Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

Abstract

Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I1- and I2-imidazoline drugs

“… + : protein expression is present; ( -) : protein immunoreactivity blocked in the adsorption test (antigenic peptide). * Anti-NISCH (IRAS): detection of nischarin (I 1 -IR, the mouse homologue of IR antisera-selected, IRAS); other anti-IRAS antibodies also detected >167 kDa full-length proteins (e.g. Piletz et al 2000b, Alahari et al, 2000, Wang et al 2009). Anti-nischarin K-15: detection of nischarin (I 1 -IR).…”

“…↓or↑: modest decrease or increase (<20 %); ↓↓or↑↑: marked decrease or increase (>30 %); ≈: no significant change. Abbreviations. corr: correlation between Bmax and IR protein; Mox: moxonidine; Efa: efaroxan; Ida: idazoxan Selected references: 1 (Ruiz et al, 1993), 2 (Escribá et al, 1994), 3 (García-Sevilla et al, 1995a,b), 4 (Escribá et al, 1995), 5 (Sastre et al, 1996), 6 (Escribá et al, 1996), 7 (García-Sevilla et al, 1996a,b), 8 (Ivanov et al, 1998a,b), 9 (Boronat et al, 1998), 10 (García-Sevilla et al, 1998), 11 (García-Sevilla et al, 1999), 12 (Piletz et al, 1999), 13 (Barturen et al, 1999), 14 (Halaris et al, 1999), 15 (Olmos et al, 1999a), 16 (Piletz et al, 2000a,b), 17 (Alahari et al, 2000), 18 (El-Ayoubi et al, 2002), 19 (Tyacke et al, 2002), 20 (Sano et al, 2002), 21 (García-Sevilla and Ferrer-Alcón, 2003), 22 (Piletz et al, 2003), 23 (Ma et al, 2003), 24 (El-Ayoubi et al, 2003), 25 (Dontenwill et al, 2003a,b), 26 (Chen et al, 2003), 27 (El-Ayoubi et al, 2004), 28 (Lim and Hong, 2004), 29 (Zhang and Abdel-Rahman, 2006), 30 (Sun et al, 2007), 31 (Zhang and Abdel-Rahman, 2008), 32 (Wang et al, 2009), 33 (Li and Zhang, 2011), 34 (Ding et al, 2013), 35 (Wang et al, 2013), 36 (Mar et al, 2013), 37 (Aceros et al, 2014), 38 (Chen et al, 2014). …”

“…17 (Alahari et al, 2000), 18 (El-Ayoubi et al, 2002), 19 (Tyacke et al, 2002), 20 (Sano et al, 2002), 21 (García-Sevilla and Ferrer-Alcón, 2003), 22 , 23 (Ma et al, 2003), 24 (El-Ayoubi et al, 2003), 25 (Dontenwill et al, 2003a,b), 26 (Chen et al, 2003), 27 (El-Ayoubi et al, 2004, 28 (Lim and Hong, 2004), 29 (Zhang and Abdel-Rahman, 2006), 30 (Sun et al, 2007), 31 (Zhang and Abdel-Rahman, 2008), 32 (Wang et al, 2009), 33 (Li and Zhang, 2011), 34 (Ding et al, 2013), 35 (Wang et al, 2013), 36 (Mar et al, 2013), 37 (Aceros et al, 2014), 38 (Chen et al, 2014).…”

“…In cell systems, IRAS/nischarin antibodies were shown to detect a protein of high molecular mass (about 170 kDa) that was postulated as a functional I 1 -IR (with nanomolar affinity for moxonidine; Piletz et al, 2000b; Sun et al, 2007; Zhang and Abdel-Rahman, 2006), an IR precursor (Piletz et al, 1999) or a membrane-associated mediator of I 1 -IR signaling (see protein Q9Y2I1 at http://www.uniprot.org/uniprot/Q9Y2Il). Moreover, other human IRAS monoclonal antibodies have been generated that have detected full-length IRAS (about 170–190 kDa) in cell systems (Wang et al, 2009; Zhang et al, 2013).…”

Characterization of the Specificity of Imidazoline I-1 Receptor Antibody for Subtype of Imidazoline Receptors In Vitro

“…The specifi city of anti-NISCH antibody against imidazoline receptors can thus be confi rmed. Recently, another antibody has been developed [ 33 ] , but it is still not commercialized and thus, it is hard to identify the specifi city of this antibody. To date, there are 3 subtypes of imidazoline receptors, imidazoline I-2 and I-3 receptors in addition to I-1 sites [ 34 ] .…”