Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples

Abstract

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…In efforts to avoid animal suffering, a multitude of BoNT detection methods has been developed with minimal use of animals. These include in vivo simulation assays, such as the hemidiaphragm assay and local injection assays [57,59,62,[78][79][80][81][82], numerous in vitro assays using immunological detection methods, endopeptidase assays, or a combination of the two such as the endopeptidase-mass spectrometry assay [83][84][85][86][87][88], and cell-based assays [89].…”

“…The MBA can lack the necessary sensitivity to detect the low levels of BoNTs often found in clinical samples from botulism patients, with data indicating that the MBA was negative in 30-40% of positively confirmed botulism cases [92][93][94][95]. Several in vitro assays with increased sensitivity [84,87,88], some of which are completed within hours, are useful for this application. However, these assays may not detect novel BoNTs and BoNTs for which the assays have not been validated, and require careful and thorough validation, ideally between independent laboratories.…”

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…Two classic plate-bound sandwich ELISAs and one lateral flow assay proved to be successful in detecting and quantifying BoNT/A, B, and E ( [42]; Table 2). In one ELISA-based strategy, six mouse mAb directed against the 50 kDa C-terminal fragment HC of the BoNT heavy chain were used in three independent ELISAs to detect BoNT/A, B, or E, respectively.…”

“…Four of the six mAb were also used in an LFA to detect BoNT/B and E, whereas two other anti-BoNT/A mAb were specifically selected for their sensitivity in the LFA format, all recognizing both the corresponding holotoxin and BoNT complex. In the other ELISA-based strategy, three combinations of mAb and/or pAb targeting BoNT/A, B, or E holotoxin were successfully employed ( Table 2, [42]). The Endopep-ELISA, a combination of classic immunoassay and functional assay, presents plate-coated peptide substrates of synaptosome associated protein of 25 kDa (SNAP-25) and vesicle associated membrane protein 2 (VAMP-2) to BoNT/A-, B-, and E-containing samples.…”

“…A BoNT PT organized by the EQuATox consortium including 23 laboratories allowed the comparison of various methods and their performance [41]. In this exercise, high purity BoNT/A-F materials were generated and [21,[24][25][26] In vitro cytotoxicity tests Measurement of cell death in vitro (endpoint and real-time assays) [21,27] BoNT/A, B and E Immunological assays Sandwich ELISA: Detection of antigen by a combination of mAb and/or pAb, i) four combinations of two pAb each from goat and/or horse targeting BoNT/A, B, E, or F; detection of holotoxin [40] ii) three combinations of two mAb targeting BoNT/A (mAb TA18 + mAb TA13), BoNT/B (mAb TB6 + mAb TB17), or BoNT/E (mAb TE81 + mAb TE49); detection of HC-domain [42,107] iii) three combinations of mAb and/or pAb targeting BoNT/A (mAb A1688 + mAb HcA78), BoNT/B (mAb B279 + horse trivalent pAb), or BoNT/E (rabbit pAb KE97 + mAb E136); detection of holotoxin [41,42,106] Lateral Flow assay: Sandwich ELISA on a paper strip i) three combinations of two mAb targeting A (mAb TA12 + mAb TA9), B (mAb TB6 + mAb TB17), or E (mAb TE81 + mAb TE49); detection of HC-domain [41,42] (continued on next page) Chromatographic methods…”