Clostridium botulinum type A toxin is the most prevalent cause of naturally 
occurring outbreaks of human botulism in the world. The active dichain 
neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the 
carboxy-terminal end consisting of a receptor-binding (HC) domain, while the 
amino-terminal (HN) domain is linked by a critical disulfide bond to a light 
chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally 
used to confirm the presence of toxin in serum or food, its sensitivity is 
insufficient to detect low toxin levels in approximately 30 to 60 % of botulism 
patients. A novel FDC (functional dual coating) microtitre plate 
immuno-biochemical assay, which quantifies botulinum toxicity by measuring the 
HC domain linked with L-chain endopeptidase activity, was modified to allow 
human serum (lysed or unlysed) to be tested without interference from the 
matrix, with toxin detection down to 0.03 mouse LD50 per ml serum or 0.13 pg 
ml(-1) using just 100 µl of clinical samples. The assay was specific for type A 
toxin and could additionally be applied to whole blood and food samples. Low 
levels of 1 to 2 mouse LD50 per ml serum of type A toxin were quantified for the 
first time using the modified FDC assay in two severely intoxicated UK patients 
who required mechanical ventilation and antitoxin. Toxin levels in recovered 
food sample extracts were also detected and one MBA-negative sample was found to 
contain 0.32 LD50 per ml extract. The FDC assay provides a real alternative for 
public health laboratories to unambiguously confirm all cases of type A botulism 
and, due to its sensitivity, a promising new tool in toxin pharmacokinetic 
studies.