Characterizing and applying immunoglobulins in snakebite diagnostics: A simple and rapid venom detection assay for four medically important snake species in Southeast Asia.

Snakebite envenoming is a medical emergency requiring urgent and specific treatment. Unfortunately, snakebite diagnostics are scarce, time-consuming and lacking specificity. Hence, this study aimed to develop a simple, quick and specific snakebite diagnostic assay using animal antibodies. Anti-venom horse immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY) were produced against the venoms of four major medically important snake species in Southeast Asia, i.e., the Monocled Cobra (Naja kaouthia), Malayan Krait (Bungarus candidus), Malayan Pit Viper (Calloselasma rhodostoma), and White-lipped Green Pit Viper (Trimeresurus albolabris). Different capture:detection configurations of double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were constructed using both immunoglobulins, and the horse IgG:IgG-HRP configuration was found to be most selective and sensitive in detecting the corresponding venoms. The method was further streamlined to develop a rapid immunodetection assay, which is able to produce a visual color change within 30 min for discrimination between different snake species. The study shows it is feasible to develop a simple, quick and specific immunodiagnostic assay using horse IgG, which can be derived directly from antisera prepared for antivenom production. The proof-of-concept indicates it is a sustainable and affordable approach in keeping with on-going antivenom manufacturing activities for specific species in the region.

An immunodetection assay developed using cobra cytotoxin-specific antibodies: Potential diagnostics for cobra envenoming.

Cobra (Naja spp.) envenoming is a life-threatening medical emergency, and a correct diagnosis is crucial to initiating timely and appropriate antivenom treatment. However, snakebite diagnostics remain unavailable in Southeast Asia. This study, therefore, developed an immunodetection assay with a potential diagnostic application for cobra envenoming. The cytotoxin of Naja kaouthia (Thai Monocled Cobra) (Nk-CTX) was purified from its venom to produce CTX-specific antibodies in rabbits and chickens. A double-antibody sandwich enzyme-linked immunosorbent assay was developed using the purified anti-Nk-CTX antibodies (immunoglobulin G and immunoglobulin Y), and its selectivity, specificity, and sensitivity for the venoms of five major cobra species in Southeast Asia (N. kaouthia, Naja sumatrana, Naja sputatrix, Naja siamensis, and Naja philippinensis) were studied. The results showed the immunoassay discriminates cobra venoms from other species commonly implicated in snakebites in Southeast Asia, i.e., the Malayan Krait, Many-banded Krait, King Cobra, Eastern Russell's Viper, Malayan Pit Viper and White-lipped Pit Viper. The immunoassay has a high sensitivity for the five cobra venoms, with detection limits (LoD) ranging from 0.6 to 2.6 ng/ml. Together, the findings suggest the potential diagnostic application of the cytotoxin immunoassay for cobra envenoming. The immunoassay was found to exhibit high immunoreactivity toward ten Asiatic cobra venoms (absorbance > 1.5), in contrast to African cobra venoms with low immunoreactivity (absorbance < 0.9). Considering the varying CTX antigenicity between Asiatic and African cobras, the immunoassay for African cobras should utilize antibodies produced specifically from the cytotoxins of African cobra venoms.

Snake venom proteomics and antivenomics of two Sundaic lance-headed pit vipers: Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia).

Envenomation by two medically important Sundaic pit vipers, Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia), causes hemotoxic syndrome with a potentially fatal outcome. Research on the compositions and antigenicity of these pit viper venoms is however lacking, limiting our understanding of the pathophysiology and treatment of envenomation. This study investigated the venom proteomes of both species through a protein decomplexation strategy, applying C18 reverse-phase high-performance liquid chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein identification through nano-electrospray ionization liquid chromatography-tandem mass spectrometry (nano-ESI-LCMS/MS) of trypsin-digested peptides. The venom antigenicity was profiled against the Thai Green Pit Viper Antivenom (GPVAV, a hetero-specific antivenom), using indirect enzyme-linked immunosorbent assay (ELISA). The venom proteomes of T. wiroti and T. puniceus consisted of 10 and 12 toxin families, respectively. The major proteins were of diverse snake venom serine proteases (19-30% of total venom proteins), snake venom metalloproteinases (17-26%), disintegrins (9-16%), phospholipases A2 (8-28%) and C-type lectins (~8%). These were putative snake toxins implicated in hemorrhage and coagulopathy, consistent with clinical hemotoxicity. GPVAV showed strong immunorecognition toward high and medium molecular weight proteins (e.g., SVMP and PLA2) in both venoms, while a lower binding activity was observed toward small proteins such as disintegrins. Conserved antigenicity in the major hemotoxins supported toxicity cross-neutralization by GPVAV and indicated that the immunorecognition of low molecular weight toxins may be optimized for improved binding efficacy. Taken together, the study provides insights into the pathophysiology and antivenom treatment of envenomation caused by T. wiroti and T. puniceus in the region.

Toxicity and cross-neutralization of snake venoms from two lesser-known arboreal pit vipers in Southeast Asia: Trimeresurus wiroti and Trimeresurus puniceus.

The lesser-known Sundaic lance-headed pit vipers Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia) contribute to the disease burden of snakebite envenomation in Southeast Asia, but their venom toxicity and neutralization remain insufficiently investigated. This study demonstrated that both venoms were procoagulant (involving thrombin-like activity), hemorrhagic, and lethal to mice, with T. wiroti venom being more lethal (LD50 = 0.78 µg/g c.f. 1.21 µg/g). The hetero-specific antivenom from Thailand, Green Pit Viper Antivenom (GPVAV, raised against Trimeresurus albolabris) cross-reacted with T. wiroti and T. puniceus venoms with a higher efficacy of immunological binding activity for the latter. The antivenom was also effective in cross-neutralizing the procoagulant, hemorrhagic and lethal effects of the venoms. In lethality neutralization, GPVAV showed a potency of 0.79-1.05 mg venom per mL antivenom, corresponding to the complete neutralization of approximately 8-10 mg venom per unit vial of antivenom for T. wiroti and T. puniceus venoms. Taken together, it was inferred that T. wiroti, T. puniceus, and T. albolabris venoms share common toxin epitopes, thus enabling the cross-neutralization observed. These findings suggest that GPVAV may be potentially useful in the management of envenomation by T. wiroti and T. puniceus while awaiting clinical trial and validation.