Genetically Engineered Elastin-Protein A Fusion as a Universal Platform for Homogeneous, Phase-separation Immunoassay

Abstract

A simple and universal platform for competitive phase-separation immunoassay is reported based on a fusion protein composed of a temperature-responsive elastin-like polypeptide (ELP) and the antibody-binding staphylococcal protein A (SpA). The basic principle is to take advantage of the ability of SpA to bind a variety of antibodies with high affinity, allowing simple separation of antigen-antibody complex by thermal precipitation. The resulting ELP-SpA fusion was shown to preserve the ability to reversibly precipitate as well as its high affinity toward different IgGs and IgMs. As a model system, a competitive phase-separation immunoassay based on the ELP-SpA format was established for paclitaxel (taxol) with IC(50) (20.18 nM) and the lower detection limit (2.94 nM) very similar to those reported for the ELISA format. Unlike the heterogeneous interaction in ELISA, which decreases the antibody-binding activity, the reported homogeneous immunoassay not only alleviates this problem but also enables the potential for high-throughput automation. We believe that the reported ELP-SpA fusion will find applications not only as a powerful diagnostic tool for diverse analytes but also a potential useful tool for purification and immobilization of antibody.