Development of sandwich ELISA for detection and quantification of human and murine a disintegrin and metalloproteinase17
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Published on: June 14, 2018
Ahmad Trad1, Nina Hedemann, Mohammad Shomali
1Biochemical Institute of the Medical Faculty of the Christian-Albrechts-Universität at Kiel, Germany.
Abstract:
ADAM17 (a disintegrin and metalloproteinase-containing protein 17) is a membrane-bound metalloproteinase, implicates in many physiological processes, including cell migration and proliferation. Of particular note, most of the studies so far are restricted on the analysis of ADAM17 mRNA levels. In this study we generated, utilizing hybridoma technology, three monoclonal antibodies (mAbs) (A 300, A 309 and A 318) against the extracellular domain of human ADAM17 to enable quantification of protein expression. The specificity of these mAbs against ADAM17 was tested by enzyme-linked immunoadsorbent assay (ELISA), flourescence-activated cell sorting (FACS) and western blotting. In order to quantify human and murine ADAM17 expression two pairs of these mAbs (biotinylated A 309 in combination with A 300 and biotinylated A 300 in combination with A 318), were used to develop sandwich ELISA. A panel of monoclonal antibodies was generated for first time to measure mouse ADAM17 with a sensitivty of 2 ng/ml. Such systems provide a useful tool to quantify protein levels of ADAM17 and are valuable tools for diagnostic purposes.
Insights
Researchers developed novel monoclonal antibodies to accurately measure ADAM17 protein levels. These tools enable precise quantification of ADAM17 (a disintegrin and metalloproteinase-containing protein 17), aiding diagnostic applications.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- ADAM17 (a disintegrin and metalloproteinase-containing protein 17) is a key metalloproteinase involved in cell migration and proliferation.
- Current research often focuses on ADAM17 mRNA, limiting protein expression analysis.
Purpose of the Study:
- To generate and characterize monoclonal antibodies (mAbs) for quantifying ADAM17 protein expression.
- To develop sensitive assays for human and murine ADAM17 detection.
Main Methods:
- Hybridoma technology was used to produce three mAbs (A 300, A 309, A 318) targeting the extracellular domain of ADAM17.
- Antibody specificity was validated using ELISA, FACS, and Western blotting.
- Sandwich ELISA assays were developed using specific mAb pairs for quantitative analysis.
Main Results:
- Three specific mAbs against human ADAM17 were successfully generated.
- A sensitive sandwich ELISA was developed, capable of detecting mouse ADAM17 at concentrations as low as 2 ng/ml.
- Validated assays enable accurate quantification of ADAM17 protein levels.
Conclusions:
- The developed monoclonal antibodies and sandwich ELISA systems are valuable tools for quantifying ADAM17 protein expression.
- These tools facilitate further research into ADAM17's physiological roles and offer potential for diagnostic applications.
