테트로도톡신 검출을 위한 Neuro-2a 시험법의 시험 매개변수 최적화 및 실험실 간 검증 연구 = Optimization of Test Parameters and Interlaboratory Validation of Neuro-2a Assay for the Detection of Tetrodotoxin

국문 초록 (Abstract)

테트로도톡신(tetrodotoxin, TTX)은 강력한 해양생물 유 래 신경독소로, 수산물 내 TTX를 검출하기 위해 기존에 주로 사용되는 mouse bioassay (MBA)와 LC-MS/MS 기법 은 낮은 검출한계와 동물 윤리 문제 등의 한계가 있어 이 를 대체할 새로운 시험법 개발이 필요합니다. Neuro-2a assay는 대표적인 세포기반 대체 시험법으로, 이 방법은 마우스 신경모세포인 Neuro-2a 세포주에 ouabain (O)과 veratridine (V)을 처리하여 과도한 Na+ 유입으로 인한 세 포 사멸을 유도한 후, Na+ 채널 억제제인 TTX가 Na+ 유 입을 차단해 세포를 보호하는 원리를 이용해 TTX를 정량 합니다. 본 연구에서는 Neuro-2a assay를 국내 실험실 환경에 적용하기 위해 TTX 처리 조건과 O/V 농도 등의 매개변수를 최적화하였습니다. 그 결과, 최적 O/V 농도로600/60 µM를 설정하였으며, S자형 용량-반응 곡선이 도출되는 8가지 농도(50-0.195 ng/mL)를 확인하였습니다. 또한,24번의 반복 실험을 통해 데이터의 신뢰도를 평가할 수있는 6가지 data criteria를 확립하였으며, 이 중 EC50 값은 약 3.824-1.268 ng/mL로 나타났습니다. 실험실 간 변동성 비교 결과, COV+와 Bottom OD값을 제외한 모든 품질 관리 기준(quality control criteria)과 데이터 기준(datacriteria)의 변동계수(CVs)는 1.31-14.92%로 도출되어, 실험의 적정성과 재현성이 확인되었습니다. 본 연구는 국내에서 활용 가능한 TTX 검출용 Neuro-2a assay의 최적 조건과 신뢰성을 평가할 수 있는 quality control criteria와data criteria를 제시하였습니다. 아울러, TTX뿐만 아니라유사체인 4,9-anhydroTTX에 대한 TEF 값을 0.2098로 산출하여, TTX뿐 아니라 다양한 유사체의 검출이 가능함을 확인하였습니다. 향후, 본 시험법은 국내 수산물 내TTX 검출을 위한 MBA 대체법으로 활용될 것으로 기대됩니다.

다국어 초록 (Multilingual Abstract)

Tetrodotoxin (TTX) is a potent marine-derived neurotoxin. Existing detection methods for TTX, such as mouse bioassay (MBA) and LC-MS/MS, are limited by ethical concerns, and low detection thresholds, particularly in the absence of reference standards. Alternative testing methods are thus critically needed. The Neuro-2a assay is a well-established cell-based assay that uses mouse-derived Neuro-2a cells treated with ouabain (O) and veratridine (V), to induce cell death via excessive Na+ influx. This assay quantitates TTX based on its ability to inhibit Na+ influx, thereby allowing the cells to survive. In this study, we optimized parameters including TTX treatment conditions and O/V concentrations, to adapt the Neuro-2a assay for domestic laboratory conditions. The optimal O/V concentrations were determined to be 600/60 µM. We also identified eight concentration points (50-0.195 ng/mL) that generated a sigmoidal dose-response curve. By conducting 24 replicate experiments, we established six key data criteria to ensure reliability, with EC50 values ranging from 3.824 to 1.268 ng/mL. A comparison of inter-laboratory variability revealed that all quality control and data criteria values, except for COV+ and Bottom OD, showed coefficients of variation (CVs) ranging from 1.31 to 14.92%, confirming the assay's accuracy and reproducibility. In this study, we also elucidate optimal assay conditions, validated quality control and data criteria for TTX detection using the Neuro-2a assay in local laboratories. Additionally, we conducted an assay with 4,9-anhydroTTX, a TTX analog, and determined a TEF value of 0.2098, demonstrating the applicability of this method for detecting TTX and its analogs. This optimized Neuro-2a assay is expected to serve as an effective alternative to MBA for detecting TTX in domestic seafood products.

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