Significance: Oral squamous cell carcinoma (OSCC) accounts for 90% of oral cancers, and earlier detection efficiently increases the survival rate. Here, we used a comprehensive literature review to select candidates and used LC-multiple reaction monitoring-MS to qualitatively and quantitatively measure target proteins in saliva samples from individuals of the at-risk population in Taiwan. Statistical analysis of the results establishes a four-protein panel sufficient to detect 88.6% of early-stage patients and 91.6% of all patients with 80.4% specificity. This panel can also be used to evaluate the risk of malignant progression from high-risk oral potential malignant disorder. Our study offers a practical foundation for clinical trials examining the ability of this panel to enable early detection of OSCC, risk assessment for cancer development, and treatment monitoring.

Taiwan is an island located in the south Pacific, a subtropical region that is home to 61 species of snakes. Of these snakes, four species—Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Bungarus multicinctus and Naja atra—account for more than 90% of clinical envenomation cases. Currently, there are two types of bivalent antivenom: hemorrhagic antivenom against the venom of T. stejnegeri and P. mucrosquamatus, and neurotoxic antivenom for treatment of envenomation by B. multicinctus and N. atra. However, no suitable detection kits are available to precisely guide physicians in the use of antivenoms. Here, we sought to develop diagnostic assays for improving the clinical management of snakebite in Taiwan. A two-step affinity purification procedure was used to generate neurotoxic species-specific antibodies (NSS-Abs) and hemorrhagic species-specific antibodies (HSS-Abs) from antivenoms. These two SSAbs were then used to develop a sandwich ELISA (enzyme-linked immunosorbent assay) and a lateral flow assay comprising two test lines. The resulting ELISAs and lateral flow strip assays could successfully discriminate between neurotoxic and hemorrhagic venoms. The limits of quantification (LOQ) of the ELISA for neurotoxic venoms and hemorrhagic venoms were determined to be 0.39 and 0.78 ng/ml, respectively, and the lateral flow strips were capable of detecting neurotoxic and hemorrhagic venoms at concentrations lower than 5 and 50 ng/ml, respectively, in 10–15 min. Tests of lateral flow strips in 21 clinical snakebite cases showed 100% specificity and 100% sensitivity for neurotoxic envenomation, whereas the sensitivity for detecting hemorrhagic envenomation samples was 36.4%. We herein presented a feasible strategy for developing a sensitive sandwich ELISA and lateral flow strip assay for detecting and differentiating venom proteins from hemorrhagic and neurotoxic snakes. A useful snakebite diagnostic guideline according to the lateral flow strip results and clinical symptoms was proposed to help physicians to use antivenoms appropriately. The two-test-line lateral flow strip assay could potentially be applied in an emergency room setting to help physicians diagnose and manage snakebite victims. Snakebite is a public health issue that causes life-threatening medical emergencies. Rapid diagnosis of snakebite in the clinic is a critical necessity in many tropical and subtropical countries, where various venomous snakes are common. Venoms from different snake species contain distinct protein components that require treatment with different antivenoms. However, given the similarity in clinical symptoms among some snake envenomations, it is often challenging for physicians to precisely define the snake species responsible for envenomation. Thus, a reliable method or assay for rapidly diagnosing envenoming species is urgently needed. Here, we present a two-step affinity purification procedure for generating species-specific antibodies (SSAbs) from antivenom, followed by the development of a sandwich ELISA (enzyme-linked immunosorbent assay) and lateral flow strip assay using these SSAbs. This feasible and cost-effective strategy allowed us to develop workable assays for distinguishing between venom proteins from hemorrhagic and neurotoxic snakes in Taiwan. The usefulness of this strategy was demonstrated in the clinic, where both diagnostic assays were shown capable of detecting venoms in blood samples from snakebite patients. Together with the observation of clinical symptoms, the two-test-line lateral flow strip assay is potentially applicable in an emergency room setting to improve snakebite diagnosis and management.