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

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

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.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

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, ...

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

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

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.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

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 ...

Medicine

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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