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Generation and characterization of polyclonal and monoclonal antibodies to human NTPDase2 including a blocking
Julie Pelletier1, Hervé Agonsanou1,2, Ninotchska Delvalle3
1Centre de Recherche du CHU de Québec - Université Laval, CHUL, 2705 Boulevard Laurier, Office T1-49, Québec, QC, G1V 4G2, Canada.
Abstract:
Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.
Insights
Researchers developed new tools to study human Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). These tools revealed NTPDase2 is present on human enteric nervous system glial cells, suggesting a role in intestinal motility.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase regulating P2 receptor signaling via ATP hydrolysis.
- NTPDase2 expression is well-characterized in rodents but poorly understood in humans.
Purpose of the Study:
- To develop and validate specific research tools for studying human NTPDase2.
- To investigate the expression and potential function of human NTPDase2.
Main Methods:
- Generation of specific mouse monoclonal and rabbit polyclonal antibodies against human NTPDase2.
- Validation of antibody specificity using Western blot, immunocytochemistry, immunohistochemistry, and flow cytometry.
- Characterization of a neutralizing monoclonal antibody (hN2-D5s).
Main Results:
- Validated antibodies demonstrated specificity for human NTPDase2.
- NTPDase2 immunoreactivity was identified on glial cells within the human enteric nervous system.
- The antibody hN2-D5s selectively inhibited human NTPDase2 activity.
Conclusions:
- Novel antibodies provide essential tools for future research on human NTPDase2.
- Findings suggest a role for NTPDase2 in human intestinal motility.
- This work lays the foundation for understanding NTPDase2's function in human physiology and disease.