Development and characterization of a JAK-2-specific antibody suitable for immunohistochemical investigative studies.
Janus kinases (JAKs) are tyrosine kinases called JAK-1, JAK-2, JAK-3, and Tyk-2, which have been shown to participate in the signaling pathways of several cytokines that are believed to play a key role in several autoimmune-mediated disorders including rheumatoid arthritis (RA). However, the availability of JAK-specific antibodies to be used in investigative efficacy studies in RA models is very limited. Therefore, in this study we developed and characterized a JAK-2-specific antibody that was used to evaluate its immunohistochemical expression in the joints of a rat adjuvant-induced arthritis (rAIA) RA preclinical model. An immunogen peptide sequence design was used to generate JAK-2-specific mouse, rat, and human polyclonal antibodies. JAK-2 plasmid cDNA was then generated and HEK293 transfected cell lines, gel electrophoresis, immunoblotting, and immunohistochemistry were used to further characterize the generated JAK-2 antibodies. We show that the generated JAK-2 antibody exhibits specificity and lacks cross-reactivity to JAK-1 and JAK-3. In addition, marked JAK-2 expression is shown in the rAIA in mixed inflammatory cells (macrophages and neutrophils), mast cells, and bone marrow elements. In conclusion, we show the development and characterization of a JAK-2-specific antibody that can be used in investigative and mechanistic studies such as preclinical efficacy models.
Radi
Zaher
Z
Pfizer Research and Development, Drug Safety Research and Development, Cambridge, MA, USA. zaher.radi@pfizer.com
Nagappan
Mathialagan
M
Huang
Xiaoying
X
Heintz
Catherine
C
Gardner
Joseph
J
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D053614:Janus Kinase 2 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew
D000906:Antibodies; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; C507927:Jak2 protein, rat; D053614:Janus Kinase 2