An assay for botulinum toxin types A, B and F that requires both functional binding and catalytic activities within the neurotoxin.
OBJECTIVE
To develop a novel assay technique for the botulinum neurotoxin family (BoNTs) which is dependent on both the endopeptidase and receptor-binding activities of the BoNTs and which is insensitive to antigenic variation with the toxin family.


RESULTS
An endopeptidase activity, receptor-binding assay (EARB assay) has been developed which captures biologically active toxin from media using brain synaptosomes. After capture, the bound toxin can be incubated with its substrate, and cleavage detected using serotype-specific antibodies raised against the cleaved product of each toxin serotype. The EARB assay was assessed using a range of BoNT serotypes and subtypes. For BoNT/A, detection limits for subtypes A(1), A(2) and A(3) were 0.5, 3 and 10 MLD(50) ml(-1), respectively. The limit of detection for BoNT/B(1) was 5 MLD(50) ml(-1) and a novel antibody-based endopeptidase assay for BoNT/F detected toxin at 0.5 MLD(50) ml(-1). All these BoNTs can be captured from media containing up to 10% serum without loss of sensitivity. BoNT/A(1) could also be detected in dilutions of a lactose- containing formulation similar to that used for clinical preparations of the toxin. Different serotypes were found to possess different optimal cleavage pHs (pH 6.5 for A(1), pH 7.4 for B(1)).


CONCLUSIONS
The EARB assay has been shown to be able to detect a broad range of BoNT serotypes and subtypes from various media.


CONCLUSIONS
The EARB assay system described is the first convenient in vitro assay system described which is requires multiple functional biological activities with the BoNTs. The assay will have applications in instances where it is essential or desirable to distinguish biologically active from inactive neurotoxin.
Health Protection Agency, Centre for Emergency Preparedness & Response, Porton Down, Salisbury, Wilts SP4 0JG, UK. liz.evans@hpa.org.uk
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D000907:Antibodies, Bacterial / Q000276:immunology; D001905:Botulinum Toxins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D019274:Botulinum Toxins, Type A; D003470:Culture Media; D010450:Endopeptidases / Q000032:analysis; D057230:Limit of Detection; D013574:Synaptosomes / Q000378:metabolism*
D000907:Antibodies, Bacterial; D003470:Culture Media; C096323:rimabotulinumtoxinB; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096321:botulinum toxin type F