Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay

Abstract

Interferon gamma (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on singe chain variable fragment (scFv) antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 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 biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6-60 pg/mL with IC₅₀ of 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 scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis.

Keywords: affinity; detection; ic-ELISA; interferon gamma (IFN-γ); phage display; scFv.

FIGURE 1

Expression and purification of human IFN-γ in Escherichia coli and animal immunization. (A) Expression and purification of human IFN-γ. Lane M: Protein Markers; lane 1: the expressed total protein of pET28a/BL21(DE3) as negative control; lane 2: the expressed total protein of pET28a-ifn-γ/BL21(DE3); lane 3, 4: the purified protein of IFN-γ-His6. (B) Titer determination of immunized mouse.

FIGURE 2

Construction and biopanning of scFv clones against IFN-γ. (A) The input and output of recombinant phage. The blue pillar indicated the input of recombinant phage, and the green pillar indicated the output of recombinant phage. (B) The amplified PCR results of selected scFv clones from biopanning result (M: DL-2000 DNA markers; lane 1: negative control; lane 2–5: the PCR products of scFv clones). (C) The ELISA result of the selected scFv clones. Here, the results showed the four scFv antibody clones with strong binding activity to IFN-γ antigen.

FIGURE 3

Construction and expression of different scFv fusion proteins against IFN-γ. (A) Constructs of scFv with different fusion formats used in this study. MBP, maltose binding protein; Sotp, stop codon; scFv, single chain fragment. (B) Soluble analysis of the expressed protein. S, the composition of soluble cell lysates; P, the composition of insoluble cell lysates. (C) Cell lysates analysis by OD₆₀₀ nm test.

FIGURE 4

Purification and immunoassay of scFv fusion proteins against IFN-γ. (A) SDS-PAGE assay of the purified scFv proteins. Lane M: protien markers; lanes 1–3: the purified protein of scFv, MBP-scFv and MBP-Linker-scFv, respectively. (B) ELISA determination. IFN-γ were coated on 96-well plates in triplicate, and the purified scFv proteins were added to the reaction wells after blocking and washing, respectively. The binding activities of four purified proteins were determined using an anti-MBP tag antibody. (C) Western blot analysis of the binding activity of MBP-Linker-scFv to IFN-γ antigen. Left panel: SDS-PAGE result for IFN-γ; right panel: western blotting results; lane 2: IFN-γ band at 20 kDa bound by MBP-Linker-scFv.

FIGURE 5

Specificity and affinity determination of scFv against IFN-γ. (A) Specificity analysis of MBP-Linker-scFv. Cross reactivity of MBP-Liner-scFv to other antigens was tested by iELISA. (B) Affinity determination of MBP-Linker-scFv. Four different concentrations of MBP-Linker-scFv were used to determine the affinity of MBP-Linker-scFv by ELISA, and the measured data were used for the quantitative determination of the affinity constant of MBP-Linker-scFv.

FIGURE 6

Sensitivity analysis of anti-IFN-γ-scFv. (A) The standard curve was carried out by ic-ELISA. The data obtained in the presence of various inhibitor and without inhibitor are referred to as B and B0, respectively. Standard curves were generated by plotting the inhibition percentage (B/B0) versus the log of three inhibitor concentrations. (B) The linear equation is y = –0.57x+1.159, with a correction coefficient (R²) of 0.9522.