Construction of the ELISA assay to quantify Semaphorin 3A in the adult brain

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  • Rei Sugiyama
    Dept. Applied Biosci., Kanagawa Inst. Tech.
  • Lin Ke-Yi
    Chiome Bioscience
  • Nakamura Fumio
    Dept. Mol Pharmacol and Neurobiol, Grad. Sch. Med., Yokohama City Univ. Dept. Biochem, Tokyo Women's Medical Univ.
  • Sakurai Takashi
    Dept. Pharmacol., Juntendo Univ. Sch. Med.
  • Goshima Yoshio
    Dept. Mol Pharmacol and Neurobiol, Grad. Sch. Med., Yokohama City Univ.
  • Yamashita Naoya
    Dept. Applied Biosci., Kanagawa Inst. Tech. Dept. Mol Pharmacol and Neurobiol, Grad. Sch. Med., Yokohama City Univ. Dept. Pharmacol., Juntendo Univ. Sch. Med.

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Other Title
  • 成体脳におけるセマフォリン3A発現を定量するELISAシステムの確立

Abstract

<p>Extracellular soluble signals that control several aspects of neuronal development are known to play a critical role in maintaining neuronal function and homeostasis in the mature nervous system. Abnormal expression and/or secretion of these molecules are therefore thought to be associated with the onset of various types of neurological disorders. It has been reported that the expression of Semaphorin 3A (Sema3A), a secreted type of repulsive axon guidance molecule, is impaired in several neurodegenerative disorders. However, due to the lack of a reliable Sema3A antibody, our knowledge about Sema3A expression in the adult brain is still limited. Here we report the identification of a pair of Sema3A monoclonal antibodies for the sandwich ELISA assay using the Autonomously Diversifying Library system. Our Sema3A monoclonal antibodies recognize the blade 3-4 or the blade 5 of Sema domain, respectively. We can measure the concentration of recombinant human Sema3A in the range of 0-100 pM by ELISA. The specificity of this assay was confirmed by using the embryonic brains from sema3A deficient mice as a negative control. Moreover, this assay could measure Sema3A concentration in Tris buffered saline- and SDS-soluble lysate obtained from the adult mice brains. These data suggested that our ELISA assay is a reliable tool for the validation of Sema3A as a biomarker in neurodegenerative disorders.</p>

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