Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins' high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3-80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.

Simultaneous differentiation and quantification of ricin and agglutinin by an antibody-sandwich surface plasmon resonance sensor.

Ricin is one of the most toxic plant toxins known. Its accessibility and relative ease of preparation makes it a potential agent for criminal or bio-terrorist attacks. Detection of ricin from unknown samples requires differentiation of ricin from the highly homologous Ricinus communis agglutinin which is currently not feasible using immunological methods. Here we have developed a simple and sensitive surface plasmon resonance (SPR) sensing system for rapid differentiation between ricin and agglutinin done in real time. Both lectins were quantified in a sandwich immunoassay-like setting by capturing with a cross-reactive antibody (R109) binding to both proteins while differentiating by injection of a ricin-specific antibody (R18) in a subsequent enhancement step. The SPR-assay was reproducible and sensitive for different R. communis cultivars, showing no false positive results when other lectins were tested. Quantification and differentiation of both molecules was also demonstrated from a crude castor bean extract and complex matrices. For the first time, we have demonstrated how the closely related lectins can be discerned and quantified in a single assay based on immunological methods. This novel approach delivers crucial information regarding the composition, purity, concentration, and toxicity of suspicious samples containing ricin in less than 30 minutes. Furthermore, we show how enhancement injections during SPR-measurements can be used to determine the ratio of two related proteins independently of the actual protein concentration by comparing normalized enhancement response levels.