Preliminary Application Of Antibodies Against Conservative Sequence Of IgVκ Derived From Non-B Cell

Immunoglobulin (Ig) is secreted by plasma cell differenciated from B lymphocyte stimulated with antigen. Ig is presented in secretory type as antibody in body fluid and also in membranous type as B cells antigen receptor (BCR) on B cell. The antibody can bind with corresponding antigen to mediate a variety of immune function, which plays an important role in specific humoral immune. The membranous type as BCR execute the role in antigen recognition, cell activation, cell differentiation and programmed cell death. Ig is constructed of two heavy chains (H chain) and two light chains (L chain), with the structure of the typical folding. And both H and L chain include variable region (V region) that bind with antigen, and constant region(C region) that is correlated with effect function of Ig.Ig is the most classical immune molecules and derived from the B lymphocytes, which has been well accepted by academic field. However, the phenomenon that non-B cells may produce Ig molecular has gradually been confirmed and accepted by many domestic and foreign scholars in recent years. The first reporter of this phenomenon is Dr. Xiaoyan Qiu of Beijing University. She found that epithelial hyperplasia tumor cells and a part of the normal epithelial cells can also generate Ig. The team of Dr. Xiaoyan Qiu found the Ig may have the function of growth factor-like activity in their preliminary study, the further research suggests that the Ig molecules produced by cancer cells is different from the typical features of B cell derived Ig in structure and gene expression regulation. This discovery has been published in "Cancer Rearch" (2003), which was awarded as one of the top 10 news for 2004 in the medicine and hygienical fields of China.B cells can produce a lot of Igs with different binding specificity to deal with a wide variety of antigens. Ig bind with antigen by V region, thus the diversity of V region could ensure the binding with all antigens. Ig is coded by germline genes that become functional coding genes through gene rearrangement. The diversity of Ig V region depends on gene rearrangement, connecting process in B cell and high-frequency mutations in B cell stimulated with antigen. According to classical immunological theory, the Ig can only be selective rearrangement in B lymphocytes during development; whereas the genes of other cells are in germline state, or rearrange insufficiently, which is attributed to no functional rearrangement of the Ig gene and no Ig molecules generated. X Qiu found that epithelial original tumor cells including lung, breast cancer, colorectal cancer, stomach cancer, nasopharyngeal cancer, pancreatic cancer, ovarian cancer etc, and alos a part of the normal epithelial cells exist functional rearrangement of the Ig gene, which were demonstrated by laser microscopic cutting (LCM) and RT-PCR technology. The usage frequencies of VDJ fragment in heavy chain present an obvious propensity.to certain recombinations, especially in different cases and different types of cancer cells may exactly express the same or similar sequence of V region. Importantly, these highly homologous sequences can not be found in Ig database of B cell, suggesting that this kind of Ig is not derived from B cells. The Ig gene transcription has this unique genetic structure, in different tissue types of the cancer cells, especially in the light chain in all of samples cases express the same conservative sequences or the similar rearrangement pattern (individual bases replacement), namely Vκ4-1-Jκ3 Ig transcription (Patent No.200510107833.9).Since IgK with Vκ4-1-Jκ3 was found in tumor cells, it might become a target for pathogenesis research, diagnosis and therapy of tumor, and the specific antibody against Vκ4-1-Jκ3 would be one of most important tools. As a collaboration project with Dr. X Qiu in Beijing University, we have already generated some polyclonal and monoclonal antibodies (mAbs) against special sequences of Vκ4-1-Jκ3 Ig, including AL13(ASINCKSSQRVSL), QF20 (QAEDVAVYYCQQYYDTPVTF), AL13B (TQSPDSLVVSLGERASINCKSSQRVSLG, prolonged AL13), and also a prokaryotic recombinant Vκ4-1-Jκ3 fusion protein with GST tag from Dr. Xiaoyan Qiu. This study is aimed to identify the specificity of anti Vκ4-1-Jκ3 mAbs and to try to use these mAbs on establishment of sandwich ELISA system for detection of Vκ4-1-Jκ3 Ig in body fluid and screening of Vκ4-1-Jκ3 Ig expression in no-lymphocytes or tissues. In our work, ELISA, Western blot were used to characterize the mAbs, which was a part of coherent work following the preparation of anti Vκ4-1-Jκ3 Ig mAbs. By these mAbs,, we also found Vκ4-1-Jκ3 Ig expression in some normal tissue and normal serum. Since this Vκ4-1-Jκ3 Igκoriginated from the epithelial tumor cells, the one of subjects in this work is to know if any differences of Vκ4-1-Jκ3 Ig existes in epithelial neoplasm patients and the normals. Therefore, we establish the sandwich ELISA system to detecte soluble Vκ4-1-Jκ3 Ig or Igκand its biological significance. In addition, we tried to use fetal tissue chip and a part of tumor cells to search expression of Vκ4-1-Jκ3 Ig or Igκ, which may provide valuable evidence or insight for the further study. Part I Characterization of Vκ4-1-Jκ3 Ig in normal serum by monoclonal antibodiesAlthough Vκ4-1-Jκ3 of Ig was found in epithelial neoplasm in high usage frequency, its basic structure is the same as normal Ig. Therefore, we performed preliminary appraisal on expression characteristics of the molecular in normal serum by anti Vκ4-1-Jκ3 Ig mAbs. The antibodies for these detections include 1A3 and 4E9 that were prepared by immunization prokaryotic recombinant GST-Vκ4-1-Jκ3 fusion protein, and 6G5 and 3H9.2 that were prepared by immunization with a 28mer synthetic peptide AL13B-KLH. The specificity and purity of mAbs was identified by Western blot based on SDS-PAGE and native PAGE, ELISA. The purity and molecular weight of mAbs were identified as the coincidence to correct values of molecular weight in heavy chain and light chain, and as the proof of less noise and high purity in four mAbs, indicating that all of four mAbs can be used in the subsequent experiment. And the specificity of four mAbs was well proved by their specifically binding with prokaryotic recombinant GST-Vκ4-1-Jκ3 fusion protein and a synthetic 28mer peptide including Vκ4-1-Jκ3 sequence, and no cross reaction with other species of IgG. We also found Vκ4-1-Jκ3 Ig existed in normal serum by Western Blot, therefore we prepared 3H9.2 affinity chromatography column to purify Vκ4-1-Jκ3 Ig from normal serum or plasma, and the purified product was identified by ELISA as well as Western Blot. We found that the purified product show a complete Ig structure proved by Western blot, and the purified products can bind with four mAbs specifically. In addition, three isotypic classes of Vκ4-1-Jκ3, namely IgG, IgM, IgA in normal serum were proved by Western Blot. The phenomena of IgG, IgM and IgA with Vκ4-1-Jκ3 Ig in normal serum suggest the requirement of detecting the samples from patients with different diseases, as well as the biologic significans of this phenomenon in patients. Part II Establishment of sandwich ELISA system for detection of Vκ4-1-Jκ3 of Ig in serumThe main object of the work presented in this chapter is to establish a sandwich ELISA detecting system, and thereby it could be used to determine the level and assess the significance of Vκ4-1-Jκ3 Ig in tumor patient and the normal. First,the four mAbs for cross-pairing to determine the capture antibody and detection antibody for setting up a sandwich ELISA system to detect Vκ4-1-Jκ3 Ig in serum. We used four mAbs as capture antibody, respectively, and HRP-rabbit anti-human IgG, HRP-goat anti-human IgM as well as HRP-goat anti-human IgA as detection antibody to build sandwich ELISA detecting system, since these paring with four mAbs to Vκ4-1-Jκ3 are not suitable for this sandwich because of nonspecific binding. Fortunately, above pairing with specific mAb and anti-isotype antibody made us to notice the IgG, IgA and IgM with Vκ4-1-Jκ3 sequence in serum samples. During choosing the best sandwich ELISA detection system, we has also tried to use mouse anti-human IgG mAb, mouse anti-human IgM mAb, mouse anti-human Igκchain mAb, goat anti-human IgG polyclonal antibody (pAb), goat anti-human IgM pAb, goat anti-human IgA pAb as capture antibody and use HRP-1A3, HRP-4E9, HRP-6G5, HRP-3H9.2 as detection antibody, but all of pairing forms were failure. Finally, we have defined 1A3 as capture antibody and HRP-rabbit anti-human IgG, HRP-goat anti-human IgM, HRP-goat anti-human IgA as detection antibody to set up the sandwich ELISA system that show high sensitivity and low noise background. Furtherment, we determined the optimal working concentration for capture antibody and detection antibody to establish standard curve for IgG, IgA and IgM with Vκ4-1-Jκ3 sequence in human serum, in which a normal serum was used as standard substitute defined by purified Vκ4-1-Jκ3 molecules, since a purified Vκ4-1-Jκ3 molecules from normal serum is not stable compare with that in serum Moreover, we preliminarily determined the serum titers of Vκ4-1/Jκ3 IgG/IgM/IgA in samples of 200 normal and 155 patiens with different cancer, the results show that the titre of Vκ4-1-Jκ3 Ig in normal serum and epithelial neoplasm serum is significantly different, the titers of Vκ4-1-Jκ3 IgG and IgA in patients are higher than that in normal (P<0.01, P<0.001); and Vκ4-1-Jκ3 IgM in patients is lower that that in normal (P<0.001)PartⅢThe application of anti-Vκ4-1-Jκ3 Ig mAbs in cytologic researchIn view of the phenomenon that Vκ4-1-Jκ3 was found in epithelial tumor cells and the work which came from non-B cells Ig by gene sequencing, we tried to know the expression of the Vκ4-1-Jκ3 molecular expression of non-B lymphocytes and tissues by the specific antibodies.It is a classical concept that Ig is secreted by or express in B cells, so we temporarily call the expression of Ig in non-B cells as non-classical expression or non-B Ig. The trial on the expression of Vκ4-1-Jκ3 in epithelial tumor cells have been widely performed in department of immunology,Beijing university, so we choose a part of tumor cells, neutrophils and human fetal tissue to start reserches by flow cytometry, laser confocal microscope and immunohistochemistry. The results show that anti V 4-1κ-Jκ3 mAbs can be bind with human neutrophils and HL-60 cell line as well as tco-localize with the commercial anti-IgG and anti-human Igκantibodies, even bind with neutrophils of RAG2" mice that are in deletion of T cell, B cell and NK cell. The binding of anti-V 4-1κ-Jκ3 mAbs to neutrophis has not been reported in other group, which would be futher confirmed. Part IV TLR2 agonist reverses the tolerance mediated by dendritic cellsThis is a part of basic technique training in my postgraduate program, and the subject belongs to project supported by Natural Science Foundation of China (NSFC). In this work, an IL-10 transfected dendritic cell (DC) model was established, and TLR2 agonist Pam3CK was confirmed to be able to reverse immune suppression induced by IL-10 transfected murine myeloid dendritic cells.In recent years, the DC mediated immune regulation including the induction and maintain of tolerance-DC have been paid much attension on central and peripheral tolerance as well as to solve puzzles in transplant rejection and autoimmune diseases. Uner certain conditions, IL-10 transfected DCs can induce antigen specific immune tolerance, which is called as tolerance dendritic cells (Tol-DC).The IL-10 transfected murine myeloid DCs were detected by flow cytometry and ELISA, the result shows that the CDllc+ cells is more than 90% and the amount of IL-10 in supernatant of 106 IL-10 transfected DC is approximately 300 pg/ml in 48 hours, which successfully established IL-10 transfected DC model secreting IL-10. DCs, vector transfected DCs, IL-10 transfected DCs were stimulated with TLR2 ligand Pam3CK in 48 hours, then MHC II, CD80,CD86 and FasL were detected by flow cytometry, and the amount of IL-6 and TNF-a were determined by ELISA. The results show that IL-10 can inhibit the CD80, CD86 and MHCII expression of mDCs and reduce the amount of IL-6 and TNF-a secreted but promote FasL expression. However, TLR2 agonist Pam3CK improve MHC II, CD80, CD86 expression of IL-10 transfected DC and promote the secretion of IL-6 and TNF-α, but inhibit FasL expression. The results show TLR2 agonists effectively reverse immune response suppression mediated by Tol-DC.