Measuring Interleukin-6 in Human Serum from Children with a Pediatric Concussion using a Single Molecule Counting Platform

Summary

Methods are described for ultrasensitive quantification of biomarker proteins using an immunoassay system capable of single molecule counting. The platform can be used to quantify a wide array of proteins in a variety of tissues from several species. This exemplar assays interleukin-6 in human serum from pediatric concussion patients.

Abstract

Efforts to expand the precision science knowledge base and promote translation of findings to clinical care remain an important area of ongoing inquiry. Part of this effort includes quantification of proteins that can be used as biological markers (i.e., biomarkers) of pathophysiological processes and other important cellular and tissue activities. Potential applications for biological markers include disease or injury diagnosis, symptom prognosis, and therapy selection/evaluation. The increased understanding of the current and future utility of protein biomarkers, combined with the realization that many biomarkers normally exist in very low levels, has prompted efforts to develop new protein quantification systems with enhanced sensitivity, improved workflows, and shorter read times. Here, we provide an overview of an ultrasensitive immunoassay system and compatible interleukin-6 (IL-6) assays. This assay platform is bead-based, like many other commercially available systems; however, rather than quantifying the fluorescent signal in the spectral address of bead regions, the ultrasensitive immunoassay system quantifies free-floating fluorochromes using a rotating laser, charge-coupled device (CCD) camera, and avalanche photodiode (APD). This high-performance system can be used to quantify a myriad of protein biomarkers, in a variety of biological specimens collected from many species. In this article, quantification of IL-6 in human serum obtained from pediatric brain injury patients will be used as an exemplar.

Introduction

The benefits of precision care initiatives are becoming increasingly appreciated, and efforts to expand current clinical applications are underway. This increased interest is fueling inquiry into protein biomarkers that have the potential to provide important insights into physiological processes inside the body that can inform clinical care. Current applications for protein biomarkers include diagnosing conditions¹, selecting appropriate therapies², and tracking response to treatment². These and other precision care successes have led to an expansion in the number and nature of biomarkers being explored. Many putative biomarkers of interest naturally exist at levels below the limit of detection of standard methods for assaying proteins. Likewise, the relatively slow translation of biomarkers to clinical care and large number of candidate biomarkers in the validation pipeline highlights the need for improved throughput of technologies used for biomarker analysis³. These deficiencies have motivated efforts to develop new methodologies that offer ultra-sensitivity, improved workflow, and other advantages over traditional methods. The purpose of this article is to provide a protocol for using a bead-based single molecule counting technology to measure IL-6 in serum.

Traditional protein quantification methods such as western blot, enzyme-linked immunosorbent assay (ELISA), liquid chromatography, mass spectroscopy, and proprietary polymerase chain reaction (PCR) assays , are characterized by sensitivities in the nanogram (ng; 10^-9) or picogram (pg; 10^-12) per milliliter (mL) range, restricting detection of proteins at or below the femtogram (fg; 10^-15) per mL range^4,5,6,7,8. However, many proteins that may provide useful windows into important physiological processes (e.g., cytokine signaling) are low-abundance with endogenous levels falling below the detection limit of conventional protein analysis techniques^9,10. Emerging new platforms are improving sensitivity through the development of single molecule counting technologies^11,12,13. Ultrasensitive systems facilitate reduced signal-to-noise ratios and, most importantly, detection of concentrations as low as the fg/mL range. The platform is unique from other systems with respect to how it counts the single molecules. During the proprietary elution step, the fluorescent dye-labeled detection antibodies are dissociated from the immunocomplex, resulting in free-floating (i.e., in suspension) fluorochromes. A rotating laser is focused through a high-numeric aperture objective to a diameter less than 4 µm. Emitted light is captured by the same objective then passed through a series of positioned mirrors and a long-pass dichroic filter. The image is captured using a charge-coupled device (CCD) camera and individual photons are counted by an Avalanche Photodiode (APD). All digital events, defined as six standard deviations above a background threshold, are counted using a digital event algorithm across a spectrum of time series for a given standard curve; the ultimate result is highly sensitive and reproducible biomarker quantification. Other technologies exist that that allow for ultrasensitive protein quantification. The first ultrasensitive system on the market was the predecessor to the system described in this manuscript; it is a flow cytometry-based instrument that detects Alex 647-labeled detection antibody-analyte complexes in solution. The solution is aspirated into the instrument and is irradiated by a red laser that is focused through a confocal lens. Read times are more than 11 hours per 384-well plate, due to the time-consuming nature of the aspiration/detection method. Another company then launched fully automated system capable of detecting in the fg/mL. Despite the automation and perceived ease-of-use, the monthly cost of maintenance and initial procurement cost may be prohibitive, especially in academic laboratories.

The digital algorithm described above also addresses the limitation of many instruments with respect to dynamic range, especially for high concentrations^14,15. A consequence of the narrow dynamic range characteristic of conventional ELISA and other common protein quantification platforms is the reduced utility for certain clinical populations and/or a need for special preparation (e.g., multiple sample dilutions) for cases and controls. Eliminating variable sample dilutions both reduces employee workload and minimizes the effects of dilution as a potential source of error^16,17,18.

The device itself is relatively small (16” high x 14” wide x 17.5” deep / ~55 pounds) and does not require fluidics; these features make the system portable and easy to maintain. The system’s workflow is designed to improve the speed of data acquisition through automation, options to save templates, and faster sample reads via use of early-termination technology. The typical time to prepare the assay (including incubations) is approximately 3.5 h for the IL-6 kit, but may be longer for other proteins if overnight incubation is recommended. The read time is approximately 1-3 h for a 384 well plate (vs. up to 13 h for other platforms); the exact time of the assay depends on the concentration of the analyte of interest with faster reads occurring at higher concentrations. The system is able to run both bead-based and plate-based assays and sample volume requirements are as low as 5 μL (average of 10-25 μL for pre-clinical samples and 10-100μL for clinical samples). If desired, the plates can be re-assayed, because the dye that is eluted off the immunocomplex is stable if stored at 4 °C. Moreover, the system offers the option to use an optimized serum matrix to produce a standard curve that more closely simulates native levels of the analyte in serum/plasma. The inclusion of optimized serum matrix is unique and is in agreement with guidelines for immunoassays outlined by the American Association of Pharmaceutical Sciences¹⁹.

The system’s primary limitation is that debris/bubbles in the sample or scratches/damage to the underside of the plate can interfere with signal acquisition and adversely affect results, due to the ultrasensitive nature of the instrument. However, when a clean workspace is maintained and care in preparation of samples and handling of plates is exercised, extremely low levels of proteins can be precisely, accurately, and reproducibly quantified. Likewise, other manageable limitations common to all protein quantification methods (e.g., effects of pre-analysis storage^20,21) apply; best laboratory practices and manufacturer instructions should be strictly adhered to.

This article is the first to demonstrate this single molecule counting system. A large and growing number of high-quality off-the-shelf and custom kits are available for this platform. Rigorous quality control testing ensures assays are consistent with standards set forth by the World Health Organization. Because this method can be used for a wide variety of sample types and sources (e.g. species), the methods described below are applicable to a number of other clinical populations and animal models. The protocol assays interleukin-6 (IL-6) in human serum collected from a sample of pediatric concussion patients.

Protocol

All the procedures including human subjects described in this protocol have been approved by the Institutional Review Board (IRB) at the University of Texas at Austin. This protocol is specifically adjusted for targeted assay of IL-6 in serum; adjustments to the protocol should be made when assaying other protein biomarkers, consistent with manufacturer’s instructions.

1. Collect, process, and store samples

1. Collect blood and process the biological samples for serum according to the manufacturer’s protocol.
2. Store serum aliquots at -80 °C until processing for laboratory analysis.

2. Prepare the workspace and reagents

1. Use 70% isopropanol to wipe down the bench and pipettes.
2. Allow all reagents to reach room temperature (20-25 °C), including the single molecule counting High Sensitivity Human IL-6 Immunoassay Kit, Standard Analyte, Quality Controls, and 10x System Wash Buffer.
   1. Keep the kit, especially the fluorescently conjugated Detection Antibody, away from light until the time of use.
   2. Dilute the 10x wash buffer to 1x with deionized water and mix thoroughly.
   3. Resuspend IL-6 Antibody-Coated Paramagnetic Beads using a rotisserie spin rotator or manual inversion for 10-20 min.

3. Prepare samples and quality controls

1. Prepare samples and quality controls immediately prior to use.
2. Centrifuge samples and quality controls at >13,000 x g for 10 min; alternatively, use a plate filtration method, which eliminates the need to transfer samples into a new tube.
3. Pipette the supernatant fluid into a new microcentrifuge tube, using care to avoid particulates.

4. Prepare the initial standard stock and perform the serial dilution

1. Prepare the standard stock solution.
   1. Use a benchtop minicentrifuge to quickly spin the standard vial; and then mix via pipetting.
   2. Refer to the Certificate of Analysis for the initial concentration and use a three-step dilution to achieve a final concentration of 50 pg/mL for Standard 1; ensure that each transfer has a volume ≥ 10 μL to reduce error associated with low-volume micropipetting.
2. Serially dilute the remaining standards in a 12-channel reservoir using 1:3 dilutions for Standard 2 through Standard 5, then 1:2 dilutions for Standard 6 through Standard 11). Ensure Standard 12 is left as a blank containing only 500 μL of Standard Diluent.
   1. For the IL-6 assay, use concentrations of 16.67, 5.56, 1.85, 0.62, 0.31, 0.15, 0.08, 0.04, 0.02, 0.01 pg/mL for Standards 2-11, respectively.
   2. Use a 12-channel pipette to promote consistency when transferring volumes from the sample plate after serial dilution.

5. Prepare target capture and incubate in a 96-well plate

1. Pipette 75 μL of standards and samples into a 96-well polypropylene plate.
2. Gently invert the bottle of Antibody-Coated Paramagnetic Beads to resuspend.
   1. Aliquot 9 mL of the Assay Buffer into a 15 mL tube and add the entire 500 μL vial of the Antibody-Coated Beads.
   2. Ensure that all Beads have been transferred, by rinsing the bead vial with 500 μL of Assay Buffer then transfer that volume into the already diluted Antibody-Coated Beads. Repeat this step two more times to bring the total volume of diluted Beads up to 11 mL.
   3. Mix the diluted Beads using gentle inversion.
3. Transfer 100 μL of the diluted Antibody-Coated Beads into each well.
4. Cover the plate with plate sealing film and incubate at 25 °C while shaking for 2 h (use a speed setting of ~1000 rpm on the plate shaker).
5. Prepare the detection antibody when there is approximately 10 min left in the incubation.
   1. Pipette 10 μL of Detection Antibody into 90 μL of Assay Buffer.
   2. Add 75 μL of diluted Detection Antibody into 2925 μL of Assay Buffer.
   3. Filter the diluted Detection Antibody using a 0.2 μm filter and place in a clean tube.

6. Post-Capture wash, detection, and post-detection wash

1. Wash the plate manually or use an automated system (following the manufacturer’s protocol).
   1. Use the Post Capture Wash setting on the automated system.
2. Hold the 96-well plate on a magnet and add 20 μL of Detection Antibody to each well, taking care to neither disturb the pellet nor touch the sides of the polypropylene plate.
   1. Use a single set of pipette tips for the whole plate if proper technique is maintained and there is no evidence of contamination.
3. Apply sealing film to the 96-well plate using firm pressure to prevent cross-contamination and spills.
4. Incubate at 25 °C on a microplate incubator/shaker for 1 h on a speed setting of ~1000 rpm.
5. Remove the sealing film carefully, and wash manually, or use an automated system (following the manufacturer’s protocol).
   1. Use the 4 cycle Pre-Transfer wash setting on the automated system.
6. Remove the plate from the magnet or automated system and shake for 1 minute on a speed setting of ~750 rpm.
7. Return to the magnet or automated system and complete the final aspirate.

7. Elution

1. Add 10 μL of Elution Buffer B to the 96 well plate containing the microparticles using a 12-channel pipette.
2. Cover the plate with plate sealing film and incubate for 10 min at 25 °C on a microplate incubator/shaker on a speed setting of ~1000 rpm.
3. While the plate is incubating, add Buffer D to the appropriate wells of a new 384-well polypropylene plate, keeping the underside protected with the cover provided.
4. Place the original 96-well plate on the magnet for at least 2 min, until the Beads form a dense pellet.
5. Transfer 10 μL of eluate to each well in the 384-well plate, by row, using a 12-channel pipette with new tips for each transfer and exercising care to avoid disturbing the pelleted Beads.
6. Cover the top of the plate with a hard universal plate cover and spin the plate at 1,000 x g for 1 min.
7. Seal the 384-well plate tightly using an adhesive aluminum foil seal.

8. Run the assay on a biomarker quantification system

1. Set up the test template or open a saved template that designates which wells contain standards, controls, and test samples of unknown concentrations, as well as specifies the number of replicates and the dilution type/factor. If desired, users can define unique identifiers for their samples as appropriate.
2. Insert the 384-well plate into the device, and run according to the manufacturer’s instructions.
3. Wait for the run to complete.
4. Remove and discard or store the plate at 2-8 °C.

9. Complete data processing and analysis

1. Create a new file of test results that contains the raw data along with information about the standard curve group and fit. Adjust the standard curve and/or remove outliers as appropriate.
2. Export data as a comma separated value (CSV) file for further analysis in the statistical software of choice.

Results

Using a Five Parameter Logistic Curve Fit (5PL) for the standard curve generated, the Lower Limit of Quantification (LLoQ) was 1.852 pg/mL and the Limit of Detection (LoD) was 0.011 pg/mL with a 0.996 R squared coefficient. Representative data from the ultrasensitive immunoassay system exported CSV file on the n=17 samples ran can be seen in Supplemental Table 1. For this run, the mean concentrations and coefficient of variation were utilized for direct comparison as seen in Table 2.

Discussion

The use of several specific types of equipment can serve to further simplify the protocol and improve results. A rotisserie rotator can be used to promote adequate suspension of the beads and to save time and energy associated with manual inversion. By using a filtration plate over a standard 96 well plate during centrifugation of the samples, the number of plate transfers can be reduced, further saving time. When performing serial dilution of the standards, a 12-channel pipette and 12-well trough could be used to transf...

Materials

Name	Company	Catalog Number	Comments
1L Stericup Filter (0.22 μm; polyethersulfone)	MilliporeSigma	SCGPU11RE	Optional and not used in this study; used to sterilize remaining 1x wash buffer for storage (up to 1 month).
384-well SensoPlate	Griener BioScience
500 mL graduated cylinder	Any	Varies
5 mL syringe	Any	Varies
96-well V-bottom polypropylene plate (500 μL)	Axygen	781892
Centrifuge with plate rotator	Any	Varies	Must be capable of reaching a speed of 1,100 x g
Container capable of holding 500 mL	Any	Varies
De-ionized or disitilled water	N/A	N/A
Erenna 10x System/Wash Buffer	MilliporeSigma	02-0111-00
Jitterbug Plate Shaker	Boekel Scientific	70-0009-00	A suitable alternative can also be used.
Microcentrifuge	Any	Varies
Microcentrifuge tubes	Any	Varies
Modified microplate washer	BioTek	95-0004-05	The microplate washer is modified to include a sphere mag-plate assembly. A suitable alternative (automated or manual) can be used provided it can be modified to include the mag-plate assembly and also has a vacuum regulator as well as a dispense/waste system including a vacuum pump
MultiScreenHTS BV 96-well Filter Plate	EMD millipore	P-96-450V-C
Personal protection equipment	Any	Varies	Gloves, labcoat, closed-toed shoes
Reservoirs for 12-channel pipettors	Any	Varies
Rotisserie Rotator	Any	Varies
Sealing tape	Any	Varies
Single channel pipettes	Any	Varies	Must have pipettes capable of transferring 10-250 μL
SMC High Sensitivity Human IL-6 Immunoassay Kit	MilliporeSigma	03-0089-01	Human assay optimized for use with serum and EDTA plasma samples. The kit contains the following 9 items (1) Assay Buffer; (2) Beads; (3) Standard Diluent; (4) Detection Antibody; (5) Standard; (6) Control 1; (7) Control 2; (8) Control 3; (9) 10X Wash Buffer; (10) Buffer D; (11) Elution Buffer B.
SMCxPROTM Complete System	MilliporeSigma	95-0100-00	This is the device used to run the assays and quantify the biomarker; the complete kit comes with the device and the associated computer and software.
Syringe filter (0.2 μm)	Any	Varies
Titer Plate Shaker	VWR	12620-926	Optional and not used in this study; used when incubating overnight
Universal plate cover	Any	Varies