Extraction of BoNT/A, /B, /E, and /F with a Single, High Affinity Monoclonal Antibody for Detection of Botulinum Neurotoxin by Endopep-MS

Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies

Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay

Animal Botulism Outcomes in the AniBioThreat Project

Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produced by Clostridium botulinum and other Clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. Clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the Australia Group List of Biological Agents. In 2010 the European Commission (DG Justice, Freedom and Security) funded a 3-year project named AniBioThreat to improve the EU's capacity to counter animal bioterrorism threats. A detection portfolio with screening methods for botulism agents and incidents was needed to improve tracking and tracing of accidental and deliberate contamination of the feed and food chain with botulinum neurotoxins and other Clostridia. The complexity of this threat required acquiring new genetic information to better understand the diversity of these Clostridia and develop detection methods targeting both highly specific genetic markers of these Clostridia and the neurotoxins they are able to produce. Several European institutes participating in the AniBioThreat project collaborated on this program to achieve these objectives. Their scientific developments are discussed here

Attomolar Detection of Botulinum Toxin Type A in Complex Biological Matrices

BACKGROUND: A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity. CONCLUSIONS/SIGNIFICANCE: The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications

Botulism in Brazil, 2000-2008: epidemiology, clinical findings and laboratorial diagnosis Botulismo no Brasil, 2000-2008: epidemiologia, achados clínicos e diagnóstico laboratorial

Botulism is a rare and potentially lethal illness caused by Clostridium botulinum neurotoxin. We describe the findings of a laboratorial investigation of 117 suspected cases of botulism reported to the surveillance system in Brazil from January 2000 to October 2008. Data on the number and type of samples analyzed, type of toxins identified, reporting of the number of botulism cases and transmission sources are discussed. A total of 193 clinical samples and 81 food samples were analyzed for detection and identification of the botulism neurotoxin. Among the clinical samples, 22 (11.4%) presented the toxin (nine type A, five type AB and eight with an unidentified type); in food samples, eight (9.9%) were positive for the toxin (five type A, one type AB and two with an unidentified type). Of the 38 cases of suspected botulism in Brazil, 27 were confirmed by a mouse bioassay. Laboratorial botulism diagnosis is an important procedure to elucidate cases, especially food-borne botulism, to confirm clinical diagnosis and to identify toxins in food, helping sanitary control measures.<br>Botulismo é uma doença rara e potencialmente letal, resultante da ação de uma neurotoxina produzida pelo Clostridium botulinum. No presente estudo, estão descritos os resultados da investigação laboratorial de 117 casos suspeitos de botulismo notificados ao sistema de vigilância, ocorridos no Brasil no período de janeiro de 2000 a outubro de 2008. Os dados obtidos sobre as fontes de transmissão, os tipos de toxina identificados e de amostras analisadas serão discutidos. Foram analisadas 193 amostras clínicas e 81 amostras de alimentos para detecção e identificação de neurotoxina botulínica. Entre as amostras clínicas, 22 (11,4%) amostras apresentaram resultado positivo para toxina (nove do tipo A, cinco do tipo AB e em oito o tipo não foi identificado) e entre as amostras de alimentos, oito (9,9%) foram positivas (cinco do tipo A, uma do tipo AB e em duas o tipo não foi identificado). Dos 38 casos considerados positivos para botulismo, 27 foram confirmados pelo bioensaio em camundongo. O diagnóstico laboratorial de botulismo é importante para elucidação dos casos, principalmente de botulismo alimentar, para confirmação dos diagnósticos clínicos e identificação das toxinas nos alimentos, provendo subsídios para as medidas de controle sanitári