Use of a new functional dual coating (FDC) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism Russell G. A. Jones 1 3 and James D. Marks 2 Correspondence Russell G. A. Jones russellgajones@gmail.com Received 2 October 2012 Accepted 14 March 2013 1 Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Hertfordshire, UK 2 Department of Anesthesia, University of California, San Francisco, USA 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 (H C ) domain, while the amino-terminal (H N ) 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 H C 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 LD 50 per ml serum or 0.13 pg ml –1 using just 100 ml 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 LD 50 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 LD 50 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. INTRODUCTION Botulism remains a severe but rare life-threatening disease in the UK (McLauchlin et al., 2006). The most common presentations are wound, infant or food botulism. Occasionally adult intestinal toxaemia is seen as a result of ingesting spores, but this typically only affects indivi- duals with an underlying gastrointestinal condition (Sheppard et al., 2012). During food-borne botulism, ingested toxin is absorbed across the gut to reach the blood circulation and then redistributed into the interstitial fluid, where it can diffuse into synaptic clefts of neuromuscular junctions and specifically bind to the nerve terminal via the toxin’s H C (receptor-binding) domain of the heavy (H) chain (Maksymowych & Simpson, 1998; Maksymowych et al., 1999; Simpson, 2004; Ahsan et al., 2005). Following uptake into the cell, the disulfide bond linking the H N domain of the H-chain to the light (L) chain is broken by the reducing conditions with subsequent activation and release of the L-chain endoprotease within the cytosol. Specific cleavage of SNAP25 1–206 protein between amino acid residues Q 197 and R 198 (in the case of type A toxin) then inhibits neurotransmitter release, with a resultant nerve blockade (Schiavo et al., 2000; Simpson, 2004). The clinical diagnosis of botulism is usually made on the early signs and symptoms, such as dry mouth, difficulty in swallowing, slurred speech, ptosis and ophthalmoplegia, which, if left untreated, progress to full-blown botulism with a symmetrical descending paralysis requiring mech- anical ventilation. A trivalent (A, B and E) botulism antitoxin is given rapidly at the earliest indication to prevent any further progression and speed recovery (CDC, 1998; Browning et al., 2011; Vanella de Cuetos et al., 2011). The licensed antitoxin used in the UK is safely adminis- tered in the hospital setting and comprises purified despeciated equine F(ab9) 2 fragments with high neutral- izing potency (Novartis trivalent ‘Botulism Antitoxin Behring’ containing ¢187 500 IU anti-A, ¢125 000 IU 3Present address: Barnet, Greater London, UK. Abbreviations: CL, confidence limits; CP-buffer, capture phosphate buffer; FDC, functional dual coating; H-chain, heavy chain; L-chain, light chain; LoD, limit of detection; MBA, mouse bioassay; PIC, protease inhibitor cocktail. Journal of Medical Microbiology (2013), 62, 828–835 DOI 10.1099/jmm.0.053124-0 828 053124 G 2013 SGM Printed in Great Britain