Development Of Monoclonal Antibodies Against Human MIF Protein And Its Preliminary Application

Human macrophage migration inhibitory factor(hMIF)is an extremely important pleiotropic regulatory cytokine discovered in 1966 and constitutively expressed in various cellular tissues throughout the body.In addition to its basic functions as a cytokine,MIF has been found to be involved in the regulation of signaling pathways,innate and acquired immune responses,inflammatory responses,cell proliferation,apoptosis and phagocytosis,and has multiple roles similar to those of enzymes,hormones,growth factors and stress response proteins.MIF has also been implicated in the development of numerous diseases including sepsis,autoimmune diseases,rheumatoid arthritis,infectious shock,and tumors.Although hMIF monoclonal antibody(MAb)has been studied more frequently,it is still important to obtain detecting antibodies with good reactivity and to establish a high throughput and rapid method to detect MIF content.The major contents of this study are included as follows:1.Cloning and prokaryotic expression of hMIF gene:A pair of specific amplification primers was designed according to the cDNA sequence of hMIF protein published by NCBI(NCBI Reference Sequence:NM 002415.2),and the DNA fragment was amplified from human peripheral blood mononuclear cell(PBMC)by RT-PCR and cloned into the prokaryotic expression vector pET-11b.The recombinant plasmid pETllb-hMIF was transformed into Escherichia coli(E.coli)BL21(DE3),and after the expression was induced by IPTG,the recombinant target protein was purified by Ni-NTA affinity chromatography and gel filtration chromatography,and the reciprocal isoenzyme activity was measured,and SDS-PAGE gel electrophoresis showed that the recombinant target protein with high purity,soluble expression and molecular weight of 13.3 kDa was obtained.2.Preparation of anti-hMIF monoclonal antibody:The purified His-hMIF recombinant protein was used as immunogen to immunize 6-week-old female B ALB/c mice,and after three immunizations,the optimal antigen coating concentration was determined using the checkerboard square indirect enzyme linked immunosorbent assay(ELISA)method at 1.25μg/mL and the optimal serum dilution of 1:12800,followed by cell fusion of mouse spleen cells with the myeloma cells SP2/0.The hybridoma cell culture supernatant was simultaneously detected using indirect ELISA and indirect immunofluorescence(IFA)assay,and pLVX-AcGFP-Nl-hMIF overexpression vector was constructed,and human embryonic kidney(HEK)293T cells transiently overexpressing hMIF were obtained after transfection using hMIF monoclonal antibody.After subcloning by limited dilution methods for three times,three hybridoma were obtained that stably secreted anti-hMIF protein monoclonal antibodies,named 1G2,2G4 and 2G11,respectively.the specificity of the three monoclonal antibodies was identified by immunoblotting(Western blot,WB),and the results showed that 1G2 and 2G4 were able to react with the immunogen His-hMIF protein and cellular tissue MIF protein The results of IFA assay showed that monoclonal antibodies 1G2,2G4 and 2G11 specifically recognized HEK 293T cells,A549 cells and HEK 293T cells transiently overexpressing hMIF.The ELIS A titre of 1G2,2G4 and 2G11 cell supernatant was 1:6400,1:1600 and 0,respectively,and their IFA titre was 1:1600,1:400 and 1:800,respectively.The monoclonal antibodies did not cross-react with other tested proteins and were stable in antibody secretion.The subclass identification results showed that the heavy chains of monoclonal antibodies 1G2,2G4 and 2G11 belonged to IgG2a,IgG1 and IgM subtypes,respectively,and their light chains were allκ subtypes.3.Identification of antigenic epitopes and preliminary establishment of hMIF double antibody sandwich ELISA method:The antigenic epitopes of 1G2 and 2G4 were finally identified by WB as 30ATGKPPQY37 and 11PRASVPP17,respectively,through stepwise construction of truncated pGEX-6P-1-hMIF expression vector.The antibody pairing was performed to determine the capture antibody as 1G2 and the detection antibody as HRP-2G4,and the sandwich ELISA conditions were optimized for His-hMIF recombinant protein as the detection antigen.The optimal coating concentration of the capture antibody 1G2 was determined to be 2.5 μg/mL,the detection antibody HRP-2G4 was diluted 1:1600,the coating conditions were 4℃ for 16 h,the blocking solution was selected to be 5%skimmed milk powder(SMP),the blocking time was 120 min,the incubation time of the target antigen was 60 min,the incubation time of the detection antibody was 60 min and the color development reaction time was 20 min.The method was highly specific,with a linear range of 10 ng/mL12500 ng/mL(y=0.0012x+0.19165,R2=0.95063),good sensitivity,detection limit of 5.54 ng/mL,good intra-and inter-batch reproducibility,and coefficient of variation less than 10%.4.Preliminary application of hMIF specific monoclonal antibodies in hMIF overexpression and knockdown cell lines:Laser confocal technique was used to localize the presence of hMIF protein in the cytoplasm of HEK 293T cells.Transfecting HEK 293T with pLVX-AcGFP-N1-hMIF overexpression vector and pX458-hMIF knockdown vector,the knockdown and overexpression of MIF cells were obtained by flow cytometry and lentivirus packaging technique,subcloning to obtain stable transitional cell lines and the MIF levels were verified by Western blot.Stimulation of overexpressed and knockdown cells using lipopolysaccharide(LPS)revealed that hMIF positively activated ERK1/2 phosphorylation,which was consistent with the results of previous studies.