Production and characterization of a bivalent single chain Fv/alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis.
A102 is a monoclonal antibody raised against the hemocyanin of the Tunisian scorpion Androctonus australis. It is directed against the subunit Aa6 and does not cross-react when tested against a variety of similar scorpion hemocyanins. Here, we report the construction of a plasmid encoding a recombinant enzyme-linked antigen-binding protein with the antigen-binding specificity of antibody A102. The DNA fragments encoding the variable domains of A102 were inserted into a prokaryotic expression vector so as to produce a single chain antibody variable fragment (scFv) fused to the bacterial alkaline phosphatase. The fusion protein preserved the IgG binding and alkaline phosphatase activities. Immunoelectron microscopic analysis showed that the recombinant protein bound antigen bivalently as is the case for natural antibodies. Crude preparations containing the conjugate were used in a rapid visual immunoassay for the specific detection of A. australis hemocyanin, using a droplet of hemolymph removed from live animals by puncture. The simplicity of the test made it suitable for the direct identification of animals belonging to this species. It could be useful in areas where A. australis, the most dangerous African scorpion, is found with other species from which it is not easy to distinguish using morphological criteria.
Mousli
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Muséum National d'Histoire Naturelle, 57 rue Cuvier, 75231 Paris cedex 05, France.
Goyffon
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Billiald
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D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004731:Endotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006433:Hemocyanins / Q000032:analysis* / Q000276:immunology; D018796:Immunoconjugates / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D012605:Scorpions; D014416:Tunisia
D000936:Antigen-Antibody Complex; D004731:Endotoxins; D018796:Immunoconjugates; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D006433:Hemocyanins; D000469:Alkaline Phosphatase