Phage Display-Mediated Immuno-Multiplex Quantitative PCR for the Simultaneous Quantification of IFN-γ and IL-6.

Abstract

In phage display technology, exogenous DNA is inserted into the phage genome, which generates a fusion protein with the phage coat protein, facilitates expression and promotes biological activity. This approach is primarily used to screen antibody libraries owing to its high library capacity and fast technical cycle; additionally, various types of genetically altered antibodies can be easily produced. In this study, we fused the pIII structural protein of the M13K07 phage with a scFv created by connecting the VH and VL domains of an anti-IFN-γ antibody. Western blotting and phage immunoprecipitation demonstrated that the recombinant phage can specifically bind to IFN-γ. After determining the amplification efficiency of the recombinant phage, we developed a PD-IPCR-based test for the cytokine IFN-γ. The method's linear range, detection limit, blank spiking recoveries, and stability were ascertained via a standard curve; the ability of PD-IPCR to target IFN-γ was compared with that of traditional ELISA, and the results of the two assays were consistent. Once the feasibility of using PD-IPCR to target individual cytokines was verified, we established a dual PD-IPCR quantitative assay based on a Taqman probe for IFN-γ and IL-6 in the same system, demonstrating the feasibility of the phage display technology constructed herein for multiple cytokine detection.