Preparation Of Mouse Anti-human CMG2 Monoclonal Antibody And Its Preliminary Application

Capillary morphogenesis gene 2?CMG2?is a type I transmembrane protein.The full-length form consists of 489 amino acids(CMG2⁴⁸⁹)and its gene is located at 4q21.CMG2 was originally identified for its involvement in capillary morphogenesis,and subsequently identified as the second receptor for anthrax toxin?ANTXR2?.Although the exact physiological function of CMG2 is unclear,mutations in this gene have been shown to cause juvenile Hyaline Fibromatosis and Infantile Hyaline Fibromatosis.In recent years,the role of CMG2 in tumors has attracted people's attention.Our previous study has found that CMG2 is highly expressed in gastric cancer and can serve as a new functional surface marker of gastric cancer stem cells?GCSCs?,involving in the maintenance of GCSCs and the progression of gastric cancer.A study by another group of our institute also found that CMG2is highly expressed in gliomas,and its expression level is closely related to the grade and the outcome of the patients.These findings suggest that CMG2 may be an important player in tumors,such as gastric cancer and glioma,and act as a potential diagnostic indicator and therapeutic target.The role of CMG2 in tumors deserves in-depth study.However,these studies are inseparable from high specificity and high affinity anti-CMG2 antibodies.To date,most of the international and domestic commercial anti-CMG2 antibodies are polyclonal antibodies,and only Abcam supplies rabbit anti-human CMG2 monoclonal antibody.Therefore,the development of anti-human CMG2 monoclonal antibodies has a good basic research and clinical application prospects.Research purposesTo prepare highly specific and high affinity mouse anti-human CMG2 monoclonal antibody,and apply it in the determination of CMG2 expression in tumor cells and tissues by estabilishing immunoblotting,immunofluorescence,flow cytometry and immunohistochemistry with it as primary antibody.Main method1.Prokaryotic expression and purification of CMG2 ectodomain antigenic peptide.According to the sequence of CMG2?UniProtKB-P58335?and on-line epitope prediction results,antigen peptides,including CMG2 ectodomain?CMG2-ex?and dominant epitope peptides,were designed and expressed in prokaryotic expression system.The expressed products were purified by nickel column.The concentration of purified products was determined by BCA method,and the purity of purified products was detected by SDS-PAGE.2.Mouse immunization,hybridoma cell preparation and screening,ascites preparation and monoclonal antibody purification.The most immunogenic antigenic peptide was selected as the immunogen,and the mice were routinely immunized and hybridoma cells were prepared.The target clones were screened by indirect ELISA using eukaryotic expressed CMG2 ectodomain as a screening antigen.Mouse ascites was routinely prepared and the purified antibodies were obtained by affinity chromatographyaffinity with Protein A.3.Characterization of the antibodies.The subclasses and affinities of antibody were identified by indirect ELISA,and the specificity of antibody was determined by antigen competitive inhibition assays.4.Establishment of detection methods using candidate monoclonal antibody as primary antibody.Immunoblot,immunofluorescence,flow cytometry and immunohistochemistry were established using the candidate monoclonal antibody as the primary antibody.5.Preliminary application of the methods established with candidate monoclonal antibody.The gastric cancer cell line SGC7901 cells and primary gastric cancer XN0422 cells were known to express CMG2 and were used to investigate the detection effects of immunoblotting,immunofluorescence and flow cytometry methods.Immunohistochemistry that used candidate monoclonal antibody as primary antibody was performed on a microarray contained 162 gastric cancer tissues,a multi-organ tumor tissue microarray and a multi-organ normal tissue microarray.The specificity and sensitivity of staining were investigated.For gastric cancer samples wich have the pathological parameters and follow-up data,the correlation between CMG2 expression level and clinicopathological features and the prognosis of patients were statistically analyzed.6.Inhibition of gastric cancer cells by candidate monoclonal antibodies.high and low concentrations of candidate antibodies were added to the two gastric cancer cells.After 4 days of incubation,cells were counted,and the inhibitory effect was analyzed.Results1.Four antigenic polypeptides,including CMG2-ex?34-318 aa?,CMG2 epitope1?94-139aa?,CMG2-epitope2?153-200 aa?and CMG2-epitope3?261-306 aa?were designed out and expressed in prokaryotic expression systems.The purity of the antigen peptides purified by the nickel column was more than 90%,and the concentration of the purified prducts was more than 2 mg/mL,which fully met the requirements for preparing the monoclonal antibody.The results of animal immunoassay showed that CMG2-ex had the strongest immunogenicity and was used as the immunogen to prepare the monoclonal antibody.2.With CMG2-ex as the immunogen and eukaryotic expressed CMG2 ectodomain as screening antigen,three monoclonal antibodies were screened and named as 2F8,6A7 and8F3.Characterization showed that the specificity and affinity of 8F3 mAb were significantly better than 2F8 and 6A7 mAb,and its subtype was IgG1.So 8F3 was identified as the candidate antibody.3.Immunoblotting,immunofluorescence and flow cytometry established by using 8F3monoclonal antibody were used to detect the expression of CMG2 in gastric cancer cell lines.The results showed that 8F3 monoclonal antibody can be applied to immunofluorescence and flow cytometry methods,but cannot be used for immunoblotting detection,suggesting that it is directed to a conformational epitope.4.The immunohistochemical method established by using 8F3 monoclonal antibody showed good application effect and clinical application potential.The detection of CMG2expression in 162 gastric cancer tissue microarray showed that the staining was mainly in the cell membrane and cytoplasm,and no non-specific staining was observed.The expression of CMG2 in gastric cancer tissues was much higher than that in adjacent tissues,and the disease-free survival and overall survival of patients with high CMG2 expression were significantly shorter than those with low expression of CMG2.Cox univariate and multivariate analysis showed that CMG2 expression level was an independent prognostic factor for patients.These results were consistent with our previous data detected with a CMG2 polyclonal antibody of Proteintech company.In addition,the results of histochemical staining of human normal tissue microarray and human multi-cancer tissue microarray with8F3 antibody as the primary antibody showed that most normal tissues exhibited weak positive or negative reaction,and different tumor tissues showed different CMG2 expression levels.5.The 8F3 antibody had no significant inhibitory effect on gastric cancer cells,indicating that 8F3 could not be used as a neutralizing blocking antibody.ConclusionsA highly specific and high affinity 8F3 mouse anti-human CMG2 monoclonal antibody was successfully prepared and it could be used for immunofluorescence,flow cytometry and immunohistochemistry determination of CMG2 expression,but not for immunoblotting method.