Purpose: Interleukin17A (IL-17A) is disulfide-linked homo dimeric cytokine of 155 amino acids and a member of an IL-17 family of related cytokines. There is now compelling evidence that patients affected by autoimmune diseases have a higher incidence of several cardiovascular diseases. IL-17A plays a crucial role in the development of chronic inflammation and probably in the hemostatic disorders observed in patients with autoimmune diseases. The study aimed to develop and validate an ultrasensitive digital immnuoassay for quantitation of IL17-A, a low abundance biomarker in human serum.
Methods: The Simoa Human IL-17A 2.0 assay is a 3-step digital immunoassay for the quantitation of total IL-17A in human serum. The assay is designed to work with the Simoa HD-1 Analyzer which utilizes Single Molecule Array (SimoaTM) technology. All the assay steps of the immunoassay are performed by the instrument (i.e. “hands-free” operation).
In the first step, anti-IL-17A coated paramagnetic capture beads were incubated with diluted samples standards and QC samples in order to capture IL-17A. The beads were washed and incubated with a biotinylated detection antibody that binds to the captured IL-17A. Following a second wash, a conjugate of streptavidin-ß-galactosidase (SBG) was added to the sample. SBG bound to the biotinylated detection antibody, resulting in the enzymatic labeling of the captured IL-17A. Following a third wash, the capture beads were resuspended in a resorufin ß-D-galactopyranoside (RGP) substrate solution and transferred to the Simoa Disc. Individual capture beads were sealed within the microwells of the array. Captured and labeled IL-17A hydrolyzed the RGP substrate into a fluorescent product that provided the signal for measurement.
Results: Method for quantitation of IL-17A in human serum samples has been validated using the Quanterix Simoa HD-1 Analyzer which has been completely validated following guidance 21 CFR part 11. The validation of method was conducted under these assay parameters: including precision and accuracy, selectivity, linearity of dilution and stability. The assay sensitivity is 0.23438 pg/mL with MRD 1:4. The dynamic range of the method is 0.23438 - 120 pg/mL. Results for accuracy, precision, mixed samples, selectivity, stability and dilution linearity met the required acceptance criteria.
Conclusion: An ultrasensitive IL-17A Simoa assay has been successfully validated by Frontage Laboratories using a Quanterix Simoa instrument that has completed Computer System Validation with the integration with Watson LIMS. These data demonstrated that endogenous level IL-17A can be quantitated in human serum at low pg/mL, even fg/mL levels by Qanterix Simoa platform.