Botulinum

The botulinum group is part of the toxin/toxoid group at NIBSC. Our work involves:

• carrying out tests to ensure quality and safety of therapeutic products derived from bacterial toxins and antitoxins
• developing biological standards
• research focused on assay developments to reduce, refine and replace animals – the 3Rs – in regulatory activities 

Botulinum toxins are among the most poisonous naturally occurring substances. They are proteins produced by the bacterium Clostridium botulinum.

There are seven known serotypes, four of which cause botulism in humans – often by people eating food containing the toxin.

The toxins cause respiratory and muscular paralysis – and even death – by blocking nerve function.  

Because of their high toxicity, Botulinum toxins are classified as category A agents by the Centres for Disease Control (CDC) and are among the 6 agents with the highest risk of potential use as bio-weapons.

Although Botulinum toxin A is the most toxic substance on earth, it can be used safely in extremely small doses to treat painful muscle spasms and involuntary eye muscle contractions. It has been produced commercially for these and other medical purposes.

Standardisation

As a World Health Organisation (WHO) International Laboratory for Biological Standardisation, NIBSC is responsible for developing, producing and characterising international and national biological reference materials.

These standards are used to calibrate biological assays and to confirm serodiagnosis for botulinum toxin serotype. 

We work closely with formulation scientists at NIBSC to optimise formulations which we can successfully freeze-dry. Meanwhile we have produced a range of Botulinum antitoxin working standards in the last 10-15 years which are in the NIBSC catalogue.

Our laboratory has a portfolio of Botulinum standards and related standards for other non-neurotoxin clostridia that are used as standards in:

• antitoxin potency tests (traditional equine products)
• assays to determine serotype specificity and identity
• serological assays for titration of immune sera (coating antigens and reference antisera)

Control testing

In fulfilling its role as a National Control Laboratory, NIBSC performs independent batch release testing for therapeutic botulinum toxin. 

Research and development

We have wide experience in developing and validating methods to control Botulinum toxin for therapy and assessing botulinum antitoxins.

With a focus on the 3Rs, we developed methods to allow us to assess functional antibodies targeting the heavy and light chains of the botulinum toxins and to assess potentially protective antibodies in local paralysis models. These assays allowed us to take part in the EU Framework 7 collaborative project within bio-defence. NIBSC’s in vitro methods have proved highly useful in this project, which completed in April 2015 and resulted in 5 publications. 

Every batch of a therapeutic preparation of Botulinum toxin has to be tested for potency and safety, and these tests are carried out at several stages of the toxin production process, usually on mice. Through research at NIBSC since the product was licensed in the early 1990s – and with long-standing external funding – we have developed a range of methods to assess the activity of botulinum toxin which has led to the mouse test being refined and replaced. In the most recent studies we were looking at cell-based assays.

The National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3R)-funded project, completed in December 2014, explored differentiation of human neuronal cell lines, mouse and human stem cells combined with several functional read-outs relevant to the action of botulinum toxins on cells. These were:

• immunodetection of intracellular substrate
• synaptic vesicle recycling and neurotransmitter release
• microelectrode array – electrophysiology as non-invasive read-out

Whereas all the methods are capable of detecting the action of toxin on cells – to various degrees and sensitivity – the most sensitive, robust, reproducible and specific approach capitalises on botulinum toxins’ ability to break down intracellular proteins that are essential for neurotransmitter release. 

A simple immunoassay format, specifically detecting these key proteins after toxin intoxication of cells, has made this approach particularly attractive for use within quality control. Using the same principle and approach, one manufacturer has developed a US Food and Drug Administration-approved cell-based assay for testing the product’s potency.

Contact us by emailing bacvac@mhra.gov.uk for information about control testing, standardisation or potential research collaboration.