Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: Palytoxin.
Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R(2)=0.9945), showing a working range of 0.0005-500ngmL(-1). Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.
Garet
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E
Area de Inmunología, Universidad de Vigo, Edificio de Ciencias Experimentales, As Lagoas Marcosende, 36310 Vigo, Pontevedra, Spain.
Cabado
A G
AG
Vieites
J M
JM
González-Fernández
A
A
D000178:Acrylamides / Q000032:analysis / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D049872:Bivalvia / Q000737:chemistry; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000276:immunology*; D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005506:Food Contamination / Q000032:analysis; D006801:Humans; D008387:Marine Toxins / Q000032:analysis / Q000276:immunology*; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012758:Shellfish / Q000032:analysis; D000095702:Polyether Toxins
D000178:Acrylamides; D000911:Antibodies, Monoclonal; D003064:Cnidarian Venoms; D008387:Marine Toxins; D019151:Peptide Library; D011994:Recombinant Proteins; C010272:palytoxin; D000095702:Polyether Toxins