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 e....

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 .......

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