Preparation And Application Of Murine Monoclonal Antibodies Against Endosialin/TEM-1/CD248

BackgroundEndosialin,also known as CD248 or tumor endothelial marker 1(TEM-1),is a type of cell surface glycoprotein produced by stromal cells[1].The expression of CD248 is high in the embryo and progressively diminished postnatally,in the post-natal period of healthy adult,there is a little retention of CD248 expression only in kidney and endometrium.In 1992,investigators reported the identification of CD248,then studies revealed that CD248 appeared predominantly at tumor-associated stromal fibroblast and tumor-associated pericytes,and the expression of CD248 is associated with the prognosis and progression of tumor.CD248 knockout(CD248-KO)mice not only normal,but also significantly reduce the tumor volume and the ability of tumor invasion and metastasis[2,3].Recent studies have confirmed that CD248 is closely related to the development and progression of fibrosis[4].The expression level of CD248 in human kidney/liver/pulmonary fibrosis specimens was positively correlated with the degree of tissue fibrosis.In the mouse model,knockout of the CD248 gene would inhibits fibrosis.Based on the important role of CD248 in chronic inflammatory diseases such as tumor and fibrosis,the study of CD248 has attracted more and more attention[5,6].ObjectivesIn this study,our objective is to obtain monoclonal antibodies specific against CD248 by the hybridoma technique,then the application of the monoclonal antibodies specific against CD248 was evaluated in the clinical application of tumors and fibrosis samples,which will lay a foundation for the exploration of the diagnosis and therapy of the CD248 targeted tumor and fibrosis.Methods1.The recombinant protein MBP-CD248 was expressed by prokaryotic system Escherichia coli.2.The hybridoma cell lines which can secrete anti-CD248 monoclonal antibodies were obtained by hybrid technique.3.To develop an ELISA-based antibody assay of CD248 with anti-CD248 monoclonal antibodies.4.To detect the CD248 in serum and urine from malignant tumor patients and renal fibrosis patients by the antigen capture immunoassay and Western blotting.5.To detect the CD248 of renal tissues from unilateral ureteral obstruction(UUO)model of renal fibrosis and clinical renal fibrosis specimens by immunohistochemistry.Results1.The recombinant protein MBP-CD248 was successfully expressed by prokaryotic system Escherichia coli and was successfully purified.2.Nine hybridoma cell lines steadily secreting monoclonal antibodies against CD248 were obtained;The affinity constant of MAbs ranged from 1.2×1010 L/mol to 8.7×1010 L/mol;Nine MAb can bind with membrane protein CD248 from the recombinant protein MBP-CD248 and osteosarcoma SJSA-1 cells by Western blotting;Flow Cytometry identified that nine MAbs could bind with osteosarcoma SJSA-1 cells well;The competitive inhibition assay showed that the nine MAb-CD248 can recognized at least three distinct antigen epitopes of CD248.HRP labeled monoclonal antibody titer were 1:3200?1:102400.3.CD248-M7 as a solid-phase immobilized capture antibody and CD248-M41-HRP as a detecting antibody to develop a antigen capture immunoassay of CD248.This method detection limit of the recombinant protein MBP-CD248 was 9.77 ng/mL.4.The serum CD248 was no difference among healthy individuals,tumor patients and fibrosis patients,which were detected by the ELISA and Western blotting.5.The expression of CD248 from the unilateral ureteral obstruction(UUO)model of renal fibrosis and clinical renal fibrosis patients was closely related to the severity of fibrosis.6.The expression of urine CD248 from 113 clinical protein urine samples were analyzed by western blotting:the CD248 positive rate of acute non-renal disease group was 13.3%;The CD248 positive rate of chronic kidney disease(CKD)patients was above 45%,especially in the CKD-5 patients was 65%.The categorical variables among these groups using chi-square test have significant differences(?2 = 97.07,v=5,p<0.05).Conclusion1.We had successfully screened CD248 specificity monoclonal antibodies and the immunogen is recombinant protein MBP-CD248,which was obtained from the genetic engineering technique.2.An antigen capture immunoassay for CD248 was successfully developed.3.There was no significant change in content of serum CD248 between tumor patients and renal fibrosis patients which were tested using present method we had developed.4.The monoclonal antibodies specific against CD248 can be an useful tool in the detection and analysis of clinical renal fibrosis.5.The CD248 in albuminuria may be as an evaluation index of renal fibrosis.