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. 2024 Jun 28;23:100201. doi: 10.1016/j.toxcx.2024.100201

Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms

Stephen Wilson Kpordze a,f,⁎, Victor Atunga Mobegi b, Gideon Mutie Kikuvi c, Joseph Kangangi Gikunju d, Courage Kosi Setsoafia Saba e,f, Jackan Moshe d, James Hungo Kimotho g
PMCID: PMC11267070  PMID: 39050508

Abstract

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 μg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Keywords: Antivenom, Anti-D. polylepis IgY antibodies, Snake venom, Neutralizing efficacy

Highlights

  • •

    First time generation of purified Chicken-based IgY polyclonal antibodies against D. polylepis venom.

  • •

    Initial report on edema-forming activity from D. polylepis venom in mice.

  • •

    IgY antibodies, PANAF-Premium™ and VINS™ antivenoms abrogate toxic effects induced by D. polylepis venom.

  • •

    IgY antibodies as potential alternative for antivenom development.

1. Introduction

On a global basis, snakebite envenoming has a significant negative impact on mortality and disability rates (Gutiérrez et al., 2017). Snakebites were listed by the World Health Organization (WHO) as a category A disease in their list of neglected tropical diseases in 2017 due to their importance to public health (Chippaux, 2017), and the World Health Assembly in 2018 adopted a resolution on the matter (Gutiérrez and Mackessy, 2021). A global strategy to prevent and control envenomings was recently introduced by the WHO, with the goal of halving (reducing by 50%) the incidence of envenoming-related amputations and deaths by the year 2030 (WHO, 2019). One of the four pillars of this strategy is "ensuring safe, effective treatment."

In Kenya, snakebites constitute a neglected emergency, with 15,000 snakebites occurring yearly (MoH-Kenya, 2019), and an estimated 400,000–600,000 in Africa suffer snakebite envenoming annually (WHO, 2017). Thus, 1 in 15 people gets bitten by snake, and 1 in 147 people die of snakebite each year in Kenya (Otieno and Gathema, 2021). Among the most common snakes behind this incidence is the Dendroaspis polylepis (Black mamba) which is classified as Category 1 snake species (i.e. it is of medical importance and constitute the greatest threat to public health) (WHO, 2017). D. polylepis venom primarily causes systemic neurotoxicity, and its neurotoxic venom being extremely potent, making it one of the most feared snakes in the world (Nguyen et al., 2022), (Ochola et al., 2019). It has been indicated as a medically important snake in Kenya, since it causes snakebites in many areas of the region (Ochola et al., 2019), (Ochola et al., 2018). The potential of black mamba venom in the production of antivenom is one of the extensively studied area, but still attracting ongoing investigations for more future prospects.

Antivenoms, which are IgG preparations or IgG fragments made from the plasma of horses or other animals immunized with venoms of one or more snake species (i.e. monospecific or polyspecific antivenoms), are the cornerstone of treatment for snakebite envenomations (WHO, 2017). However, lack of antivenoms access is a significant public health issue in Africa, and when available, it is expensive (Potet et al., 2019), (Larson et al., 2022). Sub-Saharan Africa has only one antivenom producer, situated in South Africa (Gutiérrez et al., 2017). A large portion of the region's antivenom requirements are met by a number of producers in Europe, Asia, and Latin America. Some antivenoms according to earlier report are made from species that are not native to East Africa or Sub-Saharan Africa, and hence their effectiveness upon usage in Africa for treatment turns to be compromised (Harrison et al., 2017).

It has been established that chicken eggs that have been immunized with appropriate antigens are a reliable source of IgY antibodies. The polyclonal antibody Immunoglobulin (Ig-Y) has been taunted as a potential antivenom production system, and has comparable functions as mammalian IgGs (Lee et al., 2021). Egg IgY antibodies have been used in the past to treat bacterial, viral, and other diseases (Abbas et al., 2019), with use in humans through an oral route towards Pseudomonas aeruginosa (Nilsson et al., 2007). Numerous studies on IgY based antivenoms have demonstrated its potential as a possible alternative to traditional IgG based antivenoms (Abbas et al., 2019), (Liu et al., 2017).

Whilst the mentioned previous studies have demonstrated IgY antivenoms can be developed with efficacy against a range of species, none have thus far been attempted for D. polylepis venom. In this study, we generated chicken-based IgY polyclonal antibodies against D. polylepis, and evaluated the comparable efficacy with two commercial antivenoms (Indian-made PANAF-Premium™ and VINS™ polyvalent antivenoms) to neutralize the lethal and toxic effects induced by Black mamba venom.

2. Materials and methods

2.1. Ethical approvals

The study was approved by the Jomo Kenyatta University of Agriculture and Technology (JKUAT) Institutional Scientific and Ethics Review Committee (ISERC) (approval number JKU/ISERC/02316/0976) and the Kenya Medical Research Institute (KEMRI) Animal Care and Use Committee (ACUC) (reference number KEMRI ACUC/01.09.2023).

2.2. Reagents

Tween-20, bovine serum albumin (BSA), Freund's incomplete adjuvant (FIA) and Freund's complete adjuvant (FCA) (Sigma, St Louis, Missouri, USA), Tween-20, NHS activated sepharose 4FF, rabbit anti-chicken IgG/HRP conjugated antibody, Amicon ultra-15 centrifugal filter devices and protein marker (Invitrogen, USA), 3,3',5,5'-tetramethylbenzidine (TMB), glycine, non-fat dry milk and precast gel (12% Sodium dodecyl-sulfate polyacrylamide gel electrophoresis, SDS-PAGE) (Solarbio, China), were obtained from the manufacturers (i.e. Invitrogen, sigma and solarbio). Other reagents or chemicals used were purchased from local companies and of analytical grade.

2.3. Animals

One-month-old Isa-Brown chicks (500–700 g each) and seventeen-week-old Isa-Brown hens (weighing 1.7–1.8 kg each), in good health and laying conditions (laying 5–7 eggs per week) were purchased from a local poultry farm, and used for LD50 determination and production of IgY against D. ploylepis venom, respectively. Chicks (n = 30) were grouped into 5 per cage, whereas, hens (4) were kept in individual cages with standard water and food at the Small Animal Facility for Research and Innovation (SAFARI) on JKUAT main campus. Eight-week-old male and female Balb/c mice (28–30 g each), obtained from Kenya Institute of Primate Research (IPR), were used for venom lethality and its neutralizing determination. Mice were maintained at the Kenya Medical Research Institute with food and water provided ad libitum provided.

2.4. Venom and antivenoms

Venom from D. polylepis (Black mamba) was obtained through donation from the Kenya Medical Research Institute (KEMRI), lyophilized and stored at −20 °C until use. PANAF-Premium™ (Pan Africa, B. NO: PANAF-027; Expiry date: 08/2027) and VINS™ (African IHS, B. NO: 07AS21006; Expiry date: 07/2025) polyvalent antivenoms were procured commercially and as per the manufacturers’ instructions, reconstitution was done, respectively.

2.5. LD50 determination in chicks

The median lethal dose and LD50 dose range-finding test were conducted in accordance with WHO guidelines (WHO, 2017). Briefly, five venom doses were established (0.25, 0.30 0.40, 0.50 and 0.75 mg/kg), and one chick per group was used for the doses. Each chick's venom dose was calculated using the method outlined by Ochola and associates (Ochola et al., 2019). Thus, the predetermined dosages were prepared in PBS and 0.2 mL aliquots were administered intramuscularly at multiple sites of the breast region of each chick, and within 24-h period, survivals and deaths were recorded. For the full-scale LD50 assay, at the established doses, five chicks per group were employed. Control group received only PBS. A 24-h period was used to record the deaths and survivals, and the LD50 calculated by probit analysis (WHO, 1981) using IBM SPSS (statistics version 20.). The LD50 of D. polylepis venom in chicks was estimated by using the Miller method to generate the LogDose and probit values for the standard curve. Thus, the estimated LD50 of Black mamba venom in chicks was 0.54 (0.49–0.58) mg/kg.

2.6. Chicken immunization schedule

In the first immunization, 0.5 mL of saline (containing sub-lethal dose of 0.341 mg of snake venom, based on the LD50 of D. polylepis venom in chicks = 0.54 mg/kg) emulsified with an equal volume of Freund's complete adjuvant (FCA) was administered intramuscularly to each of the four hens at various sites in their breast region. Following the initial immunization, the booster doses were administered on the 14th, 35th, 56th, and 77th day using 0.5 mL saline (containing 0.54, 1.08, 2.16, and 2.16 mg of snake venom, respectively) emulsified with an equal volume of Freund's incomplete adjuvant (FIA). Hens in the control group received an intramuscular immunization with 0.5 mL saline only. Daily egg collection was done a week before and sustained after the initial immunization for 22 weeks, labeled individually, and stored at 4 °C until use. Weekly serum samples were collected after first immunization, and stored at −20 °C (Liu et al., 2017).

2.7. IgY isolation and purification

A modified approach by Liu et al. (2017) was used to extract IgY from preimmunized and hyperimmunized egg yolks. Briefly, egg shells were cleaned with 70% ethanol, cracked, and the yolk separated from egg white. After which, the yolk was 10-fold diluted with deionized water, and a magnetic stirrer was used to vigorously swirl the mixture for 30 min. Following a 2-fold dilution with 0.04 M acetate buffer (containing 0.06 M NaCl, pH 5.0), the resultant homogenate was once again homogenized for 30 min during which caprylic acid was added to attain 1% final concentration. The yolk suspension was stored overnight at room temperature. Water-soluble fraction (WSF), the clear supernatant containing the IgY, was collected and centrifuged at 10,000 rpm for 15 min at 4 °C, and then precipitated using a 45% saturated ammonium sulfate. Centrifugation at 10,000 rpm for 20 min at 4 °C was used to recover the salt pellet. The precipitated proteins were then dissolved in phosphate buffered saline (PBS, pH 7.4) and dialyzed against the same solution. An affinity chromatography was then applied to the partially purified antibody (crude extract).

Affinity purification followed the procedure of Liu et al. (2017) with minor modifications. Briefly, venom affinity column was prepared using activated sepharose 4FF coupled with whole D. polylepis venom (20 mg/mL) dissolved in coupling buffer, blocked unreacted groups with 0.5 M ethanolamine buffer, sepharose was washed with Tris–HCl buffer and then acetate buffer, alternating for 3-times. After loading the crude IgY extract into the affinity column, the unabsorbed proteins were removed by washing the column with PBS buffer, while bound antibodies were eluted using glycine-HCl buffer. Fractions were pooled and dialysed against PBS, and further concentrated and desalted through ultra-centrifugal filter devices, and finally stored at −20 °C. SDS-PAGE and ELISA were used to assess the preparations' purity and titer, respectively.

2.7.1. Determination of IgY protein concentration, and characterization by SDS-PAGE

The Bradford protein assay kit was used to measure the protein concentration, while following the manufacturer's instructions (ThermoFisherScientific, 2023). Analysis for the purity and molecular weight of the IgY was done using precast gels (12% SDS-PAGE) in line with the manufacturer's recommendations and previously described method (Liu et al., 2017).

2.7.2. Verification of IgY and specificity by ELISA

The activities and specificity of anti-D. polylepis IgY antibodies in the serum and yolk was assessed using indirect ELISA approach (Liu et al., 2017), (Duan et al., 2016), with some minor modifications. Briefly, a 5 μg of native D. polylepis venom in 100 μL of coating buffer (pH 9.6, 0.05 M carbonate-bicarbonate) was used to coat the microplates and left 4 °C for 16 h. Wells of plate were washed 3-times with rinse buffer (pH 7.4, PBS-0.05% Tween 20). A 200 μL of blocking buffer (rinse buffer plus 5% nonfat dry milk) was used to block the unbound sites, incubated for 1.5 h at 37 °C, and wells washed 3-times. A 200 μL of diluted serum or yolk (1:1000) in dilution buffer (PBS plus 1% nonfat dry milk) was added to the well and incubated at 37 °C for 1 h before 3-times washing. A dilution of 1:5000 for the rabbit anti-IgY peroxidase in blocking buffer was done, and 100 μL added to well, and incubated at 37 °C for 1 h. Wells were washed again, 100 μL of TMB solution added, incubated for 20 min at room temperature, and reaction terminated using 50 μL of 2 M sulphuric acid. Using ELISA plate reader, absorbance was recorded at 450 nm, and results determined by a ratio of positive sample and negative sample giving at least 2.1 (i.e. P/N > 2.1; OD of the positive sample divided by OD of the negative sample), as well as the lowest dilution at which the OD value is significantly higher than the negative control (Mean OD of naïve IgY + 2 times standard deviation) (Senji Laxme et al., 2019). Wells free of venom were used as blanks, and yolk samples from collected eggs or serum before immunization were used as negative control.

2.8. Toxic activities of D. polylepis venom in mice

2.8.1. Lethality (LD50)

This assay followed the procedure by Ochola and associates (Ochola et al., 2019), and the WHO guidelines (WHO, 2017) for LD50 determination in mice. The doses for range finding test were established at 0.2, 0.3, 0.4, 0.5, and 1.0 mg/kg with one mouse per dose. 200 μL aliquots of established doses was injected intraperitoneally (i.p) into each mouse, and both deaths and survivors recorded in 24 h. For the full scale median lethal dose (LD50), 0.20, 0.3, 0.35, 0.40, and 0.45 mg/kg were established, 5 groups of mice per dose employed, 200 μL of aliquot injected via i. p route, and observed for deaths and survivors in within 24 h. Control groups only received PBS. Observational feature for neurotoxic activity of D. polylepis venom such as neuromuscular paralysis leading to respiratory arrest, results from actions of neurotoxins at the neuromuscular junctions, and falling eye lids were observed (Gutiérrez et al., 2017), (Warrell, 2010).

2.8.2. Edematogenic assay

The Resiere et al. (2018) approach was employed to assess the edematogenic activity of D. polylepis venom. Five mice per group were used, and each group received subcutaneous injections of different doses of venom diluted in 50 μL of PBS in the left footpad. The right footpads were injected with an equivalent volume of PBS. Control mice received an equal volume of PBS in their left footpad. The mice were euthanized an hour after the challenge, and a low-pressure spring caliper was used to measure the thickness or rise in footpad volume. Measurements were also taken of the control mice's left footpads. The least amount of venom that caused a 30% increase in footpad thickness or volume after 1 h of venom inoculation in comparison to the control mice's footpad that was only given PBS was determined as the minimum edema-forming dose (MED).

2.9. Neutralization studies for the lethal and toxic effects induced by D. polylepis venom in mice

The neutralizing assay for the D. polylepis venom's lethal and induced toxic effects to determine the efficacy of IgY antibodies and two commercial antivenoms (VINS™ and PANAF-Premium™) was carried out using the WHO guidelines along with other literatures (WHO, 2017), (Resiere et al., 2018), (Segura et al., 2010). In these tests, a set dose of venom challenge was incubated for 30 min at 37 °C with various antivenom dilutions. The ensuing venom-antivenom combinations were then administered in aliquots, and the corresponding effects were assessed as previously mentioned. A 3 LD50 and 6 MED challenge dosages were employed for lethality and edematogenic assays, respectively. For positive and negative control mice groups, equivalent volumes of venom and PBS were injected, respectively. The median effective dose (ED50) range-finding tests of the IgY antibodies and the two antivenoms were used to determine the various doses for each effect. Doses were 15, 30, 45, 60 and 75 μL for the neutralization of lethality, and 20, 40, 60, 80 and 100 μL for edema neutralization, respectively. Consequently, the neutralizing effectiveness (ED50) of the IgY antibodies and two antivenoms was calculated following probit analysis using StatsDirect 3 software.

2.10. Data analysis

Data analysis was done using Microsoft Excel 2019, OriginPro 2023b version and StatsDirect 3 software. ANOVA was used to find differences in group means, followed by either an unpaired t-test or a Tukey test. The cut-off for statistical significance was set at p-value <0.05.

3. Results

3.1. IgY extraction, purification, specific activity and biochemical identification

The water-soluble extraction method was used to obtain partially purified crude extract of antibody preparation from egg yolks, and then fractionated by thiophilic affinity chromatography. Through the three steps fractions (water dilution-WSF, ammonium sulfate precipitation or salting-out-SOF, and thiophilic affinity chromatography-TACF). In the different fractions (WSF, SOF and TACF), the venom-specific activities of the IgY from 328 eggs was assayed on an ELISA for comparison. The average recovery was 19.1% for venom-specific IgY. Titer of TACF was a two-fold higher than that of SOF, and approximately 16 times (15.93) higher in comparison to WSF. The results indicated a clear enrichment of venom-specific IgY through the three purification steps (Table 1).

Table 1.

IgY fractions titer and the recovery ratio of proteins.

Fractions Titer of IgY by ELISA (x104) Total proteins in yolk-328 eggs (g) Recovery ratio of proteins (%)
WSF 8.3 79.10 100
SOF 66.1 32.38 40.94
TACF 132.2 15.09 19.10
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Key; WSF: water-soluble fraction, SOF: Salting-out fraction, TACF: Thiophilic affinity chromatography.

Also, the IgY obtained from the egg yolk under reduced conditions presented two bands approximately 67 kDa and 25 kDa, representing the heavy and light chain, respectively. However, under non-reducing conditions, they exhibited one band of about 183 kDa (Fig. 1).

Fig. 1.

Fig. 1

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Lane 1: IgY under reducing conditions (1A) and lane a, IgY under non-reducing conditions (1B) Lane M: molecular weight marker.

3.2. Monitoring the concentrations of venom-specific chicken-based IgY

The concentrations were determined using the Bradford protein assay kit (Thermo Fisher Scientific InC., USA). Least yield of extraction was found for week 1 and the highest for week 13 with 10.03 and 3,164.926 μg/mL, respectively (Fig. 2).

Fig. 2.

Fig. 2

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Concentrations of the affinity purified antibodies yield per week.

3.3. Antibody response: immune response against D. polylepis venom in Layer chicken and antibody titers determination

To evaluate the immune response for the immunization process, an ELISA assay was carried out to determine the IgY-antibody titers. Assayed samples included both pre-immune and booster immunization serum samples alongside PBS (saline)-only immunized samples. Following the primary immunization, varied immune (antibody) responses were elicited against D. polylepis venom toxins in chicken. Antibody response was detected in serum of chicken by day 7. Despite the low pre-booster response in serum after the initial booster, a sharp increase in both serum and egg yolk for antibody titer was observed. At week 3, antibodies transference which is specific to D. polylepis venom was observed from serum to egg yolk after the primary immunization. The highest level of antibody response was reached at week 4 in both serum and egg yolk (titers of OD450 = 3.381 and 3.356), and the observed secondary response was maintained afterwards for the remaining weeks by the second booster injection. At week 6, there was a sharp drop in antibody production peaks (titers of OD450 = 2.659 and 2.622) (Fig. 3). The PBS-only immunized chicken however showed no immune response.

Fig. 3.

Fig. 3

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Immune response for primary and secondary antibody in serum and egg yolk of immunized Isa-Brown chicken with D. polylepis venom. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.4. Lethal and toxic activities of D. polylepis venom

Lethal activities of D. polylepis was observed in the venom-challenged mice. The LD50 for D. polylepis venom in mice was found to be 0.34 (0.29–0.38) mg/kg. Following 1-h injection with D. polylepis venom, all the mice under investigation showed signs of edema (increase footpad thickness or volume). The induced edema formation from the venom was at a dose of 0.052 μg which is thus the minimum edema-forming dose, and considered 100% activity (Table 2). The venom showed no necrotic and hemorrhagic activities in mice. However, paralysis and ptosis (falling-eye lids) were observed.

Table 2.

D. ploylepis venom's lethal and edematogenic activities.

Venom Lethality (LD50; mg/kg)a Edema-Forming Activity (MED; μg)b
D. polylepis 0.34 (0.29–0.38) 0.052 ± 0.11
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a

Using i. p route, LD50 (median lethal dose) was determined in Balb/c mice (28–30 g); 95% CI in parenthesis.

b

The term "minimum edematogenic dose” (MED) describes the venom dosage that, after 1 h of injection, caused a 30% increase in footpad volume. Results are presented as mean ± SD (n = 5) for edema-forming activities.

3.5. Lethal and toxic activities neutralization assays

As shown in Table 3 below, the neutralizing ability in inducing D. polylepis lethality was demonstrated by both the extracted chicken-based IgY antibodies and antivenoms. VINS™ antivenom was able to neutralize the lethality at a median dose of 35.41 μL as most effective, followed by 41.36 μL for Chicken-Based IgY antobodies, and 46.6 μL for the PANAF-Premium™ (Table 3). Relatively, the neutralizing capacities observed had a means close-range effect across, and similar observations made for the edema-forming neutralization assays. There were no statistical differences among their neutralizing efficacies (P = 0.320-0.859).

Table 3.

Lethal and edematous effects of D. polylepis neutralization by IgY antibodies, PANAF-Premium™ and VINS™ polyvalent antivenomsa.

Antivenoms & Antibody ED50 of lethal activity
3 LD50/antivenom (μL)		 ED50 of lethal activity venom (mg)/antivenom (μL) Edema-forming activity antivenom (μL)/6 MED
Chicken-Based IgY 41.36 (28.57–52.01) 151.85 (119.89–185.90) 80 ± 11.55
VINS™ 35.41 (21.14–45.79) 133.48 (83.56–167.40) 60 ± 18.26
PANAF-PremiumTM 46.60 (30.27–64.03) 175.65 (127.03–234.06) 90 ± 8.16
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a

The antivenom-venom ratio (μL antivenom/challenge dosage of venom) at which a certain venom-induced activity is reduced by 50% is known as neutralization, and it is expressed as median effective dose (ED50). Results are shown as mean ± SD (n = 5) for edema-forming activities and 95% CI (in parenthesis) for lethality.

4. Discussion

The Black mamba (D. polylepis) is endemic to Sub-Saharan Africa, and it is Africa's deadliest snake, with untreated bite fatality rate of 100 percent (Quarch et al., 2017). The venom is extremely toxic, causing postsynaptic blockade of neuromuscular junctions. Despite having low concentrations of necrotic-causing proteins (Laustsen et al., 2015), the venom typically does not result in necrosis and tissue destruction (Strover, 1967), (Hilligan, 1987). It is possible for neurotoxic symptoms to appear as soon as 10 min after a Black mamba bite (Tan et al., 1991), and treatment choice is usually through the use of antivenoms. However, the production of conventional antivenom derived from the blood of immunized large animals like sheep or horses can slow down venom's toxicity and pathophysiologic effects as demonstrated in some neutralization studies (Amro et al., 2018), (Rojas et al., 2005), as well as speeding up recovery. This current research to the best of our knowledge, is the first to describe the generation of anti-D. polylepis chicken-based IgY polyclonal antibodies and assess the neutralizing efficacy vis-à-vis commercial antivenoms for the lethal and toxic effects induced by D. polylepis venom.

In accordance with other reports, the study followed the three principal steps to obtain highly purified IgY (Liu et al., 2017), (Duan et al., 2016), (Araújo et al., 2010). The total yield of 15.09 g of pure IgY, with an average recovery of 19.10% for venom-specific IgY was notably higher in our study compared to other reports; and may be due to differences in venom potency, as well as number of eggs used (Liu et al., 2017), (Duan et al., 2016). In addition, it was discovered that IgY activity in TCF was 2 and 16-times higher than that of SOF and WSF, respectively (Table 1). The antibody production response induced by the black mamba venom is similar to reports from other snake venoms (Liu et al., 2017), (Duan et al., 2016), (Araújo et al., 2010), (Amro et al., 2018), (Rojas et al., 2005), (Paul et al., 2007).

Venom from mamba species are generally potent, and neurological effects of the black mamba venom appear within 2 h of the onset of bite or disease (Aalten et al., 2021). These neurotoxic effects have been observed in several researches on mice (Ochola et al., 2019), (Ainsworth et al., 2018), (Ratanabanangkoon et al., 2020). Our revealed LD50 of 0.54 mg/kg in chicks which was a first-time report, and 0.34 mg/kg in mice (Table 2). The LD50 in mice was not consistent with some previous reports which had 0.28 (0.16–0.51) μg/g (Ratanabanangkoon et al., 2020), 6.2 μg/mouse; 0.33 μg/g (Ainsworth et al., 2018) and 0.32 (0.16–0.54) μg/g (Laustsen et al., 2015) for the same D. polylepis venom. On the other hand, our result was comparable to Ochola et al. (2019) who reported 0.341 mg/kg in mice and also fell within the range of 0.055–0.940 mg/kg that other studies have noted (Harrison et al., 2017), (Laustsen et al., 2015). It is not unusual for venoms from the same species to have different toxicity actions, and a previous research by Calvete noted that venom variability is a common phenomenon both between and within species at all taxonomic levels (Calvete, 2019). The variations in the mouse breed as well mice slightly bigger/heavier than the standard 18–20 g recommended by the WHO used in this work could be the cause of the disparities in LD50 result and those published elsewhere. Also, the observed discrepancies could be attributed to venom-composition variations, sex-specific proteome differences, collection and pooling variations of black mamba venom (Menezes et al., 2006), (Mackessy et al., 2003), (Chippaux et al., 1991), (Daltry et al., 1996).

The clinical signs and symptoms that we observed in both chicks and the mice were consistent with neurotoxic venom. This included paralysis, ptosis (drooping or falling eyelids), and respiration patterns which varied from shallow to rapid (more pronounced in the chicks with accompanying unusual sound). The observation was in agreement with a report by Ochola et al. (2019). From reports, venom of black mamba has α-neurotoxins that can attach to nicotinic receptors at the motor end plate, and cause failure in respiration (Ratanabanangkoon et al., 2020), (Blanchet et al., 2014).

Despite the trace amount of metalloproteinase found by Laustsen and colleagues (Laustsen et al., 2015) in black mamba venom, it was not sufficient in our venom samples to elicit the required effect. However, we recorded edematogenic activity (minimum edematogenic dose = 0.05 ± 0.11 μg) in mice for the black mamba venom (Table 2). This observation is the first report with edema activities found in association to black mamba venom and may be arguable just as the metalloproteinase traces report by Laustsen and colleagues in the venom. Additionally, literatures have it that among the major toxins of the elapid venoms also lies others like metalloproteinases (SVMPs), snake venom serine proteases (SVPs) and L-amino acid oxidases (LAAOs), all representing an average of 6% of elapid venom (Ullah et al., 2018), (Hiu and Yap, 2020), (Kang et al., 2011), (Ferraz et al., 2019), (Ullah, 2020), (Olaoba et al., 2020), (Oliveira et al., 2022). Besides serine protease and LAAOs not present in large quantity, it is prevalent in many snake venoms, and in myocytes, can cause edema development, hemolysis, toxicity, and platelet aggregation (Gutiérrez et al., 2021), (Hiu and Yap, 2020), (Kaur et al., 2012). Observation of edema forming activity in this study could be that our venom had significant amount of LAAO or serine protease which can elicit the edema effect (Resiere et al., 2022).

The WHO has established that neutralization of lethality at the preclinical stage is the gold standard for assessing antivenom efficacy. Further testing can be necessary depending on the venom, whether existing antivenoms are being supplied to new countries or new ones are being produced (WHO, 2017), (Durán et al., 2021). Examining the possible effects of the heterogeneity in intrageneric venom patterns among Dendroaspis on the use of antivenoms for treating snakebite victims in sub-Saharan Africa was made possible by this valuable opportunity. In order to achieve this, the study evaluated the neutralizing-efficacy of generated IgY antibodies in comparison to two commercial Dendroaspis antivenoms that are currently used in sub-Saharan Africa against the effect induced by D. polylepis venom in mice. Both antivenoms and the chicken-based IgY polyclonal antibodies showed the ability to neutralize the lethal and edematogenic effects of D. polylepis venom but with varying efficacy levels. The effectiveness of VINS™ antivenom in neutralizing the lethality of black mamba venom in mice has been documented by other researchers conducting comparable investigations (Ochola et al., 2019), (Laustsen et al., 2015). Antivenoms have been reported to be effective when they can immunocapture 20–25% of the components of venom (Calvete et al., 2014), and having an immunocapture capability of at least ≥25% of venom proteins is often correlated with a positive result in in-vivo neutralization tests, according to the WHO guidelines for the manufacture, control, and regulation of antivenoms (WHO, 2017). This result confirms the manufacturers’ inscription on antivenom containers that each mL neutralizes at a minimum of ≥25LD50 for D. polylepis venom.

Regarding the determined neutralization of venom-induced lethality, the effective doses of PANAF-Premium™, IgY antibodies, and VINS™ antivenoms; the observations of mean differences in the ED50 of the antivenoms are consistent with those from other studies on D. polylepis venom neutralization (Ochola et al., 2019), (Laustsen et al., 2015), (Ainsworth et al., 2018), (Engmark et al., 2016), (Menzies et al., 2022). It further shares in the light of research by Resieri and associates when they indicated that a new batch of an antivenom had higher efficacy compared to an earlier batch as seen by differences in the ED50 values. They speculated that the variances might be caused by differences in the protein concentrations of the batches of antivenom, among other factors (Resiere et al., 2018). In addition, antibody specificities in the venom-immunized animal may result from variations in the venoms of snakes used for the immunization, and Gutierrez et al. indicated that this could potentially provide credence to the discrepancies in efficacy between antivenoms (Gutiérrez et al., 2017). The results from our study, however, are limited to these particular batches, thus caution should be exercised when interpreting them.

According to the WHO, snakebite envenomation is under category A Neglected Tropical Diseases, meaning it is an extremely serious disease. As a result, the World Health Organization (WHO) has initiated a global campaign to cut the illness burden of snakebite envenomation in half (50%) by 2030 (Williams et al., 2019). Therefore, we anticipate a significant expansion in the advantages of IgY technology and its widespread application in medical and scientific fields. Upcoming diagnostics, immunotherapy and research are anticipated to place a greater emphasis on IgY as demonstrated by Kpordze et al. (2024).

5. Conclusions

In conclusion, immunizing chickens with D. polylepis snake venom resulted in the successful raising of IgY antibodies against D. polylepis venom toxins for the first time in egg yolk with high purity and titer. The lethal effects and edematogenic activities of D. polylepis venom toxins were effectively neutralized by the generated IgY antibodies in relative comparison as the commercial antivenoms (even a very lower concentration than the commercial antivenoms preparations recommended by the manufacturers which had higher concentrations). The IgY has proven effective and may be a useful tool for creating human or animal-specific alternative treatments for snakebite sufferers. Nonetheless, more research to acquire data on safety and efficacy in this regard is necessary.

In addition to supporting previous reports on the neutralizing effectiveness of IgY antibodies, PANAF-Premium™ and VINS™ antivenoms, our results emphasize the lethal and toxic activities of D. ploylepis venom. The study reports for the first time of raising chicken-based IgY antibodies and its efficacy against D. polylepis.

Declarations

The authors declare that none of the work reported in this study could have been influenced by any known competing financial interests or personal relationships.

Ethical statement

The authors wish to state that the study was approved by the Jomo Kenyatta University of Agriculture and Technology (JKUAT) Institutional Scientific and Ethics Review Committee (ISERC) (approval number JKU/ISERC/02316/0976) and the Kenya Medical Research Institute (KEMRI) Animal Care and Use Committee (ACUC) (reference number KEMRI ACUC/01.09.2023).

A scanned copy of these documents can be made available upon request.

Funding

The African Union Commission provided funding for this study through the Pan African University scholarship programme.

CRediT authorship contribution statement

Stephen Wilson Kpordze: Writing – original draft, Validation, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Victor Atunga Mobegi: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. Gideon Mutie Kikuvi: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. Joseph Kangangi Gikunju: Writing – review & editing, Project administration, Conceptualization. Courage Kosi Setsoafia Saba: Writing – review & editing, Project administration, Methodology, Conceptualization. Jackan Moshe: Writing – review & editing, Project administration, Conceptualization. James Hungo Kimotho: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge the immense financial support of the African Union Commission for this research, to SAFARI on the JKUAT main campus and the Kenya Medical Research Institute (KEMRI) for availing their labs/animal facilities used in this study. We also acknowledge the support of the Innovation and Technology Transfer Division (ITTD-KEMRI) for donating the venoms used in the study. A special gratitude to Mr. Omari Samuel and Ms. Faith Ndung'u (ITTD-KEMRI), Mr. Perminus Kimathi Njururi (SAFARI-animal facility), Stephen G. Kaniaru (KEMRI-animal facility), Mr. Jack Mwongera (Shalom poultry farms-Juja), Mr. Patrick Koech (PAUSTI-Molecular Biology and Biotechnology Lab technician) and O'Brian Victor (SAJOREC-Natural Products Lab, JKUAT) for their diverse assistance.

Handling Editor: Dr. Ray Norton

Data availability

All data used are in the manuscript.

References

  1. Aalten M., et al. The clinical course and treatment of black mamba (Dendroaspis polylepis) envenomations: a narrative review. Clin. Toxicol. 2021;59(10):860–868. doi: 10.1080/15563650.2021.1943427. [DOI] [PubMed] [Google Scholar]
  2. Abbas A.T., El-Kafrawy S.A., Sohrab S.S., Azhar E.I.A. IgY antibodies for the immunoprophylaxis and therapy of respiratory infections. Hum. Vaccines Immunother. 2019;15(1):264–275. doi: 10.1080/21645515.2018.1514224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ainsworth S., et al. The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms. J. Proteonomics. 2018;172:173–189. doi: 10.1016/j.jprot.2017.08.016. [DOI] [PubMed] [Google Scholar]
  4. Amro W.A., Al-Qaisi W., Al-Razem F. Production and purification of IgY antibodies from chicken egg yolk. J. Genet. Eng. Biotechnol. 2018;16(1):99–103. doi: 10.1016/j.jgeb.2017.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Araújo A.S., Lobato Z.I.P., Chávez-Olórtegui C., Velarde D.T. Brazilian IgY-Bothrops antivenom: studies on the development of a process in chicken egg yolk. Toxicon. 2010;55(4):739–744. doi: 10.1016/j.toxicon.2009.11.004. [DOI] [PubMed] [Google Scholar]
  6. Blanchet G., et al. Polypharmacology profiles and phylogenetic analysis of three-finger toxins from mamba venom: case of aminergic toxins. Biochimie. 2014;103:109–117. doi: 10.1016/j.biochi.2014.04.009. [DOI] [PubMed] [Google Scholar]
  7. Calvete J.J. Snake venomics at the crossroads between ecological and clinical toxinology. Biochemist. 2019;41(6):28–33. doi: 10.1042/BIO04106028. [DOI] [Google Scholar]
  8. Calvete J., Sanz L., Pla D., Lomonte B., Gutiérrez J. Omics meets biology: application to the design and preclinical assessment of antivenoms. Toxins. 2014;6(12):3388–3405. doi: 10.3390/toxins6123388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chippaux J.-P. Snakebite envenomation turns again into a neglected tropical disease. J. Venom. Anim. Toxins Incl. Trop. Dis. 2017;23(1):38. doi: 10.1186/s40409-017-0127-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chippaux J.-P., Williams V., White J. Snake venom variability: methods of study, results and interpretation. Toxicon. 1991;29(11):1279–1303. doi: 10.1016/0041-0101(91)90116-9. [DOI] [PubMed] [Google Scholar]
  11. Daltry J.C., Wüster W., Thorpe R.S. Diet and snake venom evolution. Nature. 1996;379(6565):537–540. doi: 10.1038/379537a0. [DOI] [PubMed] [Google Scholar]
  12. Duan H., et al. Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy. J. Venom. Anim. Toxins Incl. Trop. Dis. 2016;22(1):23. doi: 10.1186/s40409-016-0078-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Durán G., et al. Assessing a 6-h endpoint observation time in the lethality neutralization assay used to evaluate the preclinical efficacy of snake antivenoms. Toxicon X. 2021;12 doi: 10.1016/j.toxcx.2021.100087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Engmark M., et al. High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays. Sci. Rep. 2016;6(1) doi: 10.1038/srep36629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ferraz C.R., et al. Multifunctional toxins in snake venoms and therapeutic implications: from pain to hemorrhage and necrosis. Front. Ecol. Evol. 2019;7(Jun) doi: 10.3389/fevo.2019.00218. [DOI] [Google Scholar]
  16. Gutiérrez J.M., Calvete J.J., Habib A.G., Harrison R.A., Williams D.J., Warrell D.A. Snakebite envenoming. Nat. Rev. Dis. Prim. 2017;3(1) doi: 10.1038/nrdp.2017.63. [DOI] [PubMed] [Google Scholar]
  17. Gutiérrez J.M. In: Handbook of Venoms and Toxins of Reptiles. second ed. Mackessy S.P., editor. CRC Press; Boca Raton, Florida, USA: 2021. Snakebite envenomation as a neglected tropical disease: new impetus for confronting an old scourge; p. 680. [Online]. Available: [DOI] [Google Scholar]
  18. Gutiérrez J.M., et al. In vitro tests for assessing the neutralizing ability of snake antivenoms: toward the 3Rs principles. Front. Immunol. 2021;11 doi: 10.3389/fimmu.2020.617429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harrison R.A., et al. Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa. PLoS Neglected Trop. Dis. 2017;11(10) doi: 10.1371/journal.pntd.0005969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hilligan R. Black mamba bites. A report of 2 cases. SAMJ (S. Afr. Med. J.) 1987;72(3):220–221. https://journals.co.za/doi/pdf/10.10520/AJA20785135_8222 [Online]. Available: [PubMed] [Google Scholar]
  21. Hiu J.J., Yap M.K.K. Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and <scp>l</scp> -amino acid oxidase. Biochem. Soc. Trans. 2020;48(2):719–731. doi: 10.1042/BST20200110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kang T.S., et al. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J. 2011;278(23):4544–4576. doi: 10.1111/j.1742-4658.2011.08115.x. [DOI] [PubMed] [Google Scholar]
  23. Kaur P., et al. Vol. 57. 2012. (Biochemistry of Envenomation). [DOI] [Google Scholar]
  24. Kpordze S.W., Kikuvi G.M., Kimotho J.H., Mobegi V.A. Development, optimization and evaluation of a sensitive Enzyme-Linked Immunosorbent Assay (ELISA) porototype for detection of chicken-based IgY polyclonal antibodies against toxins of D. polylepis venom. Antibodies. 2024;13(3):50. doi: 10.3390/antib13030050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Larson P.S., et al. Snakebite victim profiles and treatment-seeking behaviors in two regions of Kenya: results from a health demographic surveillance system. Trop. Med. Health. 2022;50(1):31. doi: 10.1186/s41182-022-00421-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laustsen A.H., Lomonte B., Lohse B., Fernández J., Gutiérrez J.M. Unveiling the nature of black mamba (Dendroaspis polylepis) venom through venomics and antivenom immunoprofiling: identification of key toxin targets for antivenom development. J. Proteonomics. 2015;119:126–142. doi: 10.1016/j.jprot.2015.02.002. [DOI] [PubMed] [Google Scholar]
  27. Lee L., Samardzic K., Wallach M., Frumkin L.R., Mochly-Rosen D. Immunoglobulin Y for potential diagnostic and therapeutic applications in infectious diseases. Front. Imunol. 2021;12 doi: 10.3389/fimmu.2021.696003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu J., et al. Preparation and neutralization efficacy of IgY antibodies raised against Deinagkistrodon acutus venom. J. Venom. Anim. Toxins Incl. Trop. Dis. 2017;23(1):22. doi: 10.1186/s40409-017-0112-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mackessy S.P., Williams K., Ashton K.G. Ontogenetic variation in venom composition and diet of Crotalus oreganus concolor: a case of venom paedomorphosis? Copeia. 2003;2003(4):769–782. doi: 10.1643/HA03-037.1. [DOI] [Google Scholar]
  30. Menezes M.C., Furtado M.F., Travaglia-Cardoso S.R., Camargo A.C.M., Serrano S.M.T. Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings. Toxicon. 2006;47(3):304–312. doi: 10.1016/j.toxicon.2005.11.007. [DOI] [PubMed] [Google Scholar]
  31. Menzies S.K., et al. Two snakebite antivenoms have potential to reduce Eswatini's dependency upon a single, increasingly unavailable product: results of preclinical efficacy testing. PLoS Neglected Trop. Dis. 2022;16(9) doi: 10.1371/journal.pntd.0010496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. MoH-Kenya . 2019. Guidelines for Prevention Diagnosis and Management of Snakebite Envenoming in Kenya. [Google Scholar]
  33. Nguyen G.T.T., et al. High-throughput proteomics and in vitro functional characterization of the 26 medically most important elapids and vipers from sub-Saharan Africa. GigaScience. 2022;11(1):giac121. doi: 10.1093/gigascience/giac121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nilsson E., Kollberg H., Johannesson M., Wejåker P.-E., Carlander D., Larsson A. More than 10 years' continuous oral treatment with specific immunoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report. J. Med. Food. 2007;10(2):375–378. doi: 10.1089/jmf.2006.214. [DOI] [PubMed] [Google Scholar]
  35. Ochola F.O., Okumu M.O., Muchemi G.M., Mbaria J.M., Gikunju J.K. Epidemiology of snake bites in selected areas of Kenya. Pan Afr. Med. J. 2018;29:217. doi: 10.11604/pamj.2018.29.217.15366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ochola F.O., Okumu M.O., Gikunju J.K., Mbaria J.M., Muchemi G.M., Nderitu J.G. Neutralization of the lethality of the venom of Dendroaspis polylepis (black mamba) in mice by two polyvalent antivenoms used in Kenyan hospitals: results of a 2009–2011 study. Sci. African. 2019;5 doi: 10.1016/j.sciaf.2019.e00118. [DOI] [Google Scholar]
  37. Olaoba O.T., Karina dos Santos P., Selistre-de-Araujo H.S., Ferreira de Souza D.H. Snake venom metalloproteinases (SVMPs): a structure-function update. Toxicon X. 2020;7 doi: 10.1016/j.toxcx.2020.100052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oliveira A.L., Viegas M.F., da Silva S.L., Soares A.M., Ramos M.J., Fernandes P.A. The chemistry of snake venom and its medicinal potential. Nat. Rev. Chem. 2022;6(7):451–469. doi: 10.1038/s41570-022-00393-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Otieno T., Gathema B. Information Sourced from State Department on Wildlife. 2021. Most common snakes in Kenya.https://twitter.com/KerioSnakebites/status/1449347833639981072/photo/1 [Google Scholar]
  40. Paul K., Manjula J., Deepa E.P., Selvanayagam Z.E., Ganesh K.A., Subba Rao P.V. Anti-Echis carinatus venom antibodies from chicken egg yolk: isolation, purification and neutralization efficacy. Toxicon. 2007;50(7):893–900. doi: 10.1016/j.toxicon.2007.06.017. [DOI] [PubMed] [Google Scholar]
  41. Potet J., Smith J., McIver L. Reviewing evidence of the clinical effectiveness of commercially available antivenoms in sub-Saharan Africa identifies the need for a multi-centre, multi-antivenom clinical trial. PLoS Neglected Trop. Dis. 2019;13(6) doi: 10.1371/journal.pntd.0007551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quarch V., Brander L., Cioccari L. An unexpected case of Black mamba (Dendroaspis polylepis) bite in Switzerland. Case Reports Crit. Care. 2017;2017:1–3. doi: 10.1155/2017/5021924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ratanabanangkoon K., et al. A pan-specific antiserum produced by a novel immunization strategy shows a high spectrum of neutralization against neurotoxic snake venoms. Sci. Rep. 2020;10(1) doi: 10.1038/s41598-020-66657-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Resiere D., et al. Preclinical evaluation of the neutralizing ability of a monospecific antivenom for the treatment of envenomings by Bothrops lanceolatus in Martinique. Toxicon. 2018;148:50–55. doi: 10.1016/j.toxicon.2018.04.010. [DOI] [PubMed] [Google Scholar]
  45. Resiere D., Mehdaoui H., Neviere R. Inflammation and oxidative stress in snakebite envenomation: a Brief descriptive review and clinical implications. Toxins. 2022;14(11):802. doi: 10.3390/toxins14110802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rojas E., Quesada L., Arce V., Lomonte B., Rojas G., Gutiérrez J.M. Neutralization of four Peruvian Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment. Acta Trop. 2005;93(1):85–95. doi: 10.1016/j.actatropica.2004.09.008. [DOI] [PubMed] [Google Scholar]
  47. Segura Á., et al. Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub-Saharan Africa. Toxicon. 2010;55(2–3):369–374. doi: 10.1016/j.toxicon.2009.08.010. [DOI] [PubMed] [Google Scholar]
  48. Senji Laxme R.R., et al. Beyond the ‘big four’: venom profiling of the medically important yet neglected Indian snakes reveals disturbing antivenom deficiencies. PLoS Neglected Trop. Dis. 2019;13(12) doi: 10.1371/journal.pntd.0007899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Strover H.M. Report on a death from black mamba bite (Dendroaspis polylepis) Cent. Afr. J. Med. 1967;13(8):185–186. http://www.ncbi.nlm.nih.gov/pubmed/6077494 [Online]. Available: [PubMed] [Google Scholar]
  50. Tan N.-H., Arunmozhiarasi A., Ponnudurai G. A comparative study of the biological properties of Dendroaspis (mamba) snake venoms. Comp. Biochem. Physiol. C Comp. Pharmacol. 1991;99(3):463–466. doi: 10.1016/0742-8413(91)90272-U. [DOI] [PubMed] [Google Scholar]
  51. Pierce Bradford protein assay kit user guide. 2023. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011181_Coomassie_Bradford_Protein_Asy_UG.pdf
  52. Ullah A. Structure–function studies and mechanism of action of snake venom L-amino acid oxidases. Front. Pharmacol. 2020;11:110. doi: 10.3389/fphar.2020.00110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Ullah A., et al. Thrombin-like enzymes from snake venom: structural characterization and mechanism of action. Int. J. Biol. Macromol. 2018;114:788–811. doi: 10.1016/j.ijbiomac.2018.03.164. [DOI] [PubMed] [Google Scholar]
  54. Warrell D.A. Snake bite. Lancet. 2010;375(9708):77–88. doi: 10.1016/S0140-6736(09)61754-2. [DOI] [PubMed] [Google Scholar]
  55. WHO Progress in the characterization of venoms and standardization of antivenoms. 1981. https://apps.who.int/iris/bitstream/handle/10665/37282/WHO_OFFSET_58.pdf?sequence=1 [PubMed]
  56. WHO, 2017. Guidelines for the production, control and regulation of antivenom immunoglobulins. Tech. Rep., Annex 5, TRS No 1004, pp 192.https://www.who.int/publications/m/item/snake-antivenom-immunoglobulins-annex-5-trs-no-1004.
  57. WHO Snakebite envenoming: strategy for prevention and control. 2019. https://www.who.int/health-topics/snakebite#tab=tab_1 [DOI] [PubMed]
  58. Williams D.J., et al. Strategy for a globally coordinated response to a priority neglected tropical disease: snakebite envenoming. PLoS Neglected Trop. Dis. 2019;13(2) doi: 10.1371/journal.pntd.0007059. [DOI] [PMC free article] [PubMed] [Google Scholar]

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