Antivenom Antibodies Research Articles

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73 %) CB-Cdc catalytic activity (5 mol), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 mol) and reduced the CTx-Cdc (5 mol) enzyme activity by 65 %, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

Development of a gold nanoparticle-based novel diagnostic prototype for in vivo detection of Indian red scorpion (Mesobuthus tamulus) venom

Indian red scorpion Mesobuthus tamulus is responsible for substantial mortality in India and Sri Lanka; however, no specific diagnostic method is available to detect the venom of this scorpion in envenomed plasma or body fluid. Therefore, we have proposed a novel, simple, and rapid method for detecting M. tamulus venom (MTV) in the plasma of envenomed animals using polyclonal antibodies (PAb) raised against three modified custom peptides representing the antigenic epitopes of K+ (Tamapin) and Na+ (α-neurotoxin) channel toxins, the two major MTV toxins identified by proteomic analysis. The optimum PAb formulation containing PAb 1, 2, and 3 in proportion (1:1:1, w/w/w) acted synergistically, demonstrating significantly higher immunological recognition of MTV than anti-scorpion antivenom (developed against native toxins) and individual antibodies against peptide immunogens. The PAb formulation could detect MTV optimally in envenomed rat plasma (intravenous and subcutaneous routes) at 30–60 min post-injection. The acetonitrile precipitation method developed in this study to augment the MTV detection sensitivity enriched the low molecular mass peptide toxins in envenomed rat plasma, which was ascertained by mass spectrometry analysis. The gold nanoparticles conjugated PAb formulation, characterised by biophysical techniques such as Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), demonstrated their interaction with low molecular mass MTV peptide toxins in envenomed rat plasma. This interaction results in the accumulation of the gold nanoparticles, thus leading to signal change in absorbance spectra that can be discerned within 10 min. From a standard curve of MTV spiked plasma, the quantity of MTV in envenomed rat plasma could be determined by gold nanoparticle-PAb formulation conjugate.

PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST INDIAN PAPER WASP ROPALIDIA MARGINATA VENOM TOXINS AND THEIR EFFICACY IN THE REVERSAL OF TOXIC EFFECTS

Objective: In this study, albino mice were injected with a sub-lethal dosage of purified wasp Ropalidia Marginata venom toxins to assess the effectiveness of polyclonal anti-venom antibodies. Methods: To neutralize the toxic effects, polyclonal antibodies were generated by immunizing albino mice. The antibody underwent partial purification using ammonium sulphate treatment and octanoic acid precipitation. To detect the presence of antibodies in the antiserum, an immunodouble diffusion test was conducted using Ouchterlony's method (1962). This involved allowing both antigens and antibodies to diffuse radially towards each other from their respective wells. When they reached an equivalence zone, a precipitation complex of antigen and antibody became visible as a concentric band, indicating the development of the combination. To quantitatively determine the amount of antibodies in the antiserum, the equivalency zone approach was used. Results: Experimental mice were injected with a combination containing 400, 800, and 1200 µg of pure antibody, which had been treated serum biomolecules, including metabolic enzymes, completely reversed in the experimental with 40% of the LD50 of wasp venom the elevated serum parameters were glucose, pyruvic acid, lipid, protein and free amino acid, reached to normal (100%) in the treated with 40% of LD50 of the venom and polyclonal treated after 6 h of administration. Anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h. Similarly, anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h treated with 40% of LD50 of the venom. Serum ACP content was obtained as 125.35% after 40% of LD50 venom injection, which was get normalized up to 102.81% after 4 h of the anti-venom treatment. Serum ALP content of 114.8% elevation was reversed back to 102.40% after anti-venom treatment. The GPT level significantly reversed up to 102.5%, while it was 130% in the venom-treated mice. A complete reversal was obtained in GPT level, which was obtained as 104.54% in the venom-treated animal. Similarly, LDH which was elevated up to 112.45 % in venom-injected mice was successfully reversed up to 100.25% after anti-venom treatment. Similarly, Ache concentration was fully recovered after anti-venom treatment 6 h, all animals (group B-E) that had received 40% of the LD50 of venom treated with pure antiserum. Conclusion: The venom-injected group showed a complete restoration of serum protein, free amino acid, uric acid, cholesterol, pyruvic acid, total lipid, and glucose level in experimental mice.

Extração e quantificação de anticorpos entivenenos em ovos de galinha contra veneno de escorpião

ABSTRACT: Avian-derived IgY is thought to be the best therapy for scorpion bites concerning low-level side effects. The present study analyzed a hypothesis about the neutralization of scorpion venom Androcotonus australis through antibodies produced in the egg yolks of chickens. The venom used for inoculation was obtained from Androctonus australis (yellow fat-tailed scorpion) from southern Punjab, Pakistan. The lethal dose of LD50 against scorpion venom was calculated in chickens and mice. Safe doses were given to egg-laying chickens to produce IgY antibodies. The antivenom IgY antibodies were extracted from the egg yolks of immunized chicken using the polyethylene glycol (PEG) method. Moreover, IgY was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Ouchterlony double immunodiffusion assay test. The antibody titers were evaluated by the enzyme-linked immunosorbent assay (ELISA). The neutralisation capacity of extracted anti-scorpion antibodies was tested on mice. The calculated LD50 of scorpion venom for chicken and mice was 4 mg/kg and 2.5 mg/kg, respectively. SDS-PAGE and Ouchterlony double immunodiffusion confirmed the presence of IgY against scorpion venom. The maximum titer value of specific IgY produced against scorpion venom was 3.5 ug/ml. A concentration of 220 ul/LD50 was effective to neutralize 1 mg of scorpion venom. It is suggested that IgY obtained from egg yolks is safe against targeted venom and can be used as an effective alternative to equine IgG antibodies against scorpion envenoming.

Analysis of antibodies avidity for Tityus serrulatus scorpion venom in antivenom production and its potential for application as a potency test

Antivenoms are the only specific medication for neutralizing toxins present in venom of animals such scorpions and snakes through antigen-antibody binding. Several analyses are carried out throughout its production in order to ensure the quality and effectiveness of the antivenom that will be administered to the patient. One of these is the potency assay, which is performed to assess the ability of antivenoms to neutralize the toxic effects of the venom injected in mice. The substitution of in vivo for in vitro assays such as ELISA has been presented by other authors, bringing several advantages such as the reduction in the use of animals, in costs and in the duration of the assays. However, the avidity index of antivenom antibodies determined by ELISA has not yet been applied for this purpose. Therefore, the objective of this study was to evaluate the avidity of sera from hyperimmunized horses with crude Tityus serrulatus venom, a scorpion species associated with the most serious accidents in Brazil, and its potential for application as a potency test replacing the in vivo assay. The avidity ELISA proved to be interesting for monitoring the binding strength of antibodies produced by horses in hyperimmune plasma production programs. It was possible to verify oscillations in antibody avidity that occurred along the immunization cycles, differences between novice and veteran horses, maturation of antibody avidity, and correlation between avidity index and antibody titre. Similar results were obtained for crude venom and purified Ts1 toxin. In addition, the avidity ELISA apparently demonstrated potential for application as a potency test in the initial stage of antivenom production. However, more studies are necessary.

Effects of Adjuvant and Immunization Route on Antibody Responses against Naja Naja oxiana Venom.

Naja naja oxiana (NNO) is one of the important venomous species in Iran. The current snakebite treatment is antivenom therapy that deals with hyper immunization of horses with crude or fractionated snake venom plus traditional adjuvants, like Freund's adjuvant. For improvement of antivenom production, it has been suggested to use different adjuvant systems or immunization procedures. In this study, humoral immune responses against immunogenic fractions of NNO venom (NNO3 and NNO4) and crude venom have been compared by usage of different adjuvant and immunization routes. Additionally, a new indirect enzyme-linked immunosorbent assay (ELISA) was set up for the detection of specific antivenom antibodies. This study was conducted on six different groups of female Dutch rabbits that were hyperimmunized using crude and fractionated NNO venom, along with Freund's and MF59 adjuvants through subcutaneous or intramuscular route. The immunization was performed four times with 10-day intervals and the levels of specific antibodiees were detected by indirect ELISA. The statistical analysis reveals a negligible variation in the antivenom titers among the venom-inoculated groups, regardless of the adjuvant type or the immunization route. Finally, it was concluded that the fractions are efficient for antivenom production, and it is possible to use MF59 adjuvant via subcutaneous routes as an alternative to Freund's adjuvants considering its fair immunopotentiation capacity and safety in animals.

Development of sandwich ELISA and lateral flow assay for the detection of Bungarus multicinctus venom

Snakebite envenoming adversely affects human health and life worldwide. Presently, no suitable diagnostic tools for snakebite envenoming are available in China. Therefore, we sought to develop reliable diagnostic tests for snakebite management. We conducted affinity purification experiments to prepare species-specific antivenom antibody (SSAb). In brief, affinity chromatography with an antibody purification column (Protein A) was conducted to purify immunoglobulin G from Bungarus multicinctus (BM) venom hyperimmunized rabbit serum. The cross-reactive antibodies were removed from commercial BM antivenin by immune adsorption on the affinity chromatography columns of the other three venoms, Bungarus Fasciatus (FS), Naja atra (NA), and O. hannah (OH), generating SSAb. The results of western blot analysis and enzyme-linked immunosorbent assay (ELISA) showed the high specificity of the prepared SSAb. The obtained antibodies were then applied to ELISA and lateral flow assay (LFA) to detect BM venom. The resulting ELISA and LFA could specifically and rapidly detect BM venom in various samples with the limits of quantification as 0.1 and 1 ng/ml, respectively. This method could effectively detect snake venom in experimentally envenomed rats (simulating human envenomation), which could distinguish positive and negative samples within 10-15 min. This method also showed promise in serving as a highly useful tool for a rapid clinical distinguishing of BM bites and rational use of antivenom in emergency centers. The study also revealed cross-reactivity between BM and heterogenous venoms, suggesting that they shared common epitopes, which is of great significance for developing detection methods for venoms of the snakes belonging to the same family.

Pharmacological Characterization of Daboia russelii Venom and its Neutralization by Polyvalent Chicken Antibodies

Snake bite is considered as a socio-economic problem in tropical countries. Daboia russelii (Russell’s viper) is responsible for 30–40% of all snakebites and the most number of life-threatening bites. Antivenom is the only treatment available. Hyperimmune sera raised from horses were shown to produce adverse effects. In this study, chicken Ig Y was generated against the venom of Daboia russelii and its neutralization studies were carried out by in-vitro methods. Antivenom antibodies were generated in chicken and purified by DEAE cellulose column and the antibody titre was estimated in serum and egg yolk by ELISA which showed the increase in titres. Ig Y was subjected to protein estimation, and SDS- PAGE was carried out to study the banding pattern. High molecular weight band 180 KD was observed. Pharmacological characterization like coagulant activity, direct haemolytic assay, indirect haemolytic assay and proteolytic activity was carried out. Minimum coagulant dose was found to be 120 µg. Neutralization studies were carried out using column purified Ig Y antibodies. Current study suggests that Ig Y antivenoms are effective in neutralising the pharmacological activities induced by venom.

A new insight into the cellular mechanisms of envenomation: Elucidating the role of extracellular vesicles in Loxoscelism

Envenomation by the Loxosceles genus spiders is a recurring health issue worldwide and specially in the Americas. The physiopathology of the envenomation is tightly associated to the venom’s rich toxin composition, able to produce a local dermonecrotic lesion that can evolve systemically and if worsened, might result in multiple organ failure and lethality. The cellular and molecular mechanisms involved with the physiopathology of Loxoscelism are not completely understood, however, the venom’s Phospholipases D (PLDs) are known to trigger membrane injury in various cell types. Here, we report for the first time the Loxosceles venom’s ability to stimulate the production of extracellular vesicles (EVs) in various human cell lineages. Components of the Loxosceles venom were also detectable in the cargo of these vesicles, suggesting that they may be implicated in the process of extracellular venom release. EVs from venom treated cells exhibited phospholipase D activity and were able to induce in vitro hemolysis in human red blood cells and alter the HEK cell membranes’ permeability. Nonetheless, the PLD activity was inhibited when an anti-venom PLDs monoclonal antibody was co-administered with the whole venom. In summary, our findings shed new light on the mechanisms underlying cellular events in the context of loxoscelism and suggest a crucial role of EVs in the process of envenomation.

Immunological properties of the chicken egg yolk (IgY) antibodies against Vietnamese cobra Naja Naja venom

Venomous snake bite is common poisonous accident and important medical problem around the world. The snake anti-venom is only type-specific protection to each venom type that was used to immunize. In this study, the chicken egg yolk (IgY) antibodies against the Vietnamese cobra Naja Naja venom was developed and evaluated in in-vitro (ELISA and SDS-PAGE) and in-vivo (hen and mice immunization). In particularly, the LD50 of the Vietnamese cobra Naja Naja venom was determined at 1.46 g kg−1 body weight of the Swiss mice. The ISA Brown layers of 16 to 18-week-old were immunized intramuscularly with the Vietnamese cobra Naja Naja venom at 0.5 mg kg−1 body weight of the layers. The specific anti-venom antibodies were presented in both serum and egg yolk at 14 days, reached to highest antibody titer at 49 to 56 days and lasted up to 111 days post-immunization. The IgY antibodies purified from the egg yolk revealed potential neutralization and protection of Vero cells and Swiss mice from toxicity of the Vietnamese cobra Naja Naja venom. To the best of our knowledge, this study result is the first cobra snake anti-venom developed in laying hens against the Vietnamese cobra Naja Naja venom. Further study is needed to whether this specific IgY anti-venom antibodies can be used as an antibody therapy for treatment of venomous snake bites in the future.

Experimental antivenoms from chickens and rabbits and their comparison with commercially available equine antivenom against the venoms of Daboia russelii and Echis carinatus snakes

In the present work, chicken (IgY) and rabbit (IgG) antibodies were generated against the venoms of Daboia russelii and Echis carinatus snakes and efficacy was compared with commercial antivenom. Antivenom antibodies were purified and evaluated by ELISA, SDS-PAGE, Western Blot. All antivenoms neutralized a challenge dose of 3LD50. Each ml of IgY, IgG and commercial antivenom neutralized 0.3, 0.34, and 0.6 mg of E. carinatus venom and 0.2, 0.22 and 0.57 mg D. russelii venom respectively. The present study suggests the generated antivenoms as effective in neutralizing the venoms injected, implying the scope of IgY antivenoms for further screening and evaluation.

Isolation of Polyclonal Single-Chain Fragment Variable (scFv) Antibodies Against Venomous Snakes of Iran and Evaluation of Their Capability in Neutralizing the Venom.

Several species of dangerous snakes are found in Iran and, according to the Emergency Response Center of Iran from 2002 to 2011, 53,787 Iranians have suffered from snakebite. Although the mortalities caused by snakebite are very low, snakebite-related amputations are still a major concern. Currently, anti-venom polyclonal antibodies derived from animals, such as horses are used to treat snakebites; however, in some cases they can cause anaphylactic shock and serum sickness. In line with this premise, generation of recombinant anti-venom antibodies can be considered as an alternative strategy. Single-chain fragment variable (scFv) antibodies offer several advantages compared to the whole antibodies, including ease of production, high affinity and specificity. In the present study, scFv antibodies were selected against the venom of the most poisonous snakes in Iran using phage display technology. Phage particles harboring anti-venom specific scFv were separated and scFv antibodies were produced in bacteria. In-vitro assay showed that polyclonal scFvs specifically bind to the venom. Furthermore, in-vivo experiment in mice BALB/c indicated effective toxin neutralization using 20 µg of polyclonal scFv. Our study indicates the neutralizing capacity of anti-venom polyclonal scFvs, although further neutralization assays are needed to confirm their effectiveness.

Cross-reactivity and cross-immunomodulation between venoms of the snakes Bothrops asper, Crotalus simus and Lachesis stenophrys, and its effect in the production of polyspecific antivenom for Central America

A mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys is used as immunogen to produce the polyspecific Central American antivenom (PoliVal-ICP). In this work, we studied the ability of each of these venoms to modulate the antibody response induced by the other two venoms included in the immunization mixture. For that, equine monospecific, bispecific and polyspecific antivenoms were prepared and compared regarding their ability to neutralize the phospholipase A2, coagulant and lethal activities of each venom, and their anti-venom antibodies concentration. Results indicate that there is low cross-reactivity and cross-neutralization between venoms of B. asper, C. simus and L. stenophrys, hence justifying the use of all of them as immunogens for the production of the Central American antivenom. It was also found that the venom of B. asper reduces the anti-crotalic response while the venom of C. simus does not affect the anti-bothropic response. On the other hand, the venoms of B. asper and C. simus increase the anti-lachesic response, and L. stenoprhys venom reduced both the anti-bothropic and anti-crotalic responses. On the basis of these results, the immunization strategy can be adjusted by preventing or taking advantage of cross-immunomodulation between venoms, in order to maximize the antibody response towards all venoms. Immune responses can be improved by injecting horses with several immunogen mixtures, composed by one or two of the three venoms, and administering them at different times during the immunization, eventually generating a high titer against the three venoms. Our results suggest that addressing the issue of immunomodulation by venoms might improve antivenom manufacture worldwide.

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity

Snakebite envenoming is a major neglected disease related to poverty in developing countries. Treatment involves the administration of a specific antivenom serum and auxiliary therapies, if necessary. The improvement of antibodies is of great importance for the technological advancement of antivenom therapy and to reduce the morbidity and mortality associated with this medical burden. In the present study, adult hens were immunized nine times with 20μg of B. arietans or C. d. terrificus venoms at three-week intervals between immunizations. Developing antibodies presented increasing avidity and affinity to antigenic toxin epitopes along immunization, attaining a plateau after the seventh immunization. Pooled egg yolk-purified IgY antivenom antibodies, subjected to in vitro–in vivo lethality assay using Swiss adult mice, exhibited potent venom lethal neutralizing activity. Taken together, chickens under the described immunization schedule were considered alternative candidates for antivenom production. Lower maintenance costs, a simple antibody manufacturing process and immunization suffering restrictions are additional advantages.

Comparison of the adjuvant activity of emulsions with different physicochemical properties on the antibody response towards the venom of West African carpet viper (Echis ocellatus)

Adjuvant emulsions are widely used to enhance the antibody response of the animals used as immunoglobulin source for producing antivenoms. Usually, the adjuvant activity of emulsions is attributed both to their ability to trigger “danger” signals from cells in which they induce death, and to form depots from which immunogens are slowly released. However, there is contradictory evidence suggesting that adjuvant activity of emulsions is independent of the dispersion type and the rate of immunogen release. In order to test how physical properties of emulsions, composed of mineral oil and water, affect their ability to enhance the antibody response towards snake venoms, we compared water-in-oil (W/O) emulsions prepared at volume ratios of 70/30, 50/50 or 30/70, a 50/50 oil-in-water (O/W) emulsion, and a water-in-oil-in-water (W/O/W) multiple emulsion. Comparison included their droplet-size, viscosity, rate of immunogen release and ability to enhance the antibody response of mice immunized with the venom of the African viperid snake Echis ocellatus. It was found that all emulsions released a low amount of venom, and that the 50/50 (W/O) and the multiple emulsion (W/O/W) were those that induced the higher anti-venom antibody response. Our results suggest that the ability of emulsions to enhance the anti-venom response is not associated to their ability to form depots from which the venom is slowly released.

High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays.

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.

Soluble P-selectin rescues viper venom-induced mortality through anti-inflammatory properties and PSGL-1 pathway-mediated correction of hemostasis.

Venomous snakebites are lethal and occur frequently worldwide each year, and receiving the antivenom antibody is currently the most effective treatment. However, the specific antivenom might be unavailable in remote areas. Snakebites by Viperidae usually lead to hemorrhage and mortality if untreated. In the present study, challenges of rattlesnake (Crotalus atrox) venom markedly increased the circulating soluble P-selectin (sP-sel) level, but not P-selectin (P-sel, Selp−/−) mutants, in wild-type mice. Because sP-sel enhances coagulation through the P-selectin ligand 1 (PSGL-1, Selplg) pathway to produce tissue factor–positive microparticles, we hypothesized that increasing the plasma sP-sel level can be a self-rescue response in hosts against snake venom–mediated suppression of the coagulation system. Confirming our hypothesis, our results indicated that compared with wild-type mice, Selp−/− and Selplg−/− mice were more sensitive to rattlesnake venom. Additionally, administration of recombinant sP-sel could effectively reduce the mortality rate of mice challenged with venoms from three other Viperidae snakes. The antivenom property of sP-sel is associated with improved coagulation activity in vivo. Our data suggest that the elevation of endogenous sP-sel level is a self-protective response against venom-suppressed coagulation. The administration of recombinant sP-sel may be developed as a new strategy to treat Viperidae snakebites.

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy.

BackgroundSnakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens.MethodsIgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice.ResultsChickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg).ConclusionsIgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabris venom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Antigenic cross-reactivity and species-specific identification of Pseudocerastes persicus fieldi snake venom

In the present study, we recognized progressively high immunological cross-reactivity between Pseudocerastes persicus fieldi (Pf) venom and six other medically important Egyptian snake venoms belonging to families Viperidae and Elapidae. Antibodies with a range of bonding strengths were shown to be involved in such cross-reactivity. Two strategies have been tried to access specificity; (i) using affinity purified species-specific anti-Pf antivenom antibodies, (ii) conducting the assay in the presence of ammonium thiocyanate (NH4SCN). The discrimination power of the prepared species-specific antivenom was demonstrated by its ability to detect Pf venom over a range of Pf concentrations (2.5 ng–2.5 μg) in a variety of body fluids. The assay could distinguish circulating Pf antigens from other viper antigens in the whole blood of experimentally envenomed mice. What seems promising in our work is the use of the chaotrope, NH4SCN, which renders the reaction medium more favorable for the specific homologous antigen-antibody interactions, primarily via preventing lower avid antibodies to share and, to a bit lesser extent, by decreasing non-specific absorbance signals frequently encountered with ELISA assays. The ELISA described herein may be useful for clinicians for identification of snake bites inflicted by Pf snake species. Balancing between specificity and sensitivity has to be considered for best results.