Screening Of A ScFv Antibody With High Affinity Against Human IFN-γ And Establishment Of The Detection Method

Interferon(IFN)is an important signal proinflammatory cytokine secreted by immune cell.Currently,two different types of interferon were found,including type Ⅰ and type Ⅱ,respectively.The type Ⅱ IFN(IFN-γ)is also known as immune interferon.Some published papers have demonstrated that IFN-γ plays a critical role in the pathogenesis and progression of many diseases,especially in antiviral,antiproliferative,differentiation inducing,and immunoregulatory properties.Besides,it has been regarded as an important marker for determination of disease-specific immune responses.Hence,it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real samples.In the present study,we obtained and synthesized the DNA sequence encoding the matured IFN-γ from NCBI database,and the size of ifn-γ is 438 bp.After amplication by PCR,the resulted ifn-γ fragment was inserted into the plasmid pET28a through genetic engineering method,and the constructed recombinant vector pET28a-ifn-γ was transformed into the E.coli host cells for protein expression via IPTG induction.The expressed and purified IFN-γ fusion protein(about 20 kDa)was used to perform the further experiments.The spleen of immunized mouse with IFN-γ was isolated and used to extract total RNA,and the cDNA synthesized by RT-PCR was used as template to amplify the variable regions of heavy chain(VH)and light chain(VL).A short DNA fragment encoding peptide(Gly4Ser)3 as linker was used to connect VH and VL into a scFv gene by SOE-PCR.Then the assembled scFv gene was cloned into the pCANTAB-5E vector,After infecting the host bacteria TG1 with helper phage M13KO7,a capacity of 1.0×108 antibody library was constructed successfully.A scFv antibody named scFv-A8 with high specificity was obtained by phage display and bio-panning,with the affinity of 2.6×109 L/mol.Maltose binding protein(MBP)was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag,and the resulted fusion protein(MBP-LK-scFv)has high solubility and antigen binding activity.The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive ELISA(ic-ELISA)for detection of human IFN-y,and the result showed that the linear range to detect IFN-y was 6～60 pg/mL,and the IC50 was 25 pg/mL.The limit of detection was 2 pg/mL(1.3 fm),and the average recovery was 85.05%,further demonstrating that the detection method based on MBP-LK-scFv has higher recovery and accuracy.Hence,the developed ic-ELISA can be used to detect IFN-y in real samples,and it may further provide a reference’ for disease diagnosis.