Development of non-radioactive labels for a receptor binding assay for paralytic shellfish poisoning (PSP) toxin testing

Aims

This research aims to address the drawbacks of an existing in vitro test for toxin contamination in shellfish, currently preventing its uptake as a replacement for live animal assays.

Background

Paralytic Shellfish Poisoning (PSP) toxin, if consumed, causes illnesses ranging from severe food poisoning to paralysis and death. Until now, the major test for biotoxin monitoring in shellfish has been the mouse bioassay (MBA), which is associated with significant animal welfare concerns because of the severe suffering involved. Each year over 300,000 mice are used in Europe alone.

PSP toxins act by blocking voltage-dependent sodium channels in neuronal and muscle cells. An in vitro receptor based assay which uses tritiated saxitoxin, a member of the PSP toxin family, has been developed and shown to be an effective alternative to the MBA.

The requirement of a radioactive component prevents the in vitro assay’s widespread use. In many parts of the world it is either not permitted to use radioisotopes, or dealing with them is a difficult and expensive procedure. If the radioactive element could be replaced, global uptake of the in vitro assay would be much more likely.

The in vitro assay requires the use of one rat brain to provide sufficient receptor preparation for testing 150 samples in duplicate. For the same number of tests using the MBA, 300 to 450 mice would be required, depending on whether duplicate or triplicate analysis is used.

Research details and methods

A number of approaches for non-radioactive labelling of the PSP toxin molecule will be investigated, including low molecular weight fluorescence labels, conjugating saxitoxin with biotin and conjugating saxitoxin with an alternative small molecular weight compound to which antibodies are already available.

Linkage and varying properties of linker molecules such as increasing length, structure and stereochemistry between the label and saxitoxin on the binding affinity of the receptor to the saxitoxin moiety will be also be studied.

Finally, the non-radioactively conjugated toxin will be used to improve the current microtitre plate based assay. Its use as a rapid response tool for detecting toxins in shellfish or seafood will then be validated.

Sodium channel receptors from pig brains, a by-product of meat production, are also being investigated. 

There is an important need to perform detection of toxins in shellfish to safeguard the public. If consumed, illnesses ranging from severe food poisoning to paralysis and death can result. Due to factors such as climate change and increasing pollution the number of toxic episodes is markedly increasing each year. For many years the major tool for biotoxin monitoring in shellfish has been the mouse bioassay (MBA). While this has served to protect the public it has resulted in the painful deaths of over a million mice annually.

Enormous efforts are being placed into finding alternatives to the MBA and Queen’s University have had a major role in this by developing fast, reliable and low-cost rapid screening tests. The current project is aimed at advancing a receptor based test developed by another research group into a method which will gain global acceptance as a replacement to the MBA. While the receptor test developed by an American research group has been shown to be very effective in detecting a wide range of toxins it has one very major disadvantage, i.e. it currently needs a radioactive component to work. In many parts of the world it is either not permitted to use radioisotopes or dealing with them is a very difficult and expensive procedure.

The research effort will be to replace this radioactive element with a safe and easy to use label which will remove a huge hindrance to the global uptake of the receptor test. The new procedure developed will be rigorously tested to make sure it provides a high level of assurance that shellfish are safe to eat. Stakeholders from regulatory agencies and shellfish producers will be invited to a workshop to have a demonstration of the method and receive training in its use. The method will also be subject to a collaborative study.