Ultrasensitive\n“Hunter of Target”: Rabbit\nMonoclonal Antibody-Based Competitive Lateral Flow Immunoassay for\nSaxitoxin

The rapid, sensitive, and accurate detection of paralytic\nshellfish\ntoxins (PSTs), such as saxitoxin (STX), is critical for protecting\nhuman health due to the frequent occurrence of toxic red tides. In\nthis work, to address the low affinity of traditional mouse monoclonal\nantibodies (m-mAbs), rabbit monoclonal antibodies (r-mAbs) against\nSTX were produced by a single B-cell sorting culture and a cross-selection\nstrategy. The r-mAbs showed 100-fold improvement in sensitivity (IC<sub>50</sub> = 0.018 ng/mL), improved stability under strong acid/alkali\nresistance (pH = 3–12), and high tolerance to organic solvent\n(MOH, 30%) in comparison with m-mAbs. The structural analysis of PSTs\nshows that the r-mAbs primarily interacted with common functional\ngroups in PSTs, including guanidine, amide, and alcoholic hydroxyl\ngroups (−OH). R-mAbs3C3 recognized STX, GTX2/3, C1/C2, NEO,\nand dcSTX with broad specificity through hydrogen bonding facilitated\nby Asp93, Gln27, Ser27A, and Asp95 in the complementarity-determining\nregion (CDR) pocket. In contrast, r-mAbs3C7 displayed high specificity\nfor STX, mediated by Ser98, Leu96, Ser30, Tyr97, and Ser53, which\nformed hydrogen bonds and hydrophobic interactions. The lack of interaction\nwith Asp93 and Asp95 in r-mAbs3C7 likely contributed to its enhanced\nspecificity. The anti-STX r-mAbs3C3 was then used to develop an ultrasensitive\nlateral flow immunoassay (LFIA) for the detection of STX with a cutoff\nvalue of 100 pg/mL. The LFIA demonstrated recovery rates ranging from\n86.6 to 106.3% in food samples (mussels, nassariidae, bullacta exarata,\nand fresh water). Given its high target selectivity and sensitivity,\nthis study provides a sight for rapid, sensitive, and accurate monitoring\nof food toxins.