Development of sandwich ELISA and lateral flow strip assays for diagnosing clinically significant snakebite in Taiwan

PLoS Negl Trop Dis. 2018 Dec 3;12(12):e0007014. doi: 10.1371/journal.pntd.0007014. eCollection 2018 Dec.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antivenins / immunology
  • Cross Reactions
  • Enzyme-Linked Immunosorbent Assay / instrumentation
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Female
  • Humans
  • Mice, Inbred C57BL
  • Snake Bites / blood
  • Snake Bites / diagnosis*
  • Snake Bites / parasitology
  • Snakes / classification
  • Snakes / immunology
  • Snakes / physiology*
  • Taiwan

Substances

  • Antivenins

Grants and funding

This work was supported by the Ministry of Science and Technology, Taiwan (grants MOST 106-2632-B-182-002 and 106-2320-B-182-029-MY3 to JSY), by Chang Gung Memorial Hospital, Taiwan (grants CMRPG3F0241, 242 and 243 to CCLin, CIRPD3B0013, CRRPD1F0051, 52 and 53, CLRPD190018 and BMRP208 to JSY), and by the “Molecular Medicine Research Center, Chang Gung University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.