Preparation, Characterization, And Initial Application Of Monoclonal Antibodies Against MASP2-C

The pathogen susceptibility depends on the body’s resistance, and innate immunity is the first-line of host to defense infection. At present, as the innate immunity research rapid development, which tell us that research the prevention and treatment of infected disease from innate immunity may be a windfall. The lectin pathway, as the third pathway of complement activation, it is an important component of the innate immunity system, and mannan-binding lectin associated serine protease2is the key of this way.MASP2and complement Cls/Clr are members of serine proteinase subfamily, it exits as zymogen in the plasma. At present the confirmed MASP family members include3serine proteases (MASP1、MASP2、MASP3) and2non-enzymatic protein (MAp19、MAp44).Through alternative splicing, MASP2and MAp19are produced from MASP2, whereas MASP1、MASP3、MAp44are produced from MASP1, all of which can made complexes with MBL. The single peptide chain of MASP2is composed of six modules and a linker region:an N-terminal complement subcomponent Clr/Cls-like domain(CUB1), followed by an epidermal growth factor(EGF)-like domain of the Ca2+-binding type, a second CUB domain(CUB2), two contiguous complement control protein modules(CCP1and CCP2), a short linker and finally a chymotrypsin-like serine protease(SP) domain. The present study indicates that the three domains of MASP2interacted with MBL-CLR via the N-terminal three modules (CUB1-EGF-CUB2) depend on the Ca2+, and the other three domains of MASP2C-terminal is serine protease activity center, complement activate are mainly rely on the SP domain to play the role of serine protease, cleave both complement factors C4and C2, hence forming the C3convertase.In lectin pathway, MBL and ficolins circulate in complexes with MASP1/MASP2(homologs of C1r and C1s).The former recognition and combined with the mannose, N-acetyl glucose and other carbohydrate structures which on the surface of pathogenic microorganism through the CRD, thus the latter was activated, the activated MASP2cleaves complement factors C4and C2to form the C3convertase C4b2a,thus initiating the activation of the complement system. Therefore, MASP2plays a crucial role in the lectin pathway because both MBL and other ficolins(H,M and L) need it to activate complement.Recent research shows that the changes of serum MASP2concentration are closely related with infectious disease, tumor, autoimmune disease and other diseases. MASP2gene point mutation, which is in the CUB1domain-where codon GGC was found instead of GAC, resulted in the substitution of glycine for aspartic acid, which lead to significantly low levels of serum MASP-2. Moreover, the mutant MASP-2could not associate with mannan-binding lectin and thus could not form the active mannan-binding lectin-MASP complexes, so as to cause or aggravate pathogen infection.Previous studies have shown that the level of serum MASP2is an independent mark to predict colorectal cancer recurrence and survival time. It is also associated with the progress and aggressive of esophageal squamous cell carcinoma. In addition, MASP2gene polymorphism is related to susceptibility to HCV infection, which variant is associated with high plasma levels of the MASP-2in hepatitis C patients.Now the datas show that different region and ethnic populations, the MASP2gene point mutation sites has obvious difference, and mutation frequency, serum level are also have difference. But the current, we have no comprehensive report about Chinese or the peoples serum MASP2concentration, the relationship with innate immunity and related diseases.Since MASP2has the above essential functions,it is necessary to do the further study for it, and the MASP2monoclonal antibody(mAb) is one of the major tools in researching. We use the purified recombinant MASP2-C protein as the antigen to immune the BALB/C mice, prepared the mAbs against MASP2-C, and identified their properties. The sandwich ELISA to detect MASP2protein in human serum was established based on the purified mAbs. In this work, therefore, we would like to supply valuable tools for detection serum MASP2concentration and study the biological functions of MASP2and the diagnoses of diseases related to MASP2deficiency.Part I. Prokaryotic expression of MASP1,2-C proteins and identity the target proteinsThe serum concentration of MASP2is under1μg/ml, and co-existence in MBL-MASPs complexes with MASP1, MASP3, sMAp and MBL. So, it is difficult to obtain high purity natural MASP2from plasma. Therefore, we use molecular biology technology to get the target protein via prokaryotic expression system. For MASP2exits as zymogen in the plasma and can autoactivate in the body, which go against the subsequent experiments. Considering that the MASP2N terminal and C terminal each has different function, We adopt section express strategy for the preparation MASP1-C, MASP2-C protein.The human MASP1,2gene fragment was amplified by PCR from pGEM-MASP1,pGEM-MASP2plasmid which contains human MASP1,2cDNA. and then inserted into prokaryotic expression vector pET17b, which named pET17b-MASP1C and pET17b-MASP2C. After identified by PCR and sequencing, the two recombinant expression vectors were transformed into Ecoli BL21(DE3) cells and screened by ampicillin. The expressed products were purified by Ni2+-NTA agarose Affinity Chromatography. The two target proteins of Mr40000in the purified recombinant products were detected by SDS-PAGE and could be recognized by anti-6His antibody in ELISA assay. The prokaryotic cell strain that excesses efficiently recombinant human MASP1and2-C proteins and the purified MASP1and2-C proteins were successfully obtained, which provides the basis for further research.Part II. Preparation and identification of monoclonal antibody against MASP2-CSix-week-old female Balb/c mice were immunized with purified recombinant MASP2-C protein emulsified with adjuvant for3times. The mAbs to MASP2-C were generated according to the conventional protocol. In briefly, spleen cells from immunized mice were fused with myeloma cell line NS-1to form the hybridoma cells. The anti-MASP2-C antibodies in the hybridoma culture supernatants were screened by indirect ELISA with MASP2-C as antigen and unrelated protein which has His label as control to exclude the production of antibody to His. The selected hybridoma cells were injected into mice pretreated by Freund incomplete adjuvant to generate ascites including monoclonal antibodies. The mAbs from ascites were purified by precipitation with caprylic acid and anamonium sulfate. The immunoglobulin isotypes for all the15mAbs were identified as IgGl, κ light chain.Indirect ELISA shows that in addition to mAb2B11,2B3have cross reaction with MASP1-C, MASP2-N, the rest13mAbs only reacted with MASP2-C, mAb to His screening is negative. Also, SDS-PAGE and Western blot demonstrated that all of them have a better specificity. Besides, we demonstrated that mAb1D1,4C11,2C11,3G4have activity to bind with natural MASP2, and can block complement lectin pathway activity by C4b deposition assay. Part Ⅲ. The establishment of sandwich ELISA detection system for MASP2-CThe purified recombinant MASP2-C protein as the antigen was used to immune the New Zealand rabbit, prepared the polyclonal antibody against MASP2-C.We labeled4mAbs(2B11,3D4,5A1,4C8) with Biotin, and then set up the sandwich ELISA with several kinds of antibody pairs including mAb-mAb, PcAb-mAb, mAb-PcAb as capture antibody and detecting antibody, repectively. The results showed that almost all of sandwich ELISA by above antibody pairs can be used to determined MASP2-C specifically sensitively. The best antibody pairs which have higher positive value and lower negative value, sensitive to natural MASP2were screened, we establish and optimize the system. At the capture antibody concentration is1μg/ml, sealing liquid is0.25%casein, lotion is0.5%o PBST, antibody diluent is PBS, detection antibody concentration is0.5μg/ml, the system can sensitive detect the standard substance and plasma MASP2, and the standard curve has a good fitting. This detection system measurement range is10-1500ng/ml, strong specificity, good repeatability, adjust to clinic detection require.