The Study of Specificity of Antirenalase Antibody Interactions with Some Proteins
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Abstract
Renalase (RNLS) is a multifunctional protein exhibiting various intracellular and extracellular functions. RNLS levels in tissues and biological fluids are typically assessed using commercially available antibodies, which vary in their specificity to the target protein. In this study we have used Western blot analysis to investigate the interaction of several primary polyclonal and monoclonal anti-renalase antibodies with recombinant human and rat RNLS, as well as with chimeric proteins in which RNLS sequences encoded by alternative exons (9 and 10) were fused to dihydrofolate reductase. To verify interaction specificity, we have used lactate dehydrogenase preparations (LDHA and LDHB), which have recently been shown to interact with certain anti-renalase antibody preparations. All polyclonal and monoclonal antibodies tested interacted effectively with all RNLS preparations examined, including fragments generated by protease treatment of the full-length recombinant protein and fragments contained within the chimeric protein fused to dihydrofolate reductase. LDHA and LDHB preparations also reacted with anti-renalase antibodies; however, the nonspecific interaction of these antibodies with LDHA and LDHB could be significantly reduced by dilution (1:10,000 instead of the 1:1,000 recommended by the manufacturers). Notably, polyclonal antibodies interacted more effectively with full-length recombinant renalase, whereas monoclonal antibodies interacted more effectively with peptide fragments generated by treating the target protein with proteases (trypsin, chymotrypsin, and chymase).
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References
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