Development of Recombinant Secondary Antibody Mimics (rSAMs) for Immunoassays using a Monomeric Alkaline Phosphatase

Abstract

In conventional immunoassays, the amplification of the signal for the target analyte is achieved by using a target-specific primary antibody and a signal-generating secondary antibody. However, producing secondary antibodies involves immunizing animals with the Fc domain and purifying the antibodies from their serum, which is a costly and time-consuming process in the mammalian system. Consequently, there is a continuous demand for alternative options, such as signal generating antibody mimics, that can selectively bind to specific target and serve as recombinant signal generators. In this study, we introduced a monomeric alkaline phosphatase(mALP) as the enzyme to develop a recombinant antibody mimic for our target detection system. mALP does not possess the glycosylation limitations found in HRP, rendering it more suitable for recombinant protein applications. We have successfully developed recombinant secondary antibody mimics using the signal generating enzyme monomeric alkaline phosphatase (mALP), along with antibody-binders such as monomeric streptavidin (mSA2) and nanobodies specific for mouse IgG1 (MG1Nb) or rabbit IgG (RNb). The recombinant secondary antibody mimics developed in this study demonstrated superior signal strength compared to conventional ALP-conjugated secondary antibodies and streptavidin (SAs) in both indirect and sandwich-type indirect ELISAs, and Western blot analysis also demonstrated that the signal generation ability of our recombinant antibody mimic showed comparable signal generation ability to conventional methods. To further enhance the sensitivity and specificity of this recombinant signal generator in various fields, our investigation focuses on exploring the potential benefits of conjugating mALP to a bivalent nanobody. Additionally, we aim to leverage the simultaneous utilization of two different antibodies within a single construct. Next, we try to enhance the sensitivity and cost-effectiveness of detecting hTNFα, a cytokine, in a sandwich-type ELISA. To achieve this, we utilized three different types of anti-hTNFalpha nanobodies conjugated with mALP. These nanobodies can be utilized to facilitate the immobilization and detection of the target analyte within the sandwich-type ELISA format. The approaches utilized in this study have revealed the promising potential for the development of novel and specific immunoassay systems and provide valuable insights to serve as a foundation for future advancements in immunoassay technology and related applications.