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

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.

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This method can help answer key questions on the nature, regulation, and biological impact of the interferon induced responses in different disease settings, including auto-immunity and infection. The main advantage of this technique is its own presidented sensitivity. This technique has great potential for biomarker discovery to improve patient management in many diseases as it can quantify cytokines with unprecedented sensitivity.

A major step with this approach was to neutralize activities as related to patients with autoimmune polyindocrine system type one. To begin, load 280 million paramagnetic beads into a microfuge tube, taking note of the volume. Then pulse spin the beads and put the tube on a magnetic separator for a minute.

Remove and discard the diluant solution thus isolating the beads. Next, wash the beads with BWB twice and BCB twice. For each wash, add 200 microliters and vortex the tube for five seconds.

Then separate the beads from the solution using the magnetic separator. After one minute of separation, discard the diluant. Next, add 190 microliters of BCB per bead-volume.

Then vortex the tube briefly, pulse spin the tube, and put the washed beads on ice. To activate the beads, combine one milliliter of cold BCB with 10 milligrams of EDC, and vortex until the solution is homogeneous. Work quickly and safely when using EDC, due to it's inherent instability and equinus solution.

Then add 10 microliters of cold, diluted EDC per bead-volume to the bead suspension and vortex the beads briefly. Next, put the beads on a shaker at room temperature for 30 minutes. To conjugate antibodies to the beads, first vortex and puls spin the activated beads.

Then put the beads in a magnetic separator for a minute and discard the BCB supernatant. Next, remove the tube from the magnet and wash the beads twice with 200 microliters of BCB. Then vortex, pulse spin, and magnetically separate the beads to remove the buffer.

Then, quickly add 200 microliters of cold, buffer-exchanged antibody to the activated beads. After vortexing the beads into solution, put the suspension on the room temperature shaker for two hours at 1, 000 RPM. Begin with giving the antibody-coated bead suspension a pulse spin and then use the magnetic column to remove the buffer.

Next, check the concentration of the protein in the buffer. Use a low volume spectraphotometer. At this point, the antibodies are coupled to the beads and any value above zero means that the beads are saturated with antibodies.

Now, wash the beads with 200 microliters of BWB, as performed in all the previous washes. Then, transfer the supernatant to a new tube and check the protein concentration. Quantification of proteins being released from the beads allows measurement of antibody coupling.

Then, wash the beads with 200 microliters of BWB again. Next, add 200 microliters of bead-blocking buffer, vortex the tube for five seconds and transfer it to the room temperature shaker for 30 minutes. After 30 minutes the beads will be blocked.

Then, wash them once using 200 microliters of BWB, followed by washing them twice with 200 microliters of bead diluent buffer for each wash. Then, store the coated and blocked beads in 200 microliters of bead diluent buffer at four degrees Celsius. This section of the video suggests strategies for how to tweak the assay conditions using the single molecule array analyzer software in home brew configuration.

Begin with testing different combinations of capture antibody conjugated beads and biotinylation detection antibody in both antibody combinations. Next, test combinations of the three different capture antibody concentrations with two different biotinylation ratios for detection and capture antibodies and vice versa. Then choose the combination that offers the lowest level of detection and use it for further steps.

In this case, a 30 to one ration of biotin to antibody gives the best limit of detection. Next, compare a two-step configuration with a three-step configuration. Three-step configurations have three different incubation steps.

One with the capture antibody, one with the detection antibody, and a final, third one for the SBG enzyme labeling. Two-step configurations combine the capture and the detection incubations. Run the single-molecule array analyzer using the selected capture and detector antibody concentrations and rations in the two and three-step configurations, pre-configured in the analyzer, following the manufacturer's instructions.

Then choose the configuration that allows for the highest sensitivity and keep it for future steps. In this case, the two-step configuration gives slightly higher sensitivity at every test point. Next, optimize the concentrations of the detector antibody and the SBG.

Test three different concentrations of detector antibody with three different concentrations of SBG. Choose the concentrations that give the highest sensitivity and keep them for future steps. In this case, 0.3 micrograms per milliliter of antibody and 150 pica molar SBG have the optimal level of detection with low background levels with fortuitous medium amplitude.

For additional optimization steps, consult the text protocol. Therein are procedures for optimizing assay specificity and reproducibility and an assay for protein competition. Using an assay optimized to detect 13 sub-classes of interferon-alpha, plasma and serum were analyzed in SLE patients, JDM patients, and healthy controls.

High levels of interferon-alpha protein were detected in both disease cohorts. The dotted line indicates the level of detection of a conventional, commercially available Eliza which would not detect the interferon-alpha protein in these patient groups, despite the known role of this cytokine in these diseases. After watching this video, you should have a good understanding on how to develop and validate a single molecule array assay for your analyze of choice.

While attempting this procedure, it's important to remember that during the cell choice of antibodies is. Since its development, this assay has allowed a better characterization of the role of interferon-alpha in a diversity of auto-immune diseases and infections. This assay has also served to monitor responses to new therapies and to identify the cellular actors responsible for certain auto-immune disorders.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

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

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