A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin Aahl

We created a construct encoding a peptide known to mimic the binding proper ties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (Aahl). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatogra phy on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, d ot blot and mass spectrometric analyses demonstrated the integrity of the s oluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinit y (K-d = 2.3 x 10(-10) m). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifuncti onal and had a low molecular mass of 28 kDa. This enabled us to develop rap id and sensitive immunoassays for the specific detection of the toxin Aahl accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of e nvenomations.