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

1. Selection of Antibodies

1. Before beginning the protocol review all key steps (Table 1) and select optimum antibodies.
   NOTE: For this protocol high affinity anti-IFN-α antibodies – the IC50 of which are described in Table 2 were selected. Preferentially, choose a pair that works well with conventional ELISA.

2. Conjugation of Capture Antibody to Paramagnetic Beads and Testing of Different Concentrations

NOTE: Always keep Bead Conjugation Buffer (BCB) and Bead Wash Buffer (BWB) on the ice during the antibody conjugation process.

1. Capture Antibody Buffer Exchange
   1. Take initial quantities of anti-IFN-α antibody A to test 3 different bead conjugations ratios (0.038 mg/mL, 0.075 mg/mL, and 0.3 mg/mL).
      NOTE: Low antibody coating concentrations can be beneficial if the assay presents a high background signal. Initial antibody quantity should account for an estimated 30% loss during the buffer exchange process. According to manufacturer's instructions and in order to reduce initial background, concentrations of 0.05 mg/mL, 0.1 mg/mL, and 0.3 mg/mL were tested, although these exact values were often not obtained due to the aforementioned variable loss in the buffer exchange process.
   2. Add the required volume of the antibody into a filter column together with BCB, up to 500 µL.
   3. Centrifuge the columns at >10,000 x g for 5 min and discard the flow-through.
   4. Wash the column twice by adding a total volume of 450 µL of BCB followed by centrifugation at >10,000 x g for 5 min and discard the flow through.
   5. Place the filter column containing the antibody into a clean microcentrifuge tube upside down – the open part of the column facing the inside of the new tube – and centrifuge at 800 x g for 1 min.
      NOTE: This step will allow collection of the cleaned antibody. No buffer addition is required for this step.
   6. Wash the membranes with 50 µL BCB and centrifuge at 800 x g for 1 min as in 2.1.5.
   7. Measure the concentration of antibody using a low volume spectrophotometer.
   8. Add BCB to achieve the desired antibody concentration and note the final volume.
      NOTE: A volume of 200 µL will be used to describe the rest of the protocol, this volume being referred to as 2 volumes (2V). All the following reagent volumes will be adapted accordingly.
   9. Vortex and keep on ice.
2. Paramagnetic Bead Preparation
   1. Transfer 2.8 x 10⁸ beads (100 µL = 1V) into a microfuge tube.
      NOTE: The volume of beads is dependent on the final volume obtained in 2.1.8.
   2. Pulse spin and put in a magnetic separator for 1 min, discard diluent, and remove from magnet.
   3. Add 2V of BWB and vortex for 5 s.
   4. Repeat 2.2.2 and 2.2.3 two times with BWB and two more times with BCB.
   5. For 1V of beads add 190 µL BCB, vortex 5 s, pulse spin, and store the washed beads on ice.
3. Bead Activation
   1. Add 1 mL of cold BCB to a 10-mg vial of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and vortex until the solution is homogeneous.
   2. For 1V of beads add 10 µL of cold diluted EDC to the washed beads, vortex, and keep in a shaker at 1,000 rpm for 30 min at room temperature.
      NOTE: EDC preparation and addition to the beads should be done as quickly as possible due to EDC instability in aqueous solutions. Also, as EDC is highly reactive, it is important to have a homogenous bead suspension before EDC addition in order to prevent heterogeneous bead activation. After EDC addition, beads must be kept in suspension.
4. Bead Conjugation of the Antibody
   1. Vortex and pulse spin the activated beads. Now put the beads in a magnetic separator for 1 min, discard BCB supernatant, and remove the tube from the magnet.
   2. Wash the beads with 2V of BCB. Vortex, pulse spin, and put in the magnetic separator for 1 min. Then discard the BCB buffer and remove the beads from the magnet.
   3. Quickly add the cold, buffer-exchanged 200 µL (2V) of antibody to the activated beads.
   4. Vortex and keep for 2 h in the shaker at room temperature.
5. Bead Blocking and Final Clean-up
   1. Pulse spin the antibody-coated beads suspension, put in the magnetic separator for 1 min, transfer the supernatant to a new tube, and measure the concentration with a low volume spectrophotometer.
      NOTE: This step will provide information on whether the beads are saturated – any values above zero will indicate bead saturation – as detection of soluble antibody unable to bind to the beads will be measurable.
   2. Remove the conjugated beads from the magnet, add 2V of BWB, vortex for 5 s, pulse spin, and put in the magnetic separator for 1 min.
   3. Transfer the supernatant to a new tube and measure the concentration using a low volume spectrophotometer.
      NOTE: This step will provide information on whether the antibodies are stably fixed to the beads. Detectable antibody concentration in this supernatant will indicate detachment of the antibody from the beads, which happens regularly. This assay often works even when very small amounts of capture antibody remain attached to the beads.
   4. Remove the conjugated beads from the magnet, add 2V of BWB, vortex for 5 s, pulse spin, and put in the magnetic separator for 1 min.
   5. Remove conjugated beads from the magnet, add 2V of Bead Blocking Buffer, vortex for 5 s, and incubate in the shaker for 30 min at room temperature.
   6. Wash the antibody-coated and blocked beads 3x with 2V, once with BWS, and 2x with Bead Diluent Buffer (discard all supernatants).
   7. Store the coated and blocked beads at 4°C until use with 2V of Bead Diluent Buffer.
      NOTE: Just before use, beads must be washed once with Detector/Sample Diluent using a volume of 250 x Bead volume/20, using the magnetic separator.

3. Detection Antibody Biotinylation

1. Detection Antibody Buffer Exchange
   1. Take 260 µg of the two anti-IFN-α Ab clones.
      NOTE: This takes into consideration a 30% loss during buffer exchange and allows testing of two different biotin/antibody ratios.
   2. Proceed as 2.1 using Biotinylation Reaction Buffer (BRB) instead of BCB.
   3. Measure the volume and the antibody concentrations and adjust the volume to obtain a final concentration of 1 mg/mL.
      NOTE: This is a suggested starting concentration, but it can be optimized if the assay does not achieve the required sensitivity.
2. Detection Antibody Biotinylation
   1. Bring the N-hydroxysuccinimide-polyethylene-glycol4-biotin (NHS-PEG4-Biotin) to room temperature and resuspend with a total of 400 µL of distilled water to achieve a 3.4 mM concentration.
   2. Decide the biotin:antibody ratio to test and mix detection antibody and diluted biotin accordingly (60:1 ratio equals 100 µL of detection antibody and 4.5 µL of biotin).
      NOTE: To begin, test 30:1 and 60:1 ratios.
   3. Vortex the antibody-biotin mix, pulse spin and incubate for 30 min at room temperature.
      NOTE: No need to shake.
3. Biotinylated Detection Antibody Purification
   1. Transfer the biotinylated antibody to a new filter and proceed as 2.1, performing 3 washing steps with BRB (400, 450, and 450 µL) and a final collection.
   2. Measure the volume and concentration of the purified, biotinylated detection antibody and store at 4 °C until use.

4. Assay Optimization

NOTE: Use of the single molecule array analyzer software in a Homebrew configuration³⁰.
The single molecule array analyzer machine is controlled by software that allows to run assay standardizations, to perform sample quantifications, to modify external parameters (bead, detector, SBG, RGP concentrations; sample dilutions…) and internal parameters (number of steps, incubation times…) to achieve optimal assay conditions, and can also be used to calculate results (background, LOD, quantities). For the setup of the single molecule array analyzer and to calculate the reagent volumes required for each round of optimization, see manufacturer's instructions. Perform the first rounds of optimization using a 2-step configuration.

1. Test combinations of capture antibody-conjugated beads and biotinylated detection antibody in both configurations.
   1. Test combinations of the three different anti-IFN-α A capture antibody concentrations with the two different anti-IFN-α B biotinylation ratios as detection and capture antibodies, and vice versa (Figure 1) by selection of the appropriate conditions in the analyzer software.
   2. Choose the most sensitive combination, that is, the conditions that offer the lowest LOD, and use it for further steps
      NOTE: Figure 1 shows how a 0.3 mg/mL anti-IFN-α A antibody bead concentration and a biotin:antibody ratio of 30:1 with the anti-IFN-α B antibody resulted in the highest sensitivity, as evidenced by an LOD of 11.6 fg/mL. (See Supplementary Table 1). This combination will be used for all future steps. Notice that the great sensitivity achieved with this particular assay before any rounds of optimization.

Figure 1: Selection of antibody orientation, capture antibody concentration on beads and biotinylation ratio. The two possible different configurations were tested, using the anti-IFN-α A as capture antibody and anti-IFN-α B as detection antibody (A), and vice versa (B). IFNα-2c was used as antigen. Different capture antibody concentrations as well as biotin:antibody (B:A) ratios were also tested for both configurations. Dotted line shows LOD for the best condition; anti-IFN-α A 0.3 mg/mL as capture and anti-IFN-α B biotin:detector antibody ratio of 30 (LOD = 11.6 mg/mL corresponding to an AEB value of 0.017265). A 2-step configuration was used. Biotin:antibody (B:A). Please click here to view a larger version of this figure.

2. Compare a 2 vs. 3-step configuration.
   NOTE: In a 3-step configuration, there are three different incubation steps, one with the capture antibody, one with the detection antibody and a final third one for the SBG enzyme labeling. However, in a 2-step configuration, capture and detection antibodies are incubated simultaneously with the analyte of interest.
   1. Run the Single molecule array analyzer with the capture and detector antibody concentration and ratios from 4.1.2 selecting the 2 and 3-step configurations pre-configured in the analyser, following manufacturer's instructions³⁰.
   2. Choose the configuration that allows for the highest sensitivity and keep it for future steps
      NOTE: As shown in Figure 2 and Supplementary Table 2, the 2-step configuration gave slightly higher sensitivity and signal/background ratio for every concentration tested. Therefore, the 2-step configuration was kept for future steps.

Figure 2: 2 vs 3-step configuration comparison. The 2 and 3-step configurations were assessed using 0.3 mg/mL of anti-IFN-α A antibody as capture and anti-IFN-α B as detection antibody in a 30 biotin:antibody ratio. IFNα-2c was used as antigen. In a 3-step condition setting, there are three different incubation steps, one with the capture antibody, one with the detection antibody and a final third one for the SBG enzyme labelling. However, in a 2-step configuration, capture and detection antibodies are incubated simultaneously with the analyte of interest. Please click here to view a larger version of this figure.

3. Detector Antibody and SBG concentration optimization
   1. Test three different concentrations of detector antibody (0.1, 0.3, and 0.6 µg/mL) together with three different concentrations of SBG (50, 150, and 250 pM) as described above³⁰.
   2. Choose the concentrations that gives the highest sensitivity and keep them for future steps.
      NOTE: Figure 3 shows that detector 0.3 µg/mL and SBG 150 pM were the conditions that showed the optimal LOD. These conditions also gave low background levels and medium amplitude, a behavior that produces good standard deviation (SD) values (Supplementary Table 3). Typical concentrations range between 0.1 – 1 µg/mL for the detection antibody and 50 – 200 pM for SBG, although higher concentrations (e.g. up to 300 pM) of the latter might be required. It is important to avoid conditions that will render backgrounds higher than 0.02 AEB. Increasing the concentration of the detector monoclonal antibody (mAb) or SBG will enhance both signal response and background. However, if the increment in signal surmounts the change in background, the LOD will improve. A situation can arise in which a decrease in background allows better discrimination between low calibrators.

Figure 3: Detector and SBG concentration optimization. Three different concentrations of biotinylated detector antibody (0.1, 0.3, and 0.6 µg/ml) together with three different concentrations of SBG (50, 150, and 250 pM) were assessed. Dotted line shows LOD for the best condition; detector antibody at 0.3 mg/mL and SBG 150pM (LOD = 0.09 mg/mL corresponding to an AEB value of 0.017184). Please click here to view a larger version of this figure.

4. Additional optimization steps
   1. If the required sensitivity has not been achieved, test different incubation times for the different steps using the preconfigured options in the analyzer software.
   2. If the assay is not sensitivity enough, optimize the number of beads per assay using the preconfigured options in the analyzer software.
      NOTE: Once testing of biological samples begins, sample volume is an additional parameter susceptible to optimization. Make sure that samples are handled according to the health and safety procedures.

5. Assay Specificity and Reproducibility

1. Test IFN-α assay specificity, by testing the assay with different IFN-α subtypes and other related proteins (Figure 4a and 4b).

Figure 4: Specificity and sensitivity of the optimized IFNα single molecule array assay. (A) IFN-β, IFN-λ1, IFN-λ2, IFN-ω, and IFN-γ recombinant proteins were tested, together with (B) 16 subtypes of IFN-α, including three IFN-α2 types (IFN-α2a, IFN-α2b, and IFN-α2c). Adapted from Rodero et al. 2017. Please click here to view a larger version of this figure.

2. Assess the reproducibility of the optimized assay by performing three independent replicate experiments and assessing inter-assay variability (Figure 5).
3. Calculate the LOD of the assay
   NOTE: The LOD was calculated using the mean of the three blanks + 3 standard deviations (SD), thus obtaining a value of 0.23 fg/mL. As the standard sample dilution factor is 1:3, the inclusion of the dilution factor gives an LOD of 0.69 fg/mL.

Figure 5: Reproducibility of the optimized pan-IFN-α single molecule array assay. Three independent runs were performed using two different lots of beads. For the second lot, two independent experiments were performed. Each measurement was acquired in duplicates. The dotted line represents the LOD, defined by mean blank average enzyme per bead + SD of all runs. IFN-α17 was used as a reference, taking into account that the derived standard curve is representative of all other IFN-α subtypes, as shown in Figure 4b. Plot shows mean value and SD (error bars). Dotted lines show LOD for the different runs; Run 1: 0.073 fg/mL AEB = 0.006238, Run 2: 0.113 fg/mL AEB = 0.007119, Run 3: 0.043 fg/mL AEB = 0.05518). Adapted from Rodero et al. 2017. Please click here to view a larger version of this figure.

6. Protein Competition Assay

1. Perform a protein competition assay (described in Figure 6 legend) to further demonstrate the specificity of the assay.
   NOTE: Plasma samples from patients with SLE (n = 5) were used.
   1. When viruses are suspected to be present in the biological sample, prepare inactivation buffer by adding 200 µL of a non-ioinic surfactant (0.5% nonidet P40 substitute) to 40 mL Detector/Sample diluent, and vortex vigorously.
   2. Centrifuge biological samples containing the analyte of interest at 10,000 g for 15 min at 4 °C to remove cellular debris.
   3. Mix 185 µL Detector/Sample diluent or inactivation buffer with 100 µL biological sample (final dilution factor 3) and 15 µL anti-IFN-α antibody A (initial concentration 1 mg/mL, final concentration 50 ug/mL) or buffer. Incubate for 30 min at room temperature.
   4. Run samples with and without anti-IFN-α antibody in the single molecule array analyser as described above.
      NOTE: In case of signal saturation, samples should be further diluted. Also, 300 µL is the volume required for duplicate analysis.

Figure 6: Protein competition assay. Competition experiments were performed using samples from five different SLE patients. Biological samples were centrifuged at 10.000 g for 15 min at 4 °C. They were diluted 1/3 with Detector/Sample diluent containing NP40 for viral inactivation. 15µl of anti-IFN-α antibody A (initial concentration 1 mg/mL, final concentration 50 µg/mL) or buffer were added to a total volume of 300 µL. Incubation was performed at room temperature for 30 min. Control group is shown in grey and samples treated with anti-IFN-α A antibody are shown in black. Graph shows mean value and SD (error bars). Dotted line represents LOD (AEB = 0.024774, 0.8037 fg/mL). Adapted from Rodero et al. 2017 Please click here to view a larger version of this figure.