Abstract
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.
Highlights
Envenoming resulting from snakebites is a significant public health issue in many regions of the world, in tropical and subtropical countries and some poor rural communities [1]
We present a two-step affinity purification procedure for generating species-specific antibodies (SSAbs) from antivenom, followed by the development of a sandwich enzyme-linked immunosorbent assays (ELISAs) 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
Summary
Envenoming resulting from snakebites is a significant public health issue in many regions of the world, in tropical and subtropical countries and some poor rural communities [1]. The species responsible for envenomation of snakebite victims referred for medical treatment is initially identified primarily based on the shape of the wound or identification of dead snakes brought to the hospital. Some clinical symptoms caused by envenomation are similar among species, and non-venomous snakes are often responsible for the patient’s snakebite [4]. Identification of venomous snake species is important for optimal clinical management, because it allows physicians to use the correct antivenom for effective treatment, thereby improving patients’ prognosis and preventing the waste of expensive antivenoms and exposing victims to antivenom-induced adverse reactions [6]. Identification of snake species is important for the management of snakebite-related injuries worldwide, there are currently no developed standard platforms or guidelines for snakebite diagnosis globally
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