Research Article: Development and Characterization of Anti-Nitr9 Antibodies

Date Published: September 24, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Radhika N. Shah, Ivan Rodriguez-Nunez, Donna D. Eason, Robert N. Haire, Julien Y. Bertrand, Valērie Wittamer, David Traver, Shila K. Nordone, Gary W. Litman, Jeffrey A. Yoder.


The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function.

Partial Text

Mammalian natural killer (NK) cells are large, granular lymphocytes of the innate immune system that express several cell surface receptors to regulate cytotoxic function through a complex network of signaling pathways. NK cell receptors include both activating and inhibitory forms that are proficient in distinguishing neoplastic or virally infected cells from normal host cells [1, 2]. The regulation of NK cell cytotoxicity is dependent on the integration of signals from activating and inhibitory receptors [3]. Although it is postulated that NK cell receptors arose early in vertebrate phylogeny, functional data are based primarily on studies of mammalian NK cell receptors [4].

Three different transcript variants from nitr9, the single putative activating NITR gene in zebrafish, and their corresponding protein isoforms have been identified and characterized. The utility of the anti-Nitr919 and anti-Nitr990 monoclonal antibodies for detecting recombinant Nitr9 was demonstrated by indirect immunofluorescence, flow cytometry, and Western blot analyses. The antibodies exhibit profound differences in recognizing the three different Nitr9 isoforms. When employed for indirect immunofluorescence, both anti-Nitr9 antibodies bound efficiently and specifically to cells-expressing all three Nitr9 isoforms. Both anti-Nitr9 antibodies are effective for detecting cell surface expression of Nitr9L and Nitr9SS by flow cytometry. The anti-Nitr990 antibody recognized all three Nitr9 isoforms by Western blot analyses, although a higher affinity for Nitr9L is noted. When using anti-Nitr990 in Western blot analyses with high levels of protein, a nonspecific band was identified. Although the identity of this protein remains unknown, it may represent a well-conserved member of the Ig superfamily.