Development of recombinant secondary antibody mimics (rSAMs) for immunoassays through genetic fusion of monomeric alkaline phosphatase with antibody binders

In conventional immunoassays, a secondary antibody is used to amplify the signal generated by the binding of the primary antibody to the target analyte. Due to concerns regarding animal use and cost-inefficiency of secondary antibody productions, there is a significant demand for the development of recombinant secondary antibody mimics (rSAMs). Here, we developed rSAMs using a signal-generating enzyme, monomeric alkaline phosphatase (mALP), and antibody-binders, including monomeric streptavidin (mSA2) and mouse IgG1- or rabbit IgG-binding nanobodies (MG1Nb or RNb). The mALP-MG1Nb, mALP-RNb, and mALP-mSA2 were genetically constructed and produced in large quantities using bacterial overexpression systems, which reduced manufacturing costs and time without the use of animals. Each rSAM exhibited high and selective binding to its respective primary antibody, generating linear band signals corresponding to the amounts of target analytes in western blots. The rSAMs also successfully generated sigmoidal signal curves that increased as the sample concentration increased. Moreover, they generated stronger signals than conventional ALP-conjugated secondary antibodies and SA, particularly in the medium to high sample concentration range, in both indirect and sandwich-type indirect ELISAs at the same sample concentration. The rSAMs we developed here may provide new insights to develop novel immunoassay-based analytical and diagnostic tools.