Preparation,Identification And Application Of Metrnl Monoclonal Antibodies Of Mouse

The epidemic surge of obesity and its related diseases have resulted in great interest in the functional role of adipose tissue.During the previous twenty years,people have realized that adipose tissue is an important metabolic regulator as an endocrine organ not just the largest energy reservoir of the body.The biologically active peptides and proteins secreted by adipose tissue are termed as adipokines,which have now been considered to be involved in the regulation of multiple physiological functions,including metabolic homeostasis,insulin sensitivity,cardio-cerebro-vascular function,immunity,and inflammation.The link between adipose tissue and obesity related diseases still remains to be elucidated.Therefore,to identify new adipokines as well as to explore their functions during the pathophysiologic process is of great value.In 2008,our lab identified a novel adipokine Metrnl,which we also named it Subfatin because of its high expression in subcutaneous fat.Our preliminary results and the limited reports from other labs these years have revealed important roles of Metrnl in metabolic homeostasis and neural development.The effective antibody of Metrnl will be a powerful mean to further explore the physiological functions of Metrnl,providing reliable detection of this adipokine in assays including Western blot,ELISA,immunohistochemistry and immunoprecipitation and allowing for new concept of Metrnl related therapy strategy.To make the antibody available,we prepared mouse Metrnl monoclonal antibodies using hybridoma technology and validated their effectiveness.Methods 1.Preparation of mouse Metrnl monoclonal antibodies(1)Mouse Metrnl peptide epitope was designed and synthesized for the conjugation with carrier protein.The coupling of polypeptide antigen was detected by SDS-PAGE and Coomassie brilliant blue staining.(2)The mouse Metrnl gene was conjugated with the modified p ET-28 a vector.After the prokaryotic expression,SDS-PAGE and Coomassie brilliant blue staining were used to detect the expressed protein and Western blot was used to detect the carried His tag.(3)The polypeptides and full-length protein were used to immunize mice,after which the blood was collected and the serum titer was measured by ELISA.The immune system of mouse candidates for cell fusion was strengthened every day for three days.Then the spleen cells were fused with SP2/0 myeloma cells to obtain fused hybridoma cells which were then screened on the HAT medium to select proper ones to undergo following subcloning before finally being injected into mice peritoneal.The ascites from mice were collected for purification of Metrnl monoclonal antibodies.2.Generation and validation of Metrnl knockout(KO)mice(1)Metrnllox P / lox P mice were mated with Ella-Cre mice to obtain Metrnl+/-;Ella-Cre mice,which were later crossed with C57 mice to obtain Metrnl+/-mice.Finally,Metrnl-/-systemic knockout mice were obtained by Metrnl+/-selfing.The genotype of the offsprings was confirmed by PCR-based DNA identification results.(2)Metrnl expression level in colon tissue of Metrnl KO mouse was identified by Real-time PCR.(3)Metrnl protein concentration in the serum of Metrnl KO mouse was detected by ELISA.3.Validation and application of mouse Metrnl monoclonal antibodies(1)Using mouse Metrnl recombinant protein and colon tissue protein of C57,WT and KO mice,Metrnl monoclonal antibodies were validated by Western blot.(2)Different effective monoclonal antibodies were used in immunohistochemistry and co-immunoprecipitation.Results 1.A total of 14 polypeptide antigens were obtained,13 of which were successfully conjugated with carrier protein.A new polypeptide antigen was re-engineered with following successful conjugation.2.The mouse Metrnl recombinant protein containing His tag was successfully expressed in the prokaryotic expression system.3.Spleen cells of mice with greater serum titer were used to fuse with SP2/0 myeloma cells.A total of 81 strains of positive hybridoma cells were screened,including 56 strains of positive hybridoma cells prepared by mouse Metrnl polypeptide antigens and 25 strains of positive hybridoma cells prepared by mouse Metrnl full-length protein.4.All ascite antibodies were validated by mouse Metrnl full-length recombinant protein.There was no positive antibody in the 56 strains of antibodies prepared by mouse Metrnl polypeptide antigens.However,there were 12 strains of antibodies which can recognize Metrnl recombinant protein among the 25 strains of antibodies prepared by mouse Metrnl full-length protein in Western blot.5.The colon tissue showed the highest m RNA level of Metrnl among the detected tissues of C57 mice.6.Metrnl systemic knockout mouse was successfully generated and both m RNA level in tissue and protein level in circulation were identified.7.The 12 strains of selected antibodies are not suitable for mouse colon tissue detection in Western blot.8.Different effective antibodies selected from the 12 strains of antibodies can be used in immunohistochemistry and co-immunoprecipitation.In summary,81 strains of mouse Metrnl monoclonal antibodies are obtained by immunizing mice using Metrnl polypeptide fragments and full-length recombinant protein.Among the 25 strains of antibodies prepared by mouse Metrnl full-length protein,12 strains can recognize Metrnl recombinant protein in Western blot,several of which can be applied in immunohistochemistry and co-immunoprecipitation.However,the selected antibodies are not suitable for tissue detection in Western blot,evidenced by tests in colon tissues from wild-type and Metrnl knockout mice.In conclusion,this study obtains several strains of mouse Metrnl monoclonal antibodies and a strain of Metrnl knockout mice,which are able to be used in Metrnl immunohistochemistry and co-immunoprecipitation,as well as Metrnl function study in the future.