Preparation, Characterization, And Initial Application Of Monoclonal Antibodies Against Human MBL-MASPs Complexes And Human Serum Albumin

MBL-MASPs complexes are composed by two types of protein molecules, namely the mannan-binding lectin (MBL) and MBL-associated serine protease (MASPs). As a family member of collectins, MBL is an important pattern recognition receptor of innate immunnity. MASPs and complement Cls/C1r are members of serine proteinase subfamily. At present the confirmed MASP family members include 3 serine proteases (MASP-1,MASP-2,MASP-3) and 1 non-enzymatic protein (sMAP), all of which can made complexes with MBL. There is very little MBL in human serum:averagely 1.0mg/L for normal people, which exists in the form of a supramolecular complex with MASPs. Hence the difficulty to obtain high-purity MBL-MASPs complexes and MBL/MASPs protein. There are multiple MBL-MASPs complexes in blood circulation, yet the remain many unknown questions about their actual types, stoichiometric relationship, adjusting mechanism during complex formation, etc.Besides selective recognition towards carbohydrate of various pathogens, MBL can also activate the complement cascade via lectin pathway through MASP-1, MASP-2 and MASP-3, which further enhance medicating and regulatory functions. It can also combine C1qR to start opsonocytophagic process, medicate the MBL dependent cytotoxicity, and function as a major molecule in mucous membrane defense.Serum MBL defects and decreased activation can lead to opsonization deficiency, which resulting in repeated acute or chronic infection of various pathogens. MBL gene mutation has close relationship with autoimmune diseases. Even the infection, development and outcome of special pathogens like HB V and HIV had something to do with MBL defects. The defecting of MASP-2 is mainly caused by genetic polymorphism, and its susceptibility impact towards infectious diseases is scarcely known. It was reported that MASP-2 defecting is a hazardous factor for post-chemotherapy infection, and the urine sMAP level for kidney cancer patients is higher than healthy people.As MBL and MASPs have the above essential functions, it is imperative to study pathogenesis for their defects, and the MBL-MASPs monoclonal antibody (McAb) is one of the major tools in researching. At present there was reports about MBL McAb preparation in China, yet there is still no domestic commercialized production of reagents while the foreign counterparts are quite expensive. Hence the urgent demands for national production of MBL-MASPs McAb.The unique character and innovative part of this study lies in:form the preparing of one McAb, obtain McAbs of multiple purposes which corresponding to MBL-MASPs complexes and MBL, MASP1, MASP2 & constituents of various epitope. Because MBL-MASPs complexes have complicated make-up, the difficulty of this study lies in the specific identification of various McAbs obtained. Via the available equipments, our initial proposed solution is to use ELISA, Western blotting and MBL function blocking tests, and comprehensive analysis which takes into consideration of constituents molecular weights.In the process of MBL-MASPs McAb preparation and identification, we had two strains of Human serum albumin (HSA)McAb, which proves that MBL-MASPs complex antibody we used for mice immunization contains small amount of HSA. As an essential part of hematological system, HSA's functions include combining and transporting endogenous and ectogenous matter, sustain blood colloid osmotic pressure, eradicate free radicals, control functions of platelet and influence artery's osmotic traits. In clinical use, high-grade HSA are widely used as plasma volume expander in treatment of massive hemorrhage, shock, burn, cancer, polycythemia and leukocytosis, hypoalbuminemia, etc. What's more, microalbuminuria is the earliest clinical manifestation of diabetes/renal diseases, as well as a signal for markedly higher cardiovascular disease morbidity and mortality rates. Early monitoring of microalbuminuria can bring early and active individual treatment, which can prevent or slow the processing of diabetes/renal diseases turning into End-Stage diabetic /renal diseases. With the above HSA functions and traits, in order to establish a keen, fast and accurate microalbuminuria testing method and HSA purification method, as well as to further purify MBL-MASPs complex and enable the research of the complexes to be more trustworthy, we have decided to apply hybridoma technique to prepare HSA McAb.Chapter One:Preparation, identification and initial application of MBL-MASPs complex monoclonal antibodyTo study the special function traits of MBL and MASPs, as well as the pathogenesis of their defecting, we had prepared McAb with MBL-MASPs complexes as antigen, in the hope of obtaining multiple McAbs in accordance with MBL-MASPs complexes, MBL, MASP1, MASP2, MASP3, sMAP and constituents of various epitope, especially the McAb that aims at MBL-MASPs binding site or the protein structure after the two integrated. Applying hybridoma technique, we had carried out multiple cell fusion and cloning, as well as human serum albumin in subtractive screening, and finally established the hybridoma cell strain which can secrete specific McAb. By combing the use of ELISA & Western blotting with molecular weights measurement for MBL-MASPs complexes and constituents in specific identification and analysis, and further examining the specific traits by measuring the McAb's blocking function to MBL, we had acquired 22 hybridoma cell strains which can possibly secrete MBL-MASPs complexes McAbs. After subgroup identification,10 strains belong to IgM subgroup while the other 12 IgG subgroup. For the IgM subgroup, we carried out initial identification via indirect ELISA; while for the IgG subgroup, further identification were done through Western blotting and MBL blocking. The result showed that among the 12 IgG McAbs,7 might aim at MBL Dimer or MBL(Dimer)-MASP2(monomer),1 aim at MBL single chain or MBL multimer; 1 aim at MBL monomer or MBL trimer; 1 aim at MBL trimer, while the other two aim at MASP1 and MASP3 respectively. Among these, the 5A7 hybridoma strain displayed same Western blotting results as those of commercial MBL antibody. We had chosen 5A7 and 1C8 in MBL function blocking tests and found both of them can be able to restrain MBL's stimulation towards PBMC secreting cytokine IL-6, IL-2 and IFN-y, which proves the two strains' MBL specificness. For the 12 IgG subgroup members, whether there were any McAbs that aim at MBL and MASPs binding site or the protein structure after the two integrated, further identifications are still needed. Besides, we carried out Horseradish peroxidase (HRP) marking to the purified MBL-MASPs complex McAb, which prepared for establishment of future ELISA examination system for MBL-MASPs complexes and relative constitutes. To sum up, through hybridoma techniques, we had obtained multiple MBL-MASPs complexes McAbs and carried out primary identification, which basically reached our planned goal and provided important tools for subsequent research works like MBL functional traits, MBL defects pathogenesis, protein isolation and purification, etc.Chapter Two:the Preparation, identification and primary application of Albumin Human Serum Monoclonal AntibodyIn order to establish a keen, fast and accurate microalbuminuria testing method and HSA purification method, as well as to further purify MBL-MASPs complex, we have decided to apply hybridoma technique to prepare HSA McAb, hoping that HSA McAb can help to build double-antibody sandwich ELISA detection system, and to prepare antibody conjugated affinity chromatography column. Through multiple cell fusion and cloning screening, we had established specific hybridoma cell strain. By combing the use of ELISA & Western blotting in specific identification and analysis, using the method of Cross-match to choose the two suitable antibodies of ELISA, applying Chessboard titrimetric method to determine appropriate working con-centration of coating antibody and detecting antibody, making antibody conjugated affinity chromatography column with HSA McAb and CNBr actived Sepharose 4B. In the end, a total of 20 hybridoma cell strains which can secrete HSA McAbs were acquired, which were indentified as IgG subgroup. All McAbs obtained were purified through Octanoic-ammonium sulfate method, and HRP labled via Simple sodium periodate method. The indirect ELISA and Western blotting tests had demonstrated good specific examination results. We had carried out in-depth research for 1C11, 2E9,3A5 and 4H1, and initially set up 3A5-2E9 and 2E9-2E9 antibody sandwich ELISA testing systems. The standard curve for the first one is:y=0.1764Ln(x)-0.2378, R2=0.9689; while the latter one is:y=0.1676Ln(x)-0.2079, R2=0.9663. Testing scope for both system is 16-2000ng/ml with testing sensitivity 8ng/ml which conformed with requirement for microalbuminuria testing, yet there were still room for improvement and clinical test. We had successfully prepared antibody conjugate and affinity chromatography column for McAb 1C11, and Western blotting results showed that the affinity chromatography column could isolate and purify HSA in human serum and MBL-MASPs complexes. In other words, it can remove the HSA impurities in MBL-MASPs complexes. However, the functional activities of purified MBL-MASPs complexes still remain to be further examined.