MSACL 2024 Abstract

An Examination of Potential Interference in a Clinical Assay for Botulinum Neurotoxins through Mass Spectrometric Detection

Kaitlin M. Hoyt (1), John R. Barr (1), André O. Hopkins (2), Janet K. Dykes (2), Carolina Lúquez (2), and Suzanne R. Kalb (1)
1. Division for Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA 2. Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

Kaitlin Hoyt, B.S. (Presenter) Centers for Disease Control and Prevention

Relevant Financial Disclosures (within past 24 months, reported on Feb 01, 2023) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:
Botulism is a paralytic disease caused by botulinum neurotoxin-mediated cleavage of proteins in the neuromuscular junction that facilitate acetylcholine exocytosis. The current approved confirmatory assay for BoNTs in clinical specimens is the mouse bioassay. An in vitro assay to detect and differentiate BoNT, the Endopep-MS method, has been developed and validated for clinical samples using a benchtop mass spectrometric detection. Validation included potential interfering substances of the Endopep-MS method in clinical specimens with the intent of method distribution in public health laboratories while seeking U.S. Food and Drug Administration (FDA) approval.

Methods:
The Endopep-MS method detects BoNT based on the cleavage products of a peptide substrate, which mimics its native target. Serum and stool samples spiked with potential interfering substances (such as acetaminophen and bilirubin) at therapeutic levels and with BoNTs at 5 times the limit of detection for each serotype, were tested by Endopep-MS, Toxin was extracted from the sample with high-affinity monoclonal antibodies and the cleavage of a serotype specific peptide substrate determined the presence of BoNT utilizing MALDI-TOF mass spectrometry detection.

Results:
The panel of potential interfering substances was used to assess selectivity for the validation of the Endopep-MS method. The interfering substances panel was designed to test exogeneous and endogenous substances that are commonly present in clinical specimens such as serum and stool extract that could affect the possible outcome of results for the Endopep-MS method. The exogeneous and endogenous substances presented no interference in testing spiked serum and stool extract with the BoNT Endopep-MS assay, with the one exception of sodium phosphate. Stool samples spiked with sodium phosphate at a concentration of 2.5 g/L resulted negative for BoNT.

Discussion & Conclusions:
The list for interfering substances was determined from the Clinical Laboratory Standards Institute (CLSI) EP07-A2 guideline, with additions made by the FDA and the National Center for Emerging and Zoonotic Infectious Diseases epidemiologists at the Centers for Disease Control and Prevention. Sodium phosphate was the only substance to show interference with the assay. It was included as an exogenous substance because it is present in most enema solutions at ~1 g; however, patients with suspected botulism only receive sterile non-bacteriostatic water enemas for stool collection (with no sodium phosphate). Although the Endopep-MS method uses sodium phosphate in gelatin phosphate-buffered collecting fluid and 1X PBST, the limits are below the interference threshold at 6 mg/mL, 0.20 mg/mL, and 1.6 mg/mL. Therefore, although the large amounts of sodium phosphate had interference with the detection of BoNT, it would not be a substance present in stool specimens collected for botulism testing. Testing for potential interfering substances is a critical step of methods validation studies and our results demonstrate that the Endopep-MS assay for BoNT detection is not affected by 72 substances.



The findings and conclusions in this presentation have not been formally disseminated by the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry and should not be construed to represent any agency determination or policy.