Ultrasensitive “Hunter of Target”: Rabbit Monoclonal Antibody-Based Competitive Lateral Flow Immunoassay for Saxitoxin

Abstract

The rapid, sensitive, and accurate detection of paralytic shellfish toxins (PSTs), such as saxitoxin (STX), is critical for protecting human health due to the frequent occurrence of toxic red tides. In this work, to address the low affinity of traditional mouse monoclonal antibodies (m-mAbs), rabbit monoclonal antibodies (r-mAbs) against STX were produced by a single B-cell sorting culture and a cross-selection strategy. The r-mAbs showed 100-fold improvement in sensitivity (IC₅₀ = 0.018 ng/mL), improved stability under strong acid/alkali resistance (pH = 3–12), and high tolerance to organic solvent (MOH, 30%) in comparison with m-mAbs. The structural analysis of PSTs shows that the r-mAbs primarily interacted with common functional groups in PSTs, including guanidine, amide, and alcoholic hydroxyl groups (−OH). R-mAbs3C3 recognized STX, GTX2/3, C1/C2, NEO, and dcSTX with broad specificity through hydrogen bonding facilitated by Asp93, Gln27, Ser27A, and Asp95 in the complementarity-determining region (CDR) pocket. In contrast, r-mAbs3C7 displayed high specificity for STX, mediated by Ser98, Leu96, Ser30, Tyr97, and Ser53, which formed hydrogen bonds and hydrophobic interactions. The lack of interaction with Asp93 and Asp95 in r-mAbs3C7 likely contributed to its enhanced specificity. The anti-STX r-mAbs3C3 was then used to develop an ultrasensitive lateral flow immunoassay (LFIA) for the detection of STX with a cutoff value of 100 pg/mL. The LFIA demonstrated recovery rates ranging from 86.6 to 106.3% in food samples (mussels, nassariidae, bullacta exarata, and fresh water). Given its high target selectivity and sensitivity, this study provides a sight for rapid, sensitive, and accurate monitoring of food toxins.