A Cell Line for Detection of Botulinum Neurotoxin Type B

Abstract

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

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

“…Similarly, in a diagnostic setting endogenous receptor binding could be an alternative for antibody extraction of toxin from complex matrices. This principle has been explored for selected serotypes by usage of recombinant receptor molecules, receptor fragments 57,83,85,86 , or synaptosomes for BoNT/A, B, or F 87–90 . However, the implementation of the BoNT receptor interaction of all serotypes in vitro is highly challenging due to the molecular structure of the BoNT receptors.…”

Rapid Detection of Botulinum Neurotoxins—A Review

“…Therefore, at least 4–6 days are needed to carry out this assay. This assay is considered to be very sensitive, detecting down to 10 pg/mL, and it is also able to detect functionally active toxin, which is in direct contrast with a vast number of other immunological methods [6,39,41]. It does, however, have several disadvantages, including the cost and ethical issues of live animal research, as well as the amount of time taken to conduct the assay [49,50].…”

“…Around 1% of people develop antibodies that make subsequent treatments ineffective [38]. Due to the fact that the toxin is a biological product which intrinsically leads to variability between production batches, each set has to be tested for safety, potency and stability before it can be used on humans [39]. The testing must be conducted at multiple steps in the manufacturing timeline, this is a mandatory requirement to be able to meet regulatory protocols, leading to marketing consent being approved [40].…”

Commentary: A Cell Line for Detection of Botulinum Neurotoxin Type B

“…The work of Rust and colleagues, describing an alternative cell-based assay for serotype B BoNT, presents a sound, scientifically determined methodology that could be applied to the single commercial type B product now available (Rust et al, 2017 ). The authors conclude that “the assay may well be suited for measuring potency of pharmaceutical BoNT/B products and antitoxin antibodies in toxin neutralization testing.” Unfortunately, there are key issues that must be addressed before any such assay is brought into use by a commercial BoNT company.…”

“…Rust et al ( 2017 ) are not the first to engineer a cell line for such an application. The US company BioSentinel, Inc. has, for some years, marketed an engineered cell line and accompanying technology as a replacement assay for BoNT potency determinations (Atapattu et al, 2013 ).…”