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.
Gandecha
A
A
Department of Botany, University of Leicester, United Kingdom.
Owen
M R
MR
Cockburn
W
W
Whitelam
G C
GC
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007118:Immunoassay / Q000379:methods*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D010834:Phytochrome / Q000032:analysis* / Q000276:immunology; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000235:genetics
D007128:Immunoglobulin Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C055966:immunoglobulin Fv; D010834:Phytochrome; D000469:Alkaline Phosphatase