Nanobody-based lateral flow assays for the detection of α-bungarotoxin in Bungarus snake venoms

Abstract Accurate snake identification is critical for effective antivenom treatment following snakebite envenoming, but remains a major diagnostic challenge. Here, we developed a prototype lateral flow immunoassay (LFIA) designed to distinguish krait ( Bungarus ) from cobra ( Naja ) envenoming in Southeast Asia. To enhance point-of-care utility, the assay employs variable domains of heavy-chain-only antibodies (V H Hs). The V H Hs target α-bungarotoxin (α-BuTx), a neurotoxin unique to Bungarus , with negative selection strategies to minimize cross-reactivity with α-cobratoxin in Naja venom. Our prototype LFIA detected α-BuTx with a visual limit of detection of 0.5–5 ng/mL in running buffer and spiked urine and serum samples. Additionally, the assay detected Bungarus whole venom at 50–500 ng/mL in running buffer. No cross-reactivity with Naja venom was observed at clinically relevant concentrations, with signals detected only at supraphysiological concentrations (≥50,000 ng/mL). These results support the feasibility of using V H H-based LFIAs for guiding timely, targeted antivenom administration in resource-limited settings.