivity of the assay. The user must also take into consideration the type of instrumentation available for reading the plate at the end of the experiment when picking the type of substrate to use, along with its corresponding enzyme-conjugated antibody.
Commonly used chromogenic substrates for HRP include 2,2&#39;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) and 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB), whereas p-Nitrophenyl Phosphate (PNPP) is used for AP. ABTS and TMB produce water-soluble green and blue colored reaction products, respectively. The green ABTS product has two major absorbance peaks, 410 and 650 nm, while the blue TMB product is best detected at 370 and 652 nm. The colors of ABTS and TMB change to yellow upon the addition of an acidic stop solution, which is best read at 450 nm. Color development for ABTS is slow, while it is fast for TMB. TMB is more sensitive than ABTS, and may produce a higher background signal if the enzymatic reaction proceeds too long. PNPP produces a yellow water-soluble product after AP conversion that absorbs light at 405 nm.

Source: Whitney Swanson1,2, Frances V. Sjaastad2,3, and Thomas S. Griffith1,2,3,4
1 Department of Urology, University of Minnesota, Minneapolis, MN 55455
...

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Immunology

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

Source: Bego, M. G. et al., <a href="https://app.jove.com/v/51207">Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.</a> J. Vis. Exp. (2015)
This video demonstrates secreted embryonic alkaline phosphatase assay using HEK-Blue interferon-&#945;/&#946; reporter cells to quantify the type-I interferons (IFNs) released following stimulation of plasmacytoid dendritic cells by HIV-1 Infected CD4+ T cells.

Source: Bego, M. G. et al., Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture ...

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

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