A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A.

Fourteen monoclonal antibodies (mAbs) against Clostridium botulinum type A neurotoxin were raised in mice using a carboxy terminal fragment of the toxin for immunization. A two-site immunometric assay was developed which allowed detection of 8 LD(50)/ml (40 pg/ml) botulinum neurotoxin A and an accurate quantification close to 25 LD(50)/ml (150 pg/ml). No cross-reactivity was observed with other toxinotypes.

Solid-phase immobilized tripod for fluorescent renewable immunoassay. A concept for continuous monitoring of an immunoassay including a regeneration of the solid phase.

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively.