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Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α
Published on: June 14, 2018
Single molecule array (Simoa) assay with optimal antibody pairs for cytokine detection in human serum samples
Danlu Wu1, Milena Dumont Milutinovic, David R Walt
1Department of Chemistry, Tufts University, Medford, MA 02155, USA. david.walt@tufts.edu.
Insights
This study developed ultrasensitive Simoa assays for ten cytokines, achieving femtomolar detection limits. These new methods enable precise measurement of cytokine levels in healthy individuals, crucial for diagnostics and therapeutic monitoring.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Cytokine concentrations in bodily fluids indicate patient health status, aiding in disease diagnosis and treatment monitoring.
- Current detection methods struggle to quantify low cytokine levels found in healthy individuals, limiting diagnostic capabilities.
Purpose of the Study:
- To develop ultrasensitive assays for ten key cytokines using the Simoa platform.
- To establish reference concentration ranges for these cytokines in healthy human serum.
Main Methods:
- Selected antibody pairs for single molecule array (Simoa) assays.
- Quantified ten cytokines: GM-CSF, TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, and IL-10.
- Determined limits of detection (LODs) ranging from 90 aM to 6 fM.
Main Results:
- Achieved ultra-low LODs for the ten targeted cytokines, significantly surpassing conventional ELISA sensitivity.
- Successfully detected and quantified cytokines in healthy human serum samples at sub-picomolar concentrations.
- Provided detailed antibody pair information and reference concentration data for healthy serum.
Conclusions:
- The developed Simoa assays offer unprecedented sensitivity for cytokine detection.
- This advancement facilitates the measurement of low-level cytokines, supporting early disease detection and personalized medicine.
- The generated reference data is valuable for future ultrasensitive immunoassay development and clinical applications.
Abstract:
Concentrations of cytokines in bodily fluids reflect the physiological or pathological state of the patient and can be used for prognosis, disease diagnosis or for monitoring therapeutic efficacy. However, in the bodily fluids of healthy or sub-healthy individuals, many cytokines are present at concentrations that are near or below the detection limits of current methods. Here we selected antibody pairs to be employed in the single molecule array (Simoa) assay for ten cytokines including GM-CSF, TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, and IL-10. The limits of detection (LODs) obtained were as low as 90 aM-6 fM. These assays allow detection of cytokines in healthy human serum samples at levels significantly below the detection limits of conventional ELISA assays. We provide detailed antibody pair information as well as the concentration profiles of ten cytokines in healthy human serum to serve as reference data for further ultrasensitive immunoassay development and future clinical applications.
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