Enzyme immunoassays for detection and quantification of venoms of Sri Lankan snakes: Application in the clinical setting

Abstract

Background Detection and quantification of snake venom in envenomed patients' blood is important for identifying the species responsible for the bite, determining administration of antivenom, confirming whether sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Currently, snake venom detection is not available in clinical practice in Sri Lanka. This study describes the development of enzyme immunoassays (EIA) to differentiate and quantify venoms of Russell's viper (Daboia russelii), saw-scaled viper (Echis carinatus), common cobra (Naja naja), Indian krait (Bungarus caeruleus), and hump-nosed pit viper (Hypnale hypnale) in the blood of envenomed patients in Sri Lanka. Methodology / Principal findings A double sandwich EIA of high analytical sensitivity was developed using biotin-streptavidin amplification for detection of venom antigens. Detection and quantification of D. russelii, N. naja, B. caeruleus, and H. hypnale venoms in samples from envenomed patients was achieved with the assay. Minimum (less than 5%) cross reactivity was observed between species, except in the case of closely related species of the same genus (i.e., Hypnale). Persistence/ recurrence of venom detection following D. russelii envenoming is also reported, as well as detection of venom in samples collected after antivenom administration. The lack of specific antivenom for Hypnale sp envenoming allowed the detection of venom antigen in circulation up to 24 hours post bite. Conclusion The EIA developed provides a highly sensitive assay to detect and quantify five types of Sri Lankan snake venoms, and should be useful for toxinological research, clinical studies, and forensic diagnosis.

A Strategy for Efficient Preparation of Genus-Specific Diagnostic Antibodies for Snakebites

“…The major issue with snakebite diagnosis is the cross-reactivity among venoms, as some proteins in each venom overlap and detection devices are rarely species-specific, instead detecting a range of species when using immunological techniques (24). To date, most diagnostic antibodies exhibit poor specificity (12)(13)(14)(15), thus hindering the correct selection and dosage of antiserum. We showed here that the principles of venom similarities and the underlying cross-reactivities of the same genus could be applied to develop universal genus-specific diagnosis tools for snakebite (11).…”

“…The application of specific antibodies in the diagnosis of snakebite can accurately judge the type of snakebite and prevent unwarranted and wasteful administration of antivenom ( 11 ). To date, most of the diagnostic antibodies were polyclonal antibodies with poor specificity and often saturate at a high concentration of snake venom, for example, the polyclonal diagnostic antibodies for distinguishing hematotoxin and neurotoxin and the polyclonal diagnostic antibodies for individual snake species ( 12 ), while the contents of snake venom in the plasma of different patients, bitten by snakes of the same species, vary up to 100-folds (from a few nanograms to hundreds of nanograms) ( 12 , 13 ). Although monoclonal antibodies have been reported, they cannot be used in the clinic because of their low specificity ( 14 , 15 ).…”

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom

“…Even so, the universal presence of this protein in both elapid and viper venoms raise questions on its utility as a marker for species-specific venom identification. Polyclonal antibody-based enzymatic-or radioactivity-assays can detect venom up to 1-5 ng/mL in the sample [28,44]. These assays PLOS NEGLECTED TROPICAL DISEASES require long detection time and expertise which is undesirable for rapid point-of-care testing of envenomation.…”

“…Traditionally, diagnosis for snake envenomation relied primarily on visual examination followed by polyclonal sera-based detection strategies such as, immunodiffusion, immunofluorescence, hemagglutination, immunoelectrophoresis, radioimmunoassay; which in turn were limited by their tendency to crossreact with the closely related venoms, the reduced assay sensitivity, poor scalability for industrial production, and a prolonged-time of detection [21][22][23]. With advancing technologies, portable ELISA based kits, optical immunoassay, dot-blot assay, and family-specific protein identification tests were developed for detection of the specific venom using a three-step affinity-purified polyclonal antibody [24][25][26][27][28]. Affinity-purified polyclonal antibody raised against a cocktail of selective venoms is limited by its scalability for industrial production.…”

Clinico-epidemiology and management of hump-nosed pit viper (Hypnale spp.) bites in dogs

“…In addition, a venom quantification study was able to demonstrated venom antigen in circulation up to 24 h post bite in human due to scarcity of specific antivenom for Hypnale spp. envenoming 20 .…”