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.
Highlights
To effective regulate immune response, would healing, and tissue regeneration, a large of different cytokines were released by immune cells, including T cells, phagocyte and NK cells (Liu et al, 2012)
The purified recombinant IFN-γ protein was used as an immunogen to immune the mice for activation of immune response, and the titer of antiserum extracted from immunized mice was tested by enzyme-linked immunosorbent assay (ELISA)
The calculated affinity constant of Maltose binding protein (MBP)-Lk-scFv was 2.6 × 109 L/mol (Figure 5B), belonging to a high affinity antibody. These results indicated that the expressed and purified MBP-Lk-scFv has high affinity and specific to IFN-γ, and could be used as an antibody reagent to detect IFN-γ
Summary
To effective regulate immune response, would healing, and tissue regeneration, a large of different cytokines were released by immune cells, including T cells, phagocyte and NK cells (Liu et al, 2012). Interferon gamma (IFN-γ) is an important cytokine secreted mainly by immune cells under antigens stimulation, playing a key role in antiviral, antiproliferative, differentiation inducing, and immunoregulatory properties (Liu et al, 2010; Shi et al, 2017). IFN-γ detection was regarded as an important biomarker and employed to diagnose latent tuberculosis in clinic (Liu et al, 2012; Chegou et al, 2013; Olivieri et al, 2016). Dysregulation of IFN-γ secretion is associated with various diseases, such as inflammatory disease (Zoller et al, 2011), human virus infections (Delannoy et al, 1999; Cimini et al, 2017), Breast tumors (Yuliatun et al, 2017), Latent trypanosome brucei gambiense infection (Ilboudo et al, 2016) and Early Lyme disease (Callister et al, 2016). In view of the vital biological function of IFN-γ and important clinic detection significance, it is necessary to develop an accurate and feasible method to detect IFN-γ for investigating the vigor of the immune response as well as for the diagnosis of potential diseases (Zamani et al, 2017)
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