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.

Design and Production of Bispecific Antibodies

“…Besides the above methods to tackle solubility issue, there are other approaches exist that may in the future be applied to facilitate the production of bispecific scFvs in bacterial platforms. For example, expression of scFv as a fusion protein with solubility enhancing tags such as MBP, NusA, and TRx can promote and facilitate correct protein folding [38,97,125,126], despite that these tags need to be removed afterward to allow normal antibody usage. Furthermore, studies have shown that co-expression of molecular chaperons such as Skp, OmpH, HlpA and FkpA, and folding modulators/catalysts such as disulfide bond metabolizing enzymes can effectively tackle protein aggregation and misfolding problems, and the “cocktails” approach has been an increasingly common expression strategy that involves the simultaneous usage of various chaperons or folding catalysts [32,127,128,129,130].…”

A functionally neutral single chain antibody to measure beta‐1 integrin uptake and recycling

“…We designed MBP-scFv K20 to retain its β1 integrin-specific binding capa- While scFvs remain widely used, they are prone to aggregation and thus often require additional genetic tags to mitigate protein instability. 51,54 Although the addition of MBP increases both the solubility and stability of our scFv, it does effectively double the size of our probe. We therefore suggest that future integrin antibody-based probe design limit increasing overall molecular size.…”

“…To increase expression and solubility, the scFv is genetically fused to a TEV-cleavable Maltose Binding Protein (MBP). 50,51 Additionally, the construct encodes an amino-terminal GP64 secretion signal sequence and a 6x-Histidine (6xHis) tag for affinity purification. scFv K20 also contains a C-terminal FLAG epitope for recognition by commerciallyavailable anti-FLAG antibodies, and a SortaseA (SrtA*) recognition motif for in vitro site-specific labeling.…”

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…Several reports have also suggested that the use of fusion partners such as GST [ 118 ], maltose-binding protein (MBP) [ 119 , 120 ], small ubiquitin-related modifier (SUMO) [ 87 , 121 ] and thioredoxin (Trx) [ 122 ] to the target proteins, especially to scFv fragments, results in solubility-enhancing properties and in increased yields of soluble and active products. However, the fusion partners must be cleaved as big tags usually interfere with the folding of the target protein and with its activity.…”