Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates.