Preliminary Application Of Antibodies Against Conservative Sequence Of Igκ Derived From Non-B Cell And The Initial Establishment Of System For The Induction Of Donor Specific Tolerance By ECDI-SPs

Classical immunoglobulin (Ig) is an important immune molecule which is secreted by B lymphocytes when stimulated by lymphocytes and then proliferating into plasma cells. Secretory Ig the form of antibody existing in body fluids, whose specificity combined with corresponding antigens and played a variety of immune function. Therefore, it plays an important role in specific humoral immunity; model Ig is existing on the surface of B cells in the form of the B cell antigen receptor (BCR) in living cells plays an important role of antigen recognition, differentiation and program cell death. Ig was mainly composed of two heavy chains and two light chains, with a typical folding domains. Each peptide chain includes variable and constant region and variable region can recognize and bind antigen. The effects of constant region and Ig function are related.According to the current immunological theory in normal body, immunoglobulin gene protein only in B lymphocyte development in the process of selective restructuring, and the gene in other somatic cells in germline status, is the characteristic product of B lymphocytes. However, the phenomenon of non B lymphocytes producing Ig molecules has been recognized by scholars at home and abroad. In 2003, Professor Xiaoyan Qiu, Peking University, first discovered that epithelial tumor cells and some of the proliferation of normal epithelial cells can produce Ig. Professor Qiu’s research group further the study and found that the Ig may be a growth factor like activity, functioning mainly on tumor growth and proliferation. Also, they found that the cancer cells producing the Ig molecule gene expression regulation mechanism and structure and source of B lymphocytes Ig typical characteristics are completely different.B lymphocytes can produce a variety of different specific Ig to cope with a variety of antigens. According to the theory, Ig is the combination of the variable region (V region) with the corresponding antigen, and the diversity of V region one the basis of the theory of response, recognition or binding. Immunoglobulin Ig by germline gene encoding, and gene by gene rearrangement into functional gene encoding. The source of diversity in the variable regions of Ig in B cell genome rearrangements occurring in combination and connected with the antigen stimulated B lymphocytes induced by high frequency mutation. According to the traditional theory of immunology, Igs only exsit in the process of selective rearrangementin of B cell development. Professor Qiu’s research team found that breast cancer, lung cancer, ovarian cancer, colon cancer, nasopharynx carcinoma, gastric cancer, pancreatic cancer and other epithelial tumor cells and normal epithelial cells in the presence of a functional Ig gene rearrangement.Exactly the same sequence of the variable region of expression of different types of tumor cells, the same individual and different individuals that tumor derived Ig sequences expressed are highly homologous. The study found that Ig database expressed in B cells were not found in these sequences, suggesting that the non Ig production by B lymphocytes is derived from tumor cells. Tumor derived from Ig gene transcription has the unique gene structure in the form of different types of tumor cells, especially light chain in all test cases were expressed in very similar rearrangement patterns or with a conserved sequence, namely Vκ4-1-Jκ3 Ig transcription the (patent application number is 200510107833.9).Considering that the Vκ4-1-Jκ3 Igs was found in tumors, it embodies the possibilities that it is a target for cancer research, diagnosis and treatment, which is essential for Vκ4-1-Jκ3 structure specific antibody. As a cooperative team belong to Beijing University, our team used bioinformatics method analysis and predicted Vκ4-1-Jκ3 Ig two specific B cell epitopes previously, with an intention to synthesize of three short peptides, namely QF20 QAEDVAVYYCQQYYDTPVTF, AL13 (ASINCKSSQRVSL), AL13B (TQSPDSLVVSLGERASINCKSSQRVSLG, also called the AL13 extended sequence), and have obtained the prokaryotic expression of Vκ4-1-Jκ3 Ig fusion protein (GST-Vκ4-1-Jκ3) from Professor Qiu. The synthetic peptide AL13B-KLH,and GST-Vκ4-1-Jκ3 immunized mice monoclonal antibodies. The anti Vκ4-1-Jκ3 Ig specific epitope antibodies on non B cells we have got proved to be an effective tool for the expression of Vκ4-1-Jκ3 Ig in tissue fluid and blood.The main purpose of this study is to identity monoclonal antibody that is against Vκ4-1-Jκ3 Ig specific epitope and to establish a detection system and its application of double antibody sandwich ELISA. We have used specific ELISA and Blot antibody Western to identify the specificity of ELISA and Blot antibody Western, which is a continuation of previous work done in anti Vκ4-1-Jκ3 Ig monoclonal antibody. Vκ4-1-Jκ3 Ig was earliest found in epithelial tumor cells and we intend to establish a double monoclonal antibody sandwich ELISA detection system, to find out whether observed differences can be found in total soluble secretary Vκ4-1-Jκ3 n normal people and cancer patients serum Vκ4-1-Jκ3 Ig expression.I Establishment and application of double antibody sandwich ELISA for detection of Vκ4-1-Jκ3 Ig systemCharacterization of anti-VK4-1-Jκ3 epitode monoclonal antibodies by GST-Vκ4-1-Jκ3 Specific Vκ4-1-Jκ3 Ig was found first in epithelial tumors, sharing the same basic structure with normal Ig. Previous work have used GST-Vκ4-1-Jκ3 immunized mice to obtain monoclonal antibody 4E9 and 1A3; In order to obtain monoclonal antibody 6G5,5D3 and 3H9.2 synthetic peptide was used in mice immunized with AL13B-KLH. Method of caprylic acid ammonium sulfate precipitation to purificate monoclonal antibody, and ELISA was used to identify purified monoclonal antibody and biotin labeled monoclonal antibody. The results showed that the anti Vκ4-1-Jκ3 Ig monoclonal antibody can specifically combine with protein GST-Vκ4-1-Jκ3 and no cross reaction was detected in the combination of human IgG and mouse IgG. Therefore,5 kinds of anti Vκ4-1-Jκ3 type Ig monoclonal antibody and biotin labeled monoclonal antibody can be used for the subsequent establishment of double antibody sandwich ELISA system.Establishment and application of double antibody sandwich ELISA for detection of Vκ4-1-Jκ3 Ig system The main purpose of this chapter is to establish the double antibody sandwich ELISA detection system, and the detection of tumor Vκ4-1-Jκ3 Ig level in normal people and patients. The preliminary work of our research team have observed significant difference in serum Vκ4-1-Jκ3 shows 3 types Ig content in normal people and cancer patients with single polyclonal antibody sandwich ELISA detection system. More specific, the performance for tumor patients serum soluble Vκ4-1-Jκ3 IgA and IgG content was significantly higher than that in normal; and Vκ4-1-Jκ3 IgM was significantly lower than that of normal people. Based on this, we have tried to establish double monoclonal antibody sandwich ELISA system for the detection of soluble Vκ4-1-Jκ3 of nuclear factor Igκ3 experimental screening to 1A3,4E9 as to capture antibody, PQ,6G5 and 5D3 as detection antibody. The results showed that double monoclonal antibody sandwich ELISA system specifically combined with GST-Vκ4-1-Jκ3 and it can also be combined with normal and tumor serum Vκ4-1-Jκ3 Ig. The reason that no significant differences are detected in serum Vκ4-1-Jκ3 total Ig expression between normal people and cancer patients proved the fault of the system.ECDI,abbreviation for 1-ethyl-3-(3’-dimethylaminopropyl)-carbodiimide, is a hygroscopic, water soluble chemical drug, widely used in peptide synthesis. Coupling with peptide and protein, ECDI could promote the activation of carboxyl group and the formation of covalent peptide mainly through the activation of the free carboxyl group. In 1979, Miller et al first proposed that ECDI coupled with antigen presenting cell has the potential to induce tolerance and confirmed that ECDI-APC can induce effector T cell anergy. ECDI-SPs coupling antigens in autoimmune diseases, such as multiple sclerosis, autoimmune diabetes, and experimental autoimmune meningitis can induce autoimmune tolerance.In the past few years, the short-term survial rate of patients with organ transplantation was significantly rasied, but the long-term survival rate beyond 10 years is still unsatisfactory. Side effects of graft rejection and long-term administration of immunosupressive drugs (non-specific) are the main reasons for this situation. Therefore, how to induce donor specific immune tolerance in transplant patients, and ultimately avoid rejection, disable, and even without immunosuppressive drug is one of the important goals of transplantation researches. One of the strategies to induce donor specific immune tolerance is to expose the donor antigen to the recipient before transplantation.The key point of this method is to determine the specific conditions of exposure to ensure that the induced receptor resistance is not induced by receptor sensitization. In a similar vein, the "child vaccination" project has been carried out in many experiments since the 1980’s,but it was excluded from clinical applications in the end because of the final cause of sensitization and pro thrombosis and other factors. Recently, some scholars applied a kind of ECDI to autoimmune disease related peptides or proteins cross-linked to spleen cells, and the infusion of autoimmune disease animal model successfully experimental autoimmune encephalitis (EAE) and autoimmune diabetes model induced by antigen toleranceXunrong Luo who was the first applicate the strategy into the field of transplantation tolerance with ECDI treated donor spleen cells (ECDI-SPs). In the mismatched MHC mice islet transplantation successfully induced islet grafts survive indefinitely. The study doesn’t rely on any immune inhibitor, which does not require transient cell deletion, antibody mediated blockade of the costimulatory signal or the use of immunosuppressive drugs before transplantation, etc. Further studies shows how ECDI-SPs infusion through a variety of mechanism can affect host allogeneic response, including cloning, clearance, and immune regulation, which worked in an alignment way to induce efficient through the synergy to transplantation immune tolerance. Except that in mice allogenic and xenogenic islet cell transplantation model was observed, ECDI-SPs long-term graft tolerance can be induced and before infusion delete B cells can effectively induce derived islet xenografts indefinite survival. The study also showed that ECDI-SPs could significantly prolong the survival of cardiac transplantation. Also, short term use of rapamycin to induce cardiac allograft survival in the recipient heart.This thesis was based on the previous studies, with an improvement on experiments of mixed lymphocyte in vitro, which consists of infusing ECDI-SPs and splenic lymphocytes to SCID mice (Severe combined immunodeficient mice, T and B cell deficient) and establishing, an in vivo mouse models. These experiments elucidate the ECDI-SPs (ECDI coupling spleen cells) induced by human lymphocytes at varous subsets of allogeneic response of dynamic change rule and mechanism. Stimulating allogeneic transplantation environment, which used ECDI-SPs to induce human lymphocyte antigen specific immune tolerance and explore the mechanism of donor specific immune tolerance.I The initial establishment of system for the induction of donor specific tolerance by ECDI-SPsProliferation and apotosis in the response of human recipient lymphocytes induced by ECDI-SPs The study has adopted mixed lymphocyte experimental flow type to testify the effect of human ECDI-SPs on the activation, proliferation, differentiation, incompetence, apoptosis, the dynamic changes of allogeneic T lymphocyte subsets on by the effects of lymphocyte and changes of cytokines in vitro. The results showed that ECDI-SPs induced programs could specifically inhibit T cell subsets proliferation. ECDI can induce apoptosis of human spleen lymphocytes.Establishment of an in vivo animal model with NOD-SCID mice The presence of human lymphocytes in mice in vivo was observed during a month period by infusing human spleen lymphocytes to NOD-SCID into mice in the initial stages. Studies have shown that liposome in NOD-SCID mice have cleared macrophages and after processing like 300cGy radiation,intraperitoneal injection of 3×107 spleen lymphocytes at different time points can be detected in human spleen lymphocytes. The initial establishment of an vivo animal model with NOD-SCID mice was proved. Later experiment could base on this model and optimize previous through vivo experiments.