A paper microfluidic device based colorimetric sensor for the detection and discrimination of elapid versus viper envenomation.

Snake bites are a neglected tropical disease, causing mortality and severe damage to various vital organs like the nervous system, kidneys and heart. There is increasing interest in designing new antivenom treatments that are more specific to particular groups (either taxonomic or regional) of species, given the increasing evidence that current polyvalent Indian antivenom is ineffective in many situations. Under these circumstances, being able to detect the species, or a group of species, responsible for the envenomation becomes important. Unfortunately, no such diagnostic tool is available in the Indian market. Such a tool will need to be rapid, sensitive and affordable. To address this need, we have combined the power of nanotechnology and paper microfluidics and herein report a device that has the ability to detect and differentiate viper venom from elapid and scorpion venom. In principle, this assay is based on the release of the dye from the stimuli-responsive glutaraldehyde cross-linked methylene blue-loaded gelatin (GMG) nanoparticles in the presence of snake venom metalloproteases and serine proteases. The developed equipment-free assay can detect and discriminate viper venom from that of elapids and scorpions. The low-end detection limit of the sensor is ∼3.0 ng for the saw-scaled viper Echis carinatus, while the same for Russell's viper Daboia russelii is ∼6.0 ng. The performance of the sensor remains unaltered for different batches of GMG nanoparticles. Altogether, this finding establishes the role of nanotechnology and paper microfluidics in the rapid and accurate detection of viper venom.

Assessment of quality and pre-clinical efficacy of a newly developed polyvalent antivenom against the medically important snakes of Sri Lanka.

Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL (Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7-97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab')2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Rapid and selective detection of experimental snake envenomation - Use of gold nanoparticle based lateral flow assay.

In this study, we have developed a gold nanoparticle based simple, rapid lateral flow assay (LFA) for detection of Indian Cobra venom (CV) and Russell's viper venom (RV). Presently, there is no rapid, reliable, and field diagnostic test available in India, where snake bite cases are rampant. Therefore, this test has an immense potential from the public health point of view. The test is based on the principle of the paper immunochromatography assay for detection of two snake venom species using polyvalent antisnake venom antibodies (ASVA) raised in equines and species-specific antibodies (SSAbs) against venoms raised in rabbits for conjugation and impregnation respectively. The developed, snake envenomation detection immunoassay (SEDIA) was rapid, selective, and sensitive to detect venom concentrations up to 0.1 ng/ml. The functionality of SEDIA strips was confirmed by experimental envenomation in mice and the results obtained were specific for the corresponding venom. The SEDIA has a potential to be a field diagnostic test to detect snake envenomation and assist in saving lives of snakebite victims.

Comparison of Montanide adjuvants, IMS 3012 (Nanoparticle), ISA 206 and ISA 35 (Emulsion based) along with incomplete Freund's adjuvant for hyperimmunization of equines used for production of polyvalent snake antivenom.

The use of adjuvant is of fundamental importance in vaccines formulations and antisera production. Currently selection and use of adjuvant systems in snake antivenom preparation has become a major issue in terms of animal welfare as well as economics. In order to minimize disadvantages associated with traditionally used Freund's adjuvant (FA) in equines and to produce potent polyvalent antivenom against four Indian snake venoms in minimum possible period, a comparison was made between various commercially available non-emulsion/emulsion based adjuvants like IMS 3012, ISA 206 and ISA 35 with Incomplete Freund's adjuvant (IFA) for their immunopotentiation capacity and safety in donor animals. The present study was conducted in 33 new horses, randomly divided into four groups and hyperimmunized using crude mixture of snake venoms, viz.; Cobra venom (CV), Russell's viper venom (RV), Krait venom (KV) and Saw-scaled viper (EV) along with four above mentioned adjuvants through subcutaneous (s.c.) route at intervals of two weeks. Periodic standard safety assessments were done. Immunopotentiation ability of each adjuvant group in terms of percent responders were estimated at 14th, 21st, 30th and 43rd week. The neutralization activity (ED(50)) of pooled sera samples by 43(rd) week, obtained with IMS 3012 group for CV, RV, KV and EV venoms were 0.133, 0.143, 0.070 and 0.270 mg venom/ml of serum respectively. The antivenom potency with IMS 3012 and overall responding horses (100%) even against weak immunogen like CV was significantly higher (p<0.05) than other three adjuvants studied. The horses of IMS 3012 group showed minimum local reactions at injection site, while horses from other three groups exhibited moderate (++) reactions; 66.7% in ISA 206, 12.5% in ISA 35 and 14.3% in IFA respectively, however these were transient and reabsorbed or healed subsequently. Finally, we conclude that, nanoparticle adjuvant IMS 3012 could be a possible alternative to the emulsion adjuvants for primary phase of immunization in antivenom preparation considering its better immunopotentiation capacity and safety in donor animals.