Establishment Of Double-Antibody Sandwich ELISA To Detect Endosialin/TEM 1(CD248)

BackgroundMalignant tumor is an important problem in human health. According to the different origination, tumor was named carcinoma or sarcoma respectively. Carcinoma is derived from epithelial cell. Sarcoma originate from connective tissue or bone which are derived from embryonic mesoderm. There are over 100 types of sarcoma. Liposarcoma and leiomyosarcoma are the most common type of sarcoma. Most of sarcoma occur in adults over 55 years, such as leiomyosarcoma, malignant fibrous tissue sarcoma, synovial sarcoma and liposarcoma. Some types of sarcoma predominantly occur in children, such as Ewing’s sarcoma, rhabdomyosarcoma and osteosarcoma. Compared with carcinoma, the incidence of sarcomas in adult malignant tumors was 1%, but sarcomas have higher grade malignancy, early blood metastasis and high mortality, nearly 50% patients with sarcoma indicate incurability. Sarcoma has a lot of different physiological and molecular types. Currently, diagnosis of sarcoma rely on X-rays, CT scans, magnetic resonance imaging to imaging (MRI), positron emission tomography (PET) and other imaging methods, then they will be confirmed by pathological biopsy.Early screening and diagnosis is important for tumor. Tumor marker is an effective role in tumor diagnosis. Tumor markers will contribute to early diagnosing and effective monitoring of the tumor. Therefore, PSA, CEA, AFP, hCG, CYFRA21-1, SCC, NSE, CA19-9, CA125, TPS-A, etc. a wide variety of serologic tumor markers have been used for diagnosis for many years in clinical. Following the study, new tumor markers have been found. Leukopenia, ESR, LDH, ALP and other serum markers were used in sarcomas, but there had poor specificity in these tests. New serum markers are necessary for early and specific diagnosis in sarcoma.Accumulated evidences suggested that endosialin/TEM1(CD248) will be a promising diagnostic marker for sarcoma. CD248 is a type I transmembrane glycoprotein which comprises an 80.9 kDa protein core, containing 757 amino acids. Its extracellular part consists of a mucin-like region and five globular domains (N-terminal C-type lectin domain, sushi-like domain and three epidermal growth factor (EGF)-like repeats). Its extracellular structure is complex, and the intracellular domain is only 51 amino acids short, contains three potential phosphorylation sites and a C-terminal PDZ binding end. Through a transmembrane, the extracellular part connect with the intracellular part. CD248 was expressed mainly in embryo period, and progressively diminished after birth, almost no expression in all kind of organ except kidney in the healthy adult. CD248 was first identified as a tumor vascular endothelial antigen in the early 1990s, however, it was later reported to be a marker of stromal fibroblasts and a subset of pericytes associated with tumor vessels as well as human mesenchymal stem cells, but not tumor endothelium. Then the studies in other groups showed that CD248 is expressed in the almost kinds of sarcoma cells., and when sarcoma cells were cultured in vitro, the CD248 in culture supernatant can be detected as well. CD248 is confirmed express in the mesangium and a subset of interstitial cells in healthy kidney. CD248 expression was upregulated during fibrosis, and correlated with progression of human renal disease.CD248 was also overexpressed in patiens with some types of cancer and patients with arthritis.Therefore, in this study, we want to found a ELISA method to detect CD248 in serum. This new marker will help better track and treat for sarcoma,CKD and arthritis patients.Objectives1. To construct a recombinant prokaryotic expression vector and acquired CD248 p rotein;2. To generate rabbit or mouse polyclonal antibodies for CD248;3. Establish a double-antibody sandwich ELISA assay for Endosialin/TEM1 (CD248) detecting.4. To evaluate the established double-antibody sandwich ELISA assay for Endosialin/TEM1(CD248) detecting primarily.Method1. The prokaryotic expression of CD248The primers were designed refering to the human CD248 gene sequence in pGEX-2TK vector and the multiple cloning site in pMAL-5x, then the fragment of CD248 was clone and insert the pMD19-T vector by TA clone method. The gene sequence was confirmed by sequencing. Then pMAL-5x and pMD19-CD248 were double digested with NdeⅠ and EcoRI. The fragment of CD248 and pMAL-5x were purified and conneted with DNA ligase, then the recombinant expression plasmid pMAL-5x-CD248 was constructed with the ligation product. The protein CD248 expression was induced IPTG, and was confrimed by SDS polyacrylamide gel electrophoresis display. After isolation of the inclusion bodies, the protein CD248 purification and renaturation was studied.2. Preparation anti-CD248 polyclonal antibodies from rabbit and mouseBALB/c mice were divided into three groups with different CD248 antigen concentration with 32.05μg,64μg,128.2μg. The antiserum titer was deteced in 7 days after the fourth immunization. Then the different group was boosted with 64.1μg MBP-CD248,64.1μg GST-CD248 and 5 x 105 active human osteosarcoma SJSA-1 cells respectively at the fifth immunization,10 days latter, all blood were collected and serums were separated. New Zealand white rabbits were immunized using 897.4μg MBP-CD248 protein in the first three times, then one rabbit was boosted with 1 x 107 SJSA-1 cells. Seven days after the fourth immunization, blood was collected from rabbit carotid artery and stored at-20℃. The antibody titers was detected with Indicted ELISA assay.3. Establishment of double-antibody sandwich ELISA methodDifferent concentrations of rabbit anti-CD248 polyclonal antibodies were coated on ELISA plate as the capture antibody, the mouse anti-CD248 polyclonal antibody was used to be the detection antibody. The optimization of conditions were confirmed with purified CD248 protein.4. Evaluation of the established double-antibody sandwich ELISA assayThe sensitivity of this new ELISA method was analysised by diluted CD248 protein solution. Moreover, ELISA method was evaluated by detected the CD248 in culture supernatants of active human osteosarcoma SJSA-1 cells and CKD patients serum.Results1. The prokaryotic expression vector pMAL-5xCD248 was successfully constructed. The molecular weight of MBP-CD248 was about 75KDa. and the MBP-CD248 was expressed with 1mM IPTG induced for 3h at 30℃. Expressed protein CD248 existed in the form of inclusion body. The inclusion body was washed with denaturation buffer firstly,then followed by denaturation and refolding in proper buffer to harvest CD248 protein.2. After immunized with different concentration of purified MBP-CD248 protein, the antbody titer in low-dose group were 1:102400; 1:204800 and 1:409600; the antbody titer in middle-dose group were 1:204800,1:204800 and 1:102400; the antbody titer in high-dose group were 1:204800,1:204800,1:204800. Antbody titer in rabbits were 1:1024000.3. The best concentration of purified rabbit anti-CD248 polyclonal antibody to capture was 15.12μg/ml. The detection antibody used in 1:16000 titer. The HRP labelled antibody used in 1:20000 titer.4. The minimum detective MBP-CD248 protein was 18.31 ng/ml and GST-CD248 protein was 585.94ng/ml with the new ELISA assay. CD248 in cultured supernatant can be deteced with human osteosarcoma SJSA-1 cells cultured in 108h.Part of CKD patients could be distinguished from healthy people.Conclusion1. Acquired the CD248 protein by prokaryotic expression systerm;2. Preparate anti-CD248 polyclonal antibodies from rabbit and mouse;3. Successfully establish a double-antibody sandwich ELISA assay for Endosialin/TEM1/CD248.