Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay

Abstract

Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell’s viper venom or Australian elapid venom measured by EIA. In confirmed Russell’s viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell’s viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell’s viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom.

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

“…Hence, snake venom detection is not possible in these cases due to complete neutralization of venom by antivenom. Maduwage et al (2016) described a method to dissociate venom-antivenom complexes, thus allowing venom detection [31]. This protocol detected D. russelii and H. hyupnale venom in post-antivenom samples in our study, indicating a further use of EIA in clinical practice.…”

“…Venom antigens in serum samples from patients that had been treated with antivenom were detected after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 95˚C [31]. Treated serum samples underwent venom EIA as described above [31].…”

“…Few clinical studies have detected venom of Sri Lankan snakes by Enzyme Immunoassays (EIA), but they have not been introduced in the routine clinical practice [29][30][31][32][33]. The most commonly used method of venom detection in biological samples is EIA owing primarily to its high analytical sensitivity.…”

Clinical and Pharmacological Investigation of Myotoxicity in Sri Lankan Russell’s Viper (Daboia russelii) Envenoming

“…Measurement of venom in the samples was carried out using a Russell’s viper venom specific Enzyme-Immunoassay (EIA) which has been previously described [6,34,35]. In brief, rabbit IgG was raised against Russell’s viper venom from Sri Lanka.…”

“…The lower limit of detection for the assay was 2.5 ng/ml. In cases where no venom was detected in pre-antivenom samples or pre-antivenom samples were not available, the post-antivenom samples were subjected to heat dissociation treatment and then tested for venom using the same EIA [35]. …”