Identification Of Six Kinds Of Monoclonal Antibodies Against Rat Nogo-A Molecule In Immunofluorescent Histochemical Staining And Identification Of Their Epitopes

The central nervous system （CNS） of adult mammal after injury is limited to recovery because of the inability of damaged axons to reconnect to their physiological structure and function. The factors which influence axon regeneration are considered as neural cell autonomous, the astroglial scar, promotion and inhibition factors in vivo or in vitro, CNS inflammation and others.In the recent 20 years’research, it has found some inhibition factors sourced from CNS myelin, such as Nogo-A, MAG, Omgp, netrins and so on. Necessarily, the structure, location and function of Nogo-A, Nogo receptor （NgR） and the Nogo Receptor complex components (p75^NTR/NgR1/LINGO-1 or TROY/NgR1/LINGO-1) have been extensively studied. They will be a topic of great research interest and clinical relevance. In the current studies, Nogo-A and NgR components can benefit in precursor migration, neurite growth and branching, synaptic stability and plasticity in the developing nervous system; At mean while, they can induces oligodendrocyte differentiation and myelination; They have potential roles in microglia/macrophages responses and the inflammation in secondary damage after SCI; They also have roles in the regulation of endoplasmic reticulum （ER） structure, processing of amyloid precursor protein and cell survival.Previous researches have suggested that Nogo-A can inhibit axons sprouting and growth of injured fibres after spinal cord or brain injury. There are three inhibitory functional areas in Nogo-A: one is Nogo-66 in C terminal of Nogo-A, others are 1-172 aa and 174-979 aa in N terminal of Nogo-A. Using function-blocking Nogo-A-specific antibodies to block Nogo-A, a soluble Nogo-66 binding fusion protein comprising domains of NgR1, antagonistic peptides,blocking Rho-A and its downstream target ROCK can help for regeneration.Nevertheless, the kinds of commercialization of anti-Nogo-A antibodies are less and their functions are limit. It had confined the research of Nogo-A and especially the research of the function of Nogo-A in the process of SCI. Although using function-blocking Nogo-A-specific antibodies can block the function of Nogo-A, the target and short peptide drugs have not been identified. There are many questions to explore in the translation from basic research to clinical application.In our initial experiments, we express 570-691 aa fragments of Nogo-A molecule and Nogo-66 fragments （1026-1091 aa） by prokaryotic expression system. After inoculating the BALB/c-nude^-/- mice, we acquired six kinds of monoclonal antibodies against rat Nogo-A molecule, named as Nogo66-1, Nogo66-2, Nogo66-3, Nogo66-4, NogoN-1, NogoN-2 mAbs from ascites. Western blot showed that all antibodies recognized the Nogo-A molecule （200 KD）. But we don’t know whether these antibodies can be used in immunohistochemical studies with CNS tissues? First, we identified the characteristics of six monoclonal antibodies against rat Nogo-A molecule in immunofluorescent histochemical staining. Then, we managed to find the epitopes of Nogo-A to which the 6 monoclonal antibodies to bind to provide convince about their functional characters, signal pathways and applications.In the first part, we prepared six kinds of monoclonal antibodies against rat Nogo-A molecule from ascites by hybridoma peritoneal injection. Then the immunofluorescence histochemical staining was used to detect the reactivity and specificity of those 6 mAbs in spinal tissue sections of rat. We acquired information about the specificity of the 6 monoclonal antibodies and distribution of the antigens, Nogo-A, in CNS.In the second part, we use constructed several truncated recombinant plasmids of Nogo-66 and Amino-Nogo-A（aa 570-691） which were translated in prokaryotic expression system. We used Western blot to identify the epitopes which could be recognized by those monoclonal antibodies. So that we can study Nogo-A or Nogo receptor function by antibodies and deeply testify Nogo-A which is a key stabilizer and negative growth regulator in the adult CNS.From above experiments, we got the results as follows:1. All 6 mAbs were double-labelled with commercial rabbit anti-Rat Nogo-A polyclonal antibody （pAb） in spinal cord sections respecitvely;2. All 6 mAbs were colocalization with MBP respectively. However Nogo66-3 and NogoN-1 could also be double-staining with GFAP respectively.3. We acquired several different length fragments of Nogo-66 and Amino-Nogo-A; we constructed recombinant plasmids successfully; we also express the fusion proteins: GST-△Nogo66_a（aa1026-1091）、GST-△Nogo66_b（aa1056-1091） GST-△NogoA-N_a（aa 570-691）、GST-△NogoA-N_b（aa 601-691）、GST-△NogoA-N_c（aa 634-691）、GST-△NogoA-N_d（aa 669-691）.4. We identified the epitopes which could be recognized by 6 mAbs through constructing several truncated Nogo-A and Nogo-66. Nogo66-1 mAb、Nogo66-2 mAb、Nogo66-3 mAb、Nogo66-4 mAb can recognize aa 1026-1055 of NogoA ; NogoA-N1 mAb、NogoA-N2 mAb can recognize aa 634-668 of NogoA.From above all, Nogo66-1、Nogo66-2、Nogo66-4 and NogoN-2 mAb can recognize Nogo-A in spinal cord specificly. They can be used in immunofluorescence hitochemical staining in future studies. Nogo66-1、Nogo66-2、Nogo66-3、Nogo66-4 mAbs can combine aa 1026-1055 of Nogo-A; NogoA-N-1、NogoA-N-2 mAbs can combine aa 634-668 of Nogo-A. These results will know the characters of immunofluorescence hitochemical staining of these mAbs clearly and sure about their epitopes.