Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F

Abstract

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the ∼50 kD C-fragments of each toxin. Standard curves were linear over 0.5–10 ng/ml (BoNT E) or 2–20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5–10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.

Introduction

The anaerobic bacterium Clostridium botulinum produces seven serologically distinct toxins. Recognized as the most potent toxins of biological origin, botulinum neurotoxins (BoNTs) are the causative agents of food-borne, infant, and wound botulism (Sakaguchi, 1983). The toxins act presynaptically at the neuromuscular junction by blocking acetylcholine release and thereby inducing a flaccid muscular paralysis and potentially death due to asphyxiation (Simpson, 1986). All serotypes (MW approximately 150 kD) consist of two polypeptide subunits joined by an intrachain disulfide bridge, which are bound to non-toxic neurotoxin-associated proteins (NAPs). These serve to protect the toxins from cleavage by proteases in the gastrointestinal tract. The mechanism of action is similar for each serotype. The heavy chain (B chain) is the binding subunit that interacts with a receptor on the presynaptic membrane. The light chain (A chain) is the catalytic subunit. Once translocated across the cell membrane, its zinc-dependent protease activity hydrolyzes specific proteins associated with synaptic vesicle docking and acetylcholine release (Schiavo et al., 1994).

Currently, the mouse bioassay is the most widely accepted method for detecting BoNTs in serum and foods. This assay has the desired sensitivity (<5 mouse lethal units/ml), but is cumbersome, time consuming (1–4 days) and involves the use of large numbers of animals (Shone et al., 1985). Enzyme-linked immunosorbent assays (ELISAs) have been reported by several laboratories (Dezfulian and Bartlett, 1984, Shone et al., 1985), but lack the required sensitivity. An enzyme-linked coagulation assay (ELCA) was reported with a sensitivity comparable to the mouse bioassay (Doellgast et al., 1994), but this assay relies upon a sophisticated amplification system utilizing a snake venom coagulation factor, and is limited by its complexity and reagent expense. Singh and Silvia (1996) developed a fiber optic biosensor to detect BoNT E which could detect toxin at about 500 pg/ml. However, it required complex, expensive equipment and highly trained operators. We report here the development of capture ELISAs for BoNT serotypes E and F that approach the sensitivity of the mouse bioassay, are simple to use, and accurately measure toxins in human serum and assay buffer.