Preparation Of Rabbit Monoclonal Antibody Against CGMP And Development Of Competitive ELISA For CGMP Determination

The second messengers Guanosine 3’,5’-cyclic monophosphate (cyclic GMP, cGMP) and Adenosine 3’,5’-cyclic monophosphate (cyclic AMP; cAMP) are important signaling molecules in many prokaryotes and eukaryotes. cGMP has the positive metabolic and functional effects in cell. It has been shown that cGMP can reduce myocardial metabolism, inotropy and function. Forthermore, NO-cGMP pathway regulates stem cell proliferation and differentiation. cGMP also take important role in down-regulates gluconeogenesis, parasympathetic nerve excited,visual signal conduction, mooth muscle,skeletal muscle,nervous system functions,platelet activation,osteoblastic growth etc al.Competitive protein-binding assay and Radioimmunoassay were the main methods for the detection of cGMP in the 70-80’s of twentieth Century. Because of radioactive immunoassay had potential harm to the human body, it had been gradually replaced by the enzyme labed method and fluorescence ELISA method. The two mehods base on rabbit polyclonal antibody or mouse monoclonal antibody. Differences between batches and bad specificity are main problems for ELISA kit based on polyclonal antibody. There is without above shortcomings for ELISA kit based on mouse monoclonal antibody.But it is difficult to obtain reproducible data for the user. This is because the absorbance value between different cGMP concentrations is very small.In this study, cGMP-conjugated antigen (cGMP-KLH) were produced by coupling cGMP with keyhole limpet hemocyanin(KLH). Finally, conjugates were proved to be successfully by direct ELISA. The immunogen was injected into New Zealand white rabbits. The hybridoma cells were obtained by fusing spleen cells of rabbit to rabbit myeloma-like cell line of 240E (Epitomics Inc., USA). The best hybridoma cell was choosen to do subclonging then to do large-scale culture by indirect ELISA and indirect competitive ELISA. The hyridoma cell culture supernatant was purified by Protein A mehod.The IgG concentration of rabbit monoclonal antibody was detected by OD280 and fortebio methods, respectively. Direct ELISA and competitive ELISA were used to detect the sensitivity and specificity of cGMP rabbit monoclonal antibody. The reaction of rabbit monoclonal antibody with carrier protein(BSA、KLH)was detected. The result showed that the RabMab had no reaction to carrier protein. The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cross reaction of rabbit monoclonal antibody with cAMP, Inosine 3’:5’-cyclic monophosphate (cIMP),Cytidine 3’,5’-cyclic monophosphate (cCMP),Adenosine 5’-monophosphate (AMP),Adenosine 5’-diphosphate (ADP),Adenosine 5’-triphosphate disodium (ATP),Guanosine 5’-monophosphate disodium (GMP),Guanosine 5’-diphosphate (GDP) and Guanosine 5’-triphosphate tris (GTP) was detected. The rabbit monoclonal antibody had 33% cross-reactivity to inosine 3’:5’-cyclic monophosphate (cMP) and little or no cross-reactivity to other compounds.The conditions of ELISA experiment was optimazed. The optimal conditions of competitive ELISA were as follows:the concentration of rabbit monoclonal antibody was 0.2μg/mL and cGMP-HRP was 1:100 times dilution of the stock. Under the optimal conditions, a sensitive and specific standard curve was with the range of detection from 1.95 ng/mL to 120 ng/mL. The limit of detection was 1.95 ng/mL. The recovery of assay was 89%-103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.