Research On The Preparation And Application Of Monoclonal Antibody Against Human IDH1 R132H

Glioma also known as primitive neuroectodermal tumors is the most frequently occurring primary brain tumors which originates from neural ectoderm,accounting for about 40%of intracranial tumors and 60%of the epithelial tissue nerve tumors.Therein more than half of them were glioblastoma multiforme(GBM),which median survival is only about one year and characterized by invasive growth and poor prognosis.The treatment is focused on three traditional methods including surgical resection,radiotherapy and chemotherapy.Due to the lack of specific molecular markers,the diagnosis,classification and identification of glioma from other disease are very difficult.Moreover,with the rapid development of advanced medical technology such as MRI,CT and needle biopsy,especially the samples taken from the tumor edge area,contain only limited diagnostic information,and the diagnosis and treatment choice almost entirely depend on histopathology.So looking for malignant gliomas specific molecular markers is important for its diagnosis and treatment.In 2008,Parsons used high-density oligonucleotide arrays to analyze 22 human tumor samples and found in～70%of low-grade gliomas(grade Ⅱ and Ⅲ),as well as secondary glioblastoma multiforme(grade IV)patients’ isocitrate dehydrogenase 1 gene(EDH1)active site G395 was mutated.The most common mutation is guanine to adenine(G395A),which amino acid is arginine to histidine(R132H),accounting for about 90%.Wild-type IDH1 patients’ average survival time is one year,which is shorter than the mutant IDHI patients.The researchers from Heidelburg University,Duke University and University of Tsukuba developed three IDH1 R132H antibodies in 2009 and 2011,which can be applied to detect IDHI R132H expression at different levels and diagnosis of glioma using immunohistochemical staining.They were also be used for the prognosis of clinical judgment and the choice of treatment.The first step is to prepare the immunogen for generating monoclonal antibodies against a peptide conaning a mutated amino acid.The methods of preparation immunogen carrying a small peptide include chemical synthesis peptide carrying a certain amino acid mutation,followed by coupling hemocyanin(KLH)or bovine serum albumin(BSA),or the expression of fusion protein containing a synthesized peptide DNA sequence linked with other gene.Finally monoclonal antibodies against a small peptide were generated using hybridoma technology.It is difficult to achieve an ideal monoclonal antibody against a small peptide by using immunogens prepared by the methods mentioned above due to their poor immune response to a small peptide.In this study we used adenovirus prime-protein boost heterologous immunization strategy(AP regimen)to prepare IDH1 R132H specific monoclonal antibody,and then the specificity of antibodies was confirmed by western blot and immunoflorsence staining in vitro,we also applied it on glioma patients’ tissue samples by immunohistochemistry analysis.The concrete research contents of this topic are as follows:(1).Construction of adenovirus E1 shuttle plasmid which co-expresses IDH1 R132H peptide and hepatitis b virus core antigen HBcAg by using conventional technology of molecular cloning methods.IDH1 R132H peptide was synthesized and subcloned to adenovirus E1 shuttle vector pAd5/E1-CMV-HBcAg.After enzyme digestion and sequence analysis,we obtained recombinant adenovirus shuttle vector pAd5/El-CMV-HBcAg-IDH1 R132H.(2).Construction of IDH1 R132H peptide modified adenovirus hexon HVR5 plasmid by one-step ligation system.IDH1 R132H peptide was inserted into the adenovirus backbone vector pAd5/E3-eGFP-Luc/Hexon HVR5-LacZ,then the recombinant adenovirus backbone vector pAd5/E3-eGFP-Luc/Hexon HVR5-IDH1 R132H backbone was obtained through the blue white spot screening,enzyme digestion and sequence analysis.(3).Construction of recombinant adenovirus vector with the HBcAg-IDH1 R132H peptide expressing cassette in E1 region and IDH1 R132H peptide modification in hexon HVR5 by two steps of homologous recombination.ScaI linearized pAd5/E1-CMV-HBcAg-IDH1 R132H and ClaI linearized pAd5/E3-eGFP-T2A-Luc/Hexon HVR5-DH1 R132H were co-transformed into E.coli BJ5183 by the homologous recombination.Recombinant adenovirus plasmid pAd5/E1-CMV-HBcAg-IDH1 R132H/E3-2TA-Luc-eGFP/Hexon HVR5-IDH1 R132H was obtained through enzyme digestion,PCR and sequence analysis.(4).Package and purification of recombinant adenovirus Ad5/E3-CMV-T2A-Luc-eGFP/Hexon HVR5-IDH1 R132H and Ad5/E1-CMV-HBcAg-IDH1 R132H/E3-Luc-T2A-eGFP/Hexon HVR5-IDH1 R132H.Two recombinant adenovirus vectors were linearized respectively by PacI and then transfected HEK 293 cells,the cells were harvested 7 to 10 days later.Amplified adenovirus was purified by CsCl density gradient method.Detection of biological activity of recombinant adenovirus by eGFP expression and Luciferase reporter assay in vivo/vitro.(5).Expression and purification of HBcAg-IDH1 R132H and Granulin E-IDH1 R132H proteins using prokaryotic expression system.Prokaryotic expression vector for HBc virus-like particles containing small peptide epitope pET-28a/HBcAg-LacZ was digested by BamH1 and SfuI,and then was ligated with IDH1 R132H peptide.pRSET-B/Granulin E prokaryotic expression vector was modified by a polyclonal site,then ligated with IDH1 R132H peptide.Both of them were respectively electrotransformed to E.coli Rossatta and BL21(DE3).The fusion protein was induced by IPTG,then collected bacterial lysate and purified by Ni-NTA affinity chromatography.(6).Other reported IDH1 R132 mutants’(C/S/G/L/V/P)peptide were synthesized and inserted into reconstructed pRSET-B/Grn E BS prokaryotic expression vector,respectively electrotransformed to BL21(DE3)and detected expression by SDS-PAGE after IPTG induction.(7).The mutant R132H was obtained using PCR method based on the pGEM-T/IDH1 plasmid.Then the mutant R132H was subcloned into pAd5/El-CMV-Flag eukaryotic expression vector,and its expression was detected in HEK293 cell lines.(8).Preparation and identification of monoclonal antibodies against IDH1 R132H.By the above mentioned immunogens,five kinds of immunization strategies were compared to determine the final immunization protocol.Balb/c mice immunizied by Ad5/E3-Luc-T2A-eGFP/Hexon HVR5-IDH1 R132H was innoculated by intramuscular routine with 2×1010 vp,after one week,the mice were immunized with Ad5/El-CMV-HBcAg-IDH1 R132H/E3-eGFP-Luc/Hexon HVR5-IDH1 R132H,then boosted with HBcAg-IDH1 R132H peptide fusion protein and freund’s complete adjuvant subcutaneous multi-point injection after grinding one week later,2 weeks later we collected vein blood to determine serum antibody titer.The mice with titer higher than 64,000 were boosted with HBcAg/IDH1 R132H fusion protein by intraperitoneal injection,then isolated B cells from spleen were fused with myeloma cell SP2/0 for producing hybridoma,Gm E-IDH1 R132H fusion protein was used as indirect Elisa screening antigen,through limited dilution assay,and finally we obtained positive hybridomas.The antibodies were diagnosed by western blot and immunofluorescence staining.Antibodies were applied on glioma patients’ samples by immunohistochemistry assay,and compared with commercial H09 antibody.Through experiments we got the following results:(1).Successfully prepared recombinant adenovirus vectors carrying IDH1 R132H peptide,which were named pAd5/El-CMV-HBcAg-IDH1 R132H/E3-Luc-T2A-eGFP/Hexon HVR5-IDH1 R132H and pAd5/E3-Luc-T2A-eGFP/Hexon HVR5-IDH1 R132H,respectively.(2).Adenovirus vaccines were obtained with viral particles of 1012 vp/ml,the infection multiplicity of recombinant adenovirus reached 109 pfu/ml,and report gene luciferase can be expressed in vivo and in vitro.(3).Successfully constructed pET-28a/HBcAg-IDH1 R132H,pRSET-B/Gm E-IDH1 R132H and pRSET-B/Grn E-IDH1 R132 mutants(C/S/G/L/V/P)prokaryotic expression plasmid and obtained IDH1 R132H peptide fusion proteins,the concentration of HBcAg-IDH1 R132H pritein is 0.52mg/ml,Gm E-IDH1 R132H is 1.6 mg/ml.Successfully constructed pAd5/El-CMV-IDH1 R132H-Flag eukaryotic expression plasmid and expressed in HEK 293 cells.(4).Through adenovirus-protein combined immunization,we prepared 5 strains hybridomas against IDH1 R132H,and confirmed by western blot and immunofluorescence staining,four strains of them are specific for IDH1 R132H(1D6,2B11,3C6 and 4F5),a strain is for IDH1 wild type(2D7).The heavy chain subtype of 1D6 is IgM,that of 2B11 and 3C6 is IgG2b,that of 4F5 is IgG3,respectively.Through further identification we found that 4F5 is cross reactive with R132L and R132V,1D6 is cross reactive with R132L,another 2 strains(2311 and 3C6)are specificly against R132H.(5).By immunohistochemical staining with clinical diagnosis of malignant glioma pathological paraffin section,the result shows that four EDH1 R132H mAbs could be applied to the diagnosis of malignant glioma compared with commercial antibody H09.These results provide specific antibodies for glioma diagnosis,and laid the foundation for investigating the mechanisms of IDH1 R132H involving in glioma.It also provides a new and effective immunization regime for the preparation of monoclonal antibody against peptide.