BACKGROUND-AIM

Anatoxin (~165 Da) is a natural alkaloid neurotoxin produced by some cyanobacteria in water. Acute anatoxin toxicitylead to respiratory arrest and rapid death in mammals. Development of a simple immunoassay based detection toolsfor anatoxin is hampered by the lack of antibodies; small size of anatoxin makes this hapten a difficult target forantibody generation. The aim of this study was to develop new antibodies against anatoxin by first generating a biotin-anatoxin conjugate (bio-anatoxin) and then using it to isolate binders from a synthetic antibody phage library designedfor the recognition of low-molecular-weight compounds.

METHODS

Bio-anatoxin conjugate was produced by means of synthetic organic chemistry and purified with HPLC. To generateanti-anatoxin binders, a synthetic scFv library was enriched for three rounds against the bio-anatoxin immobilizedon streptavidin or neutravidin coated magnetic beads. Unwanted non-specific streptavidin binders from the antibodylibrary were removed by subtractive panning on streptavidin coated wells. The enrichment of immunoreactive phagewas monitored by immunoassay. Gene pools from the third round were cloned into expression vector to produce theantibody as fusion with alkaline phosphatase (scFv-AP). The active binders were identified with immunoassay andsequencing.

RESULTS

The bio-anatoxin provided the expected mass (~934 Da) in HPLC chromatogram. The bio-anatoxin bound tostreptavidin well was recognized by an existing anti-anatoxin mAb confirming the functionality of the conjugate tobe used in the selection round. Phage immunoassay revealed the enrichment of anatoxin specific clones in the phagepopulation. Among 192 screened individual antibody clones, over 80% showed binding ability towards bio-anatoxin.Among the 20 sequenced antibodies, eight were unique.

CONCLUSIONS

A functional bio-anatoxin conjugate was successfully synthesized and used for in-vitro selection of antibodies froma synthetic antibody library. The enriched binders with 40% diversity are highly potential to be further utilized orto develop immunoassay for quantitative measurement of anatoxin from water. The study also demonstrates theusefulness of the synthetic antibody libraries, for targeting low-molecular weight compounds, as a source of bindersagainst very small haptens.