Tetrodotoxin　(TTX)　is　a　small　molecular　weight　neurotoxin　that　occludes　voltage-gated　sodium　channels　in　nerve　and　muscle　tissue,　resulting　in　respiratory　paralysis　and　death.　A　high　affinity　antibody　that　can　neutralize　the　toxicity　of　TTX　is　still　lacking,　so　it　is　very　important　to　prepare　an　antibody　for　TTX　therapy　and　detection.　In　the　present　study,　a　chemical　method　was　used　to　prepare　the　tetrodotoxin　complete　antigen,　and　a　small　amount,　repeatedly　immunity　way　was　carried　to　immunize　4　mice.　The　amplified　genes　encoding　monoclonal　antibodies　against　TTX　were　used　to　construct　the　phage　display　library.　After　six　rounds　of　biopanning,　an　antibody　named　scFv-T53　was　characterized　from　clones　showing　high　affinity　and　specific　to　TTX,　and　its　affinity　constant　was　1.1　x　106　L/mol.　Three　dimensional　structure　of　the　scFv-T53　was　constructed　by　computer　modeling,　and　Trx　was　docked　to　the　scFv-T53　model　to　obtain　the　structure　of　the　binding　complex.　Two　predicted　essential　amino　acids,　1(183　and　1189,　were　mutated　to　verify　the　theoretical　model.　Both　mutants　lost　binding　activity　significantly　against　TTX　as　predicted　by　the　theoretical　model.　Hence,　the　above　results　will　be　useful　for　screening　the　high　affinity　anti-TTX　scFv　mutants.　(C)　2014　Elsevier　Ltd.　All　rights　reserved.