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Open Access

Peer-reviewed

Research Article

Bispecific single-domain antibody (VHH) fused with human IgG1 Fc with dual specificity effectively neutralize Naja Kaouthia venom

  • Kanokporn Pothisamutyothin,

    Roles Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Visualization, Writing – original draft

    Affiliation Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

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  • Sirisak Sathorn,

    Roles Data curation, Investigation, Methodology, Resources

    Affiliation Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

    ⨯
  • Ratchana Boonmee,

    Roles Data curation, Investigation, Methodology

    Affiliation Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

    ⨯
  • Wichit Thaveekarn,

    Roles Data curation, Investigation, Methodology

    Affiliation Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

    ⨯
  • Wanatchaporn Arunmanee

    Roles Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing

    wanatchaporn.a@chula.ac.th

    Affiliations Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand, Center of Excellence in Cancer Cell and Molecular Biology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

    ⨯

Abstract

Naja kaouthia, or the monocled cobra, is one of the most medically important snakes in Thailand, responsible for approximately 17% of snakebite cases. Conventional horse-derived antivenoms are lifesaving, yet they may trigger severe allergic reactions and exhibit batch to batch variability. Nanobodies (VHH) are promising alternatives as recombinant antivenoms having demonstrated the ability to neutralize snake venom both in vitro and in vivo. However, a major challenge in developing them is the diverse and complex composition of snake venoms, which requires therapies capable of targeting multiple toxins. To address this, we developed a bispecific VHH that simultaneously targets the two main toxins in N. kaouthia venoms, α-neurotoxin (αNTX) and phospholipase A2 (PLA2), fused to a human IgG Fc domain (bispecific VHH-Fc), which was selected to prolong serum half-life and reduce the immunogenicity risks associated with animal-derived antivenoms. The bispecific VHH-Fc, along with two monospecific nanobodies (VHH-αNTX-Fc and VHH-PLA2-Fc), was expressed in Chinese hamster ovary (CHO) cells and purified from culture supernatant after 5–6 days. Immunoblotting confirmed the successful expression and Fc fusion of these constructs, as detected by anti-human IgG-Fc antibodies conjugated to horseradish peroxidase (HRP). Importantly, antigen-binding assays demonstrated that the bispecific VHH-Fc exhibited the the strongest binding signal to crude N. kaouthia venom compared to the monospecific nanobodies. In in vivo murine neutralization assays, the bispecific VHH-Fc showing higher survival than equine-derived antivenom (33%) and comparable efficacy to a VHH-Fc cocktail under the tested conditions. Complete protection was achieved at higher doses. These results demonstrate that the bispecific VHH-Fc can be efficiently produced in a mammalian expression system and possesses strong binding and neutralizing activity against N. kaouthia venom under the defined experimental conditions. Our findings support the bispecific VHH-Fc as a promising next-generation therapeutic candidate for the treatment of snakebite envenoming, while highlighting the importance of integrating binding and functional assays when evaluating antibody efficacy.

Author summary

Snakebite envenoming remains a major public health problem in many developing countries, often leading to serious complications such as muscle paralysis. The standard treatment, horse-derived antivenom, can cause severe allergic reactions and sometimes lacks the precision needed to target specific toxins in snake venom. To address these challenges, we developed a new type of treatment using engineered antibody fragments called nanobodies (VHH). In this study, we focused on a special bispecific VHH-Fc that can recognize and neutralize two of the most dangerous toxins found in monocled cobra venom which are α-neurotoxin and phospholipase A2. We produced this bispecific VHH-Fc, along with two single-target VHHs, in mammalian cells to ensure they would be effective and stable in the body. Our results showed that these VHHs, especially the bispecific version, could be produced efficiently and were able to bind to and neutralize cobra N. kaouthia venom in laboratory tests and animal models. This research suggests that bispecific VHH-Fc could offer a safer and more effective alternative to traditional antivenoms.

Citation: Pothisamutyothin K, Sathorn S, Boonmee R, Thaveekarn W, Arunmanee W (2026) Bispecific single-domain antibody (VHH) fused with human IgG1 Fc with dual specificity effectively neutralize Naja Kaouthia venom. PLoS Negl Trop Dis 20(3): e0014119. https://doi.org/10.1371/journal.pntd.0014119

Editor: Marco Aurélio Sartim, Universidade Federal do Amazonas, BRAZIL

Received: June 5, 2025; Accepted: March 5, 2026; Published: March 17, 2026

Copyright: © 2026 Pothisamutyothin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: K.P. received Ph.D. funding support from the Government Pharmaceutical Organization, Thailand. This research was supported by the Faculty of Pharmaceutical Sciences, Chulalongkorn University (Grant No. Phar2567_RG009) and the the Second Century Fund (C2F), awarded to W.A. Funder Website: https://www.pharm.chula.ac.th The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Snakebite envenoming remains a significant public health concern in tropical and developing regions, leading to mortality and morbidity. Snakebites result in an estimated 1.8-2.7 million cases of envenoming and 81,000–138,000 deaths globally with the highest burden reported in Asia [1,2]. Among the most medically important snake families in Asia are Elapidae and Viperidae, which are responsible for the majority of severe envenomation cases. In Thailand, snakes from Elapidae family are responsible for 17% of snakebite incidents, with Naja kaouthia (monocled cobra) being the leading cause of hospitalizations [3–5]. Current treatment relies on polyclonal antivenoms, typically derived from hyperimmunized animals such as horses or sheep [6,7]. Although these antivenoms can be effective, they are associated with several limitations, including high production cost, lengthy manufacturing processes, and the risk of severe hypersensitivity reactions [8–10]. Previous studies have shown that a Taiwanese freeze-dried neurotoxic antivenom (FNAV) can neutralize the lethality of N. kaouthia venom in animal models, though requiring relatively high doses and showing variation depending on the venom source [11]. Moreover, broader studies from neighboring regions, such as India, have highlighted disturbing deficiencies in current antivenoms, showing that geographic variation in venom composition often leads to poor neutralization and limited cross-protection [12]. Geographic variation in both venom composition and antivenom neutralization potency has also been demonstrated, suggesting limited cross-protection by current polyvalent and monovalent antivenoms [13,14]. These drawbacks underscore the urgent need for alternative therapeutic strategies that are safer, more cost-effective, and easier to produce.

Recent advances in antivenom research have focused on developing novel therapeutic strategies beyond traditional polyclonal antibodies, including recombinant antivenoms, and small-molecule inhibitors [15]. Among the novel antivenom therapies are nanobodies, or single-domain antibodies (VHHs), derived from Camelidae species. Nanobodies offer several advantages over conventional antibodies, including high affinity, solubility, specificity, efficacy, low immunogenicity, and remarkable stability under varying pH and temperature conditions [6,16]. Their small size enables deep tissue penetration, though it also results in a short serum half-life [17]. In addition, incorporation of human Fc domain offers an additional safety advantage by reducing the immunogenicity risks associated with animal-derived antivenoms, thereby improving their translational potential for therapeutic use [17]. The limitation can be addressed by engineering nanobodies with human IgG Fc domains or human serum albumin (HSA) fusions, both of which are established strategies to extend circulation time [18,19]. Previous studies have already provided proof-of-concept that nanobody-based constructs can neutralize venom. For instance, Chavanayarn et al., 2012 generated VHHs against PLA₂ from N. kaouthia and demonstrated that they specifically bound and inhibited enzymatic activity [20]. Richard et al. 2013 developed high-affinity llama-derived VHHs and a VHH-Fc against α-NTX, showing potent in vivo neutralization of neurotoxicity [21]. More recently, Bailon Calderon et al., 2020 reported nanobodies targeting the hemorrhagic and myotoxic components of Bothrops atrox venom, which successfully reduced local tissue damage in vivo [22]. Together, these works established that nanobodies can be engineered to target distinct venom toxin families, including αNTX and PLA₂.

One of the major challenges in developing recombinant antivenoms is the complex composition of snake venoms, which contain multiple, distinct toxin families. Since snake venoms typically contain multiple toxin families that act through different pathological mechanisms, such as αNTXs causing paralysis and PLA₂ contributing to local tissue damage and potentiating neurotoxicity, effective antivenom therapies often require a cocktail formulation to achieve broad neutralization of these diverse toxins. In the case of N. kaouthia venom, the primary components are three-finger toxins (3FTXs, 51%), PLA2s (27%) [5]. PLA2s represent the second most abundant toxin family in this venom, following 3FTXs. The main 3FTXs from N. kaouthia venom are αNTX [23,24]. The pharmacological effects of 3FTXs is mediated by αNTX, which inhibit the nicotinic acetylcholine receptor (nAChR), leading to skeletal muscle paralysis [24–26]. In parallel, PLA₂ toxins contribute to neurotoxicity by inducing calcium influx through neuronal ion channels, triggering excessive neurotransmitter release, and ultimately causing neuromuscular blockade. Beyond their neurotoxic role, PLA₂s also contribute to local tissue damage and myotoxicity, thereby exacerbating the overall clinical severity of envenoming [26]. Therefore, a therapeutic agent capable of simultaneously targeting both αNTX and PLA2 would represent a promising antivenom strategy against Thai cobra envenoming. Bispecific antibodies have emerged as an innovative therapeutic format, engineered to recognize two different epitopes or antigens within a single molecule. This strategy has already been successfully applied in oncology and infectious diseases to enhance therapeutic potency, reduce the risk of resistance, and provide synergistic effects compared to monospecific antibodies [27,28]. Applying this concept to antivenom development offers the potential to simultaneously neutralize multiple key toxins in snake venoms within a single recombinant construct, providing broader and more efficient protection than conventional antibody cocktails.

In this study, we developed monospecific nanobodies targeting α-neurotoxin (VHH-αNTX) and phospholipase A2 (VHH-PLA2), as well as a bispecific VHH designed to target both major toxin components of N. kaouthia venom. To improve their translational relevance, all VHH constructs were fused to a human IgG1 Fc to facilitate expression and dimerization in ExpiCHO-S cell; pharmacokinetic and long-term stability parameters were not directly evaluated in this study. The binding activity of VHH-αNTX-Fc, VHH-PLA2-Fc, and the bispecific VHH-Fc to crude venom were evaluated using ELISA and their neutralization efficacy was assessed in vivo. The comparative efficacy of recombinant VHH-Fc constructs, both monospecific and bispecific, in neutralizing N. kaouthia venom was evaluated. This approach aims to offer a safer, more effective, and broadly applicable alternative to traditional antivenoms.

Methods

Ethics statement

All animal experiments were conducted in accordance with the guidelines of the Thai Pharmacopoeia and the Thai Red Cross Society. All study protocols in this study were approved by the Queen Saovabha Memorial Institute Animal Care and Use Committee (Protocol No. QSMI-ACUC-05–2024)). Mice (3–4 weeks old, weighing 18–20 g) were obtained from Institute of Cancer Research (ICR), supplied by National Laboratory Animal Center, Mahidol University, Salaya Campus, Nakhon Pathom, Thailand. All animals were acclimatized for 7 days before experimentation to minimize stress and ensure physiological stability.

Construction of VHH-Fc in a mammalian expression vector for transient expression

The gene fragment encoding VHH-αNTX was previously published (Patent No. US 2013/0259864 A1, Cluster II C2) [21], while the VHH-PLA2 gene was previously reported by Chavanayarn et al., 2012 [20]. Codon-optimized gene sequences were synthesized by Twist Bioscience (USA) and cloned into the pFUSE-hIgG1-Fc expression vector (InvivoGen, USA) using the EcoRI and BglII restriction sites. Each VHH gene was inserted between the IL-2 signal sequence and the human IgG1 Fc region within the vector.

To construct a bispecific antibody consisting of both VHH-αNTX and VHH-PLA2, the two VHH sequences were linked via a (G4S)3 glycine-serine peptide linker. The pFUSE vector containing VHH-αNTX was amplified by PCR using the primers 5’-CTCTGGTCACAGTGTCATCCAGATCTGACAAAACTCACAC-3’ and 5’-GCTGCTCACGGTCACTTGAGTAC-3’. Similarly, the pFUSE vector containing VHH-PLA2 was amplified by PCR using the primers 5’-CAGCTCGTTGAATCTGGCGGGGG-3’ and 5’-GTGTGAGTTTTGTCAGATCTGGATGACACTGTGACCAGAG-3’. PCR reactions were carried out using Taq DNA polymerase (Invitrogen, USA) according to the manufacturer’s protocol. The PCR products and the (G4S)3 linker were assembled using the Gibson Assembly kit (New England Biolabs), following the manufacturer’s protocol. All recombinant plasmids were propagated in Escherichia coli (E. coli) Mach I cells and purified using the QIAGEN Plasmid Maxi kit (Qiagen, Germany). The recombinant DNA sequences were verified by Barcode-Tagged (BT) DNA sequencing (U2Bio, Korea) using pFUSE specific primers. DNA concentration and purity were assessed using a Nanodrop One microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA).

Cell culture

ExpiCHO-S cells (Thermo Fisher Scientific, USA) were cultured in HyCell TransFx-C transfection medium (Cytiva, USA) at 37 °C in an incubator with 8% CO2 and shaking at 125 rpm in vented, non-baffled Erlenmeyer shake flasks (Corning, cat. no. 431143). Cells were seeded at 2 x 105 – 3 x 105 viable cells/ml and subcultured every 3–4 days. Viable cell density and cell viability were assessed using the trypan blue exclusion method with 0.4% trypan blue solution (Gibco, cat. no. 15250061).

Transient protein expression in ExpiCHO-S cells

One day prior to transfection, ExpiCHO-S cells were seeded at a density of 5 x 105 viable cells/ml in 30 ml of culture medium in 120 ml vented, non-baffled Erlenmeyer shake flasks (Corning). After 24 hours, the cell density reached approximately 1.0 x 106 viable cells/ml, with viability exceeding 95% (Day 0). Transfections were performed at a density of 1.0 x 106 cells/ml in HyCell TransFx-C transfection medium using plasmid DNA at a final concentration of 1 µg/ml. Before transfection, DNA–polyethyleneimine (PEI-Max; Polysciences, USA) complexes were formed at various DNA/PEI-Max mass ratios. Plasmid DNA and PEI-Max were each diluted separately at 1:20 of the final culture volume. Diluted PEI-Max was added dropwise to the diluted DNA solution and incubated for 10 min at room temperature. The DNA/PEI-Max mixture was then added to the cells. Cultures were maintained at either 34.5 ∘C and 37 ∘C with 8% CO2 and shaken at 125 rpm. Cells were cultured for five to six days depending on viability.

Daily measurements of viable cell density and cell viability were carried out. At the end of the expression period, cultures were harvested by centrifugation at 5,000 x g for 30 min at 4 ∘C. Supernatants were filtered through a 0.22 µm Steritop Millipore Express PLUS filter unit (Merck, USA) and kept at 4 ∘C for purification.

Purification of VHH-Fc proteins

Following clarification of the culture medium, the crude supernatant containing VHH-αNTX-Fc, VHH-PLA2-Fc and the bispecific VHH-Fc was purified by a HiTrap rProtein A FF column (GE Healthcare Technologies, West Milwaukee, WI, USA) on an ÄKTA start fast protein liquid chromatography (FPLC) system (GE Healthcare, USA). The column was equilibrated with binding buffer (20 mM sodium phosphate, 300 mM NaCl, pH 7.0) at a flow rate of 1 ml/min. Bound proteins were eluted using 0.1 M sodium citrate buffer (pH 3.0). Eluted protein fractions were collected and immediately neutralized with 1 M Tris-HCl (pH 9.0), followed by concentration using Amicon Ultra-4 centrifugal filter units with a 10 kDa molecular weight cutoff (Merck, USA). The proteins were then buffer-exchanged into phosphate-buffered saline pH 7.4 (PBS). Protein concentrations were determined by the Bicinchoninic acid (BCA) assay using the Pierce BCA Protein Assay Kit (Thermo fisher scientific, USA).

SDS-PAGE and Western blot analysis

Purified proteins were identified by SDS-PAGE and Western blot under both reducing and non-reducing conditions. Proteins were resolved on 10% polyacrylamide gels and stained with Coomassie Brilliant Blue. For Western blotting, proteins were transferred onto the nitrocellulose membranes (Merck KGaA, Darmstadt, Germany) using a semi-dry transfer system (Trans-Blot SD System and PowerPac HC Power Supply System, Bio-Rad, Singapore). Membranes were blocked with 5% (w/v) skim milk powder (Titan Biotech Ltd, India) in PBST (PBS containing 0.05% Tween-20) and then incubated with a 1:5000 dilution of anti-Human IgG peroxidase-conjugated antibody (Sigma-Aldrich, USA) in blocking buffer. Membranes were then washed by PBST three times for 15 min each. Protein bands were detected using Immoblion Forte Western HRP Substrate (Merck, USA) for 3 min and visualized via chemiluminescence using the ImageQuant LAS4000 system (GE Healthcare, USA). To assess glycosylation, purified proteins were treated with peptide:N-glycosidase F (PNGase F) or endoglycosidase H (Endo H), according to the denaturing protocols provided by the manufacturer (New England Biolabs, USA). Deglycosylated proteins were analyzed by SDS-PAGE and visualized by Coomassie staining.

In vitro venom binding assay via Enzyme-Linked Immunosorbent Assay (ELISA)

Nunc-Immuno MaxiSorp plates (Thermo Fisher Scientific, USA) were coated with 100 ng/well of crude N. kaouthia venom (Queen Saovabha Memorial Institute) diluted in 50 mM bicarbonate buffer pH 9.6 and incubated at 4°C overnight. Plates were then blocked with 100 µl of PBS supplemented with 1% bovine serum albumin (BSA) for 1 h at 37 °C. After blocking, plates were washed three times with PBST (PBS containing 0.1% Tween-20). VHH-αNTX-Fc and bispecific VHH-Fc were serially diluted 3-fold, while VHH-PLA2-Fc was diluted 2-fold, with concentrations ranging from 5 µg/mL to 140 µg/mL, and added to the wells. Plates were incubated for 1 hour at 37 °C and then washed with 200 µl of PBST.

A 1:1000 dilution of anti-human IgG (Fc specific) peroxidase-conjugated antibody (Sigma-Aldrich, USA) in blocking buffer was added to each well and incubated for 1 h at 37 °C. After washing, 100 µl of 3,3’,5,5’-tetramethylbenzidine (TMB) chromogenic substrate (Invitrogen, USA) was added and incubated at room temperature for 20 min. The enzymatic reaction was stopped with 100 µl of 1 N H2SO4. Absorbance at 450 nm was measured using a CALIOstar microplate reader (BMG Labtech, Germany).

In vivo neutralization study

Murine Lethal Dose (LD₅₀ and LD₁₀₀) determination

The lethality of crude Naja kaouthia venom was determined in mice weighing 18–20 g according to the Thai Pharmacopoeia. For LD₅₀ determination, mice were divided into five groups (n = 4 per group) and injected intravenously via the caudal vein with 0.5 mL of graded venom concentrations, with the middle dilution expected to approximate the LD₅₀. Morbidity and mortality were monitored for 48 h. The LD₅₀, defined as the venom dose causing death in 50% of mice within 48 h, was calculated by Probit analysis from triplicate experiments.

Following LD₅₀ determination, the absolute lethal dose (LD₁₀₀) was established by testing progressively higher venom doses above the LD₅₀ using small incremental increases (1–1.3 × LD₅₀). The LD₁₀₀ was defined as the minimum venom dose that consistently produced 100% mortality under the defined experimental conditions and was subsequently used as the challenge dose in the murine neutralization assay.

Median effective dose (ED50) determination

To evaluate the protective efficacy of VHH-Fc, mice were assigned to six groups (n = 6 per group) including VHH-αNTX-Fc, VHH-PLA2-Fc, bispecific VHH-Fc, a 1:1 molar ratio cocktail of VHH-αNTX-Fc and VHH-PLA2-Fc, a positive control group receiving equine-derived anti-N. kaouthia venom, and a negative control group receiving normal saline solution (NSS). A commercially available equine-derived anti-N. kaouthia venom was produced by the Queen Saovabha Memorial Institute (Thai Red Cross Society, Bangkok, Thailand), The antivenom was provided as a purified bulk intermediate product (lot number: 23004NK) with a reported protein concentration of 5.19 g%. Prior to testing, the antivenom was diluted in PBS to a final protein concentration of 1.0 mg/mL while the cocktail of VHH-αNTX-Fc and VHH-PLA2-Fc was adjusted to 0.7 mg/mL. The antivenoms were serially diluted to final concentrations ranging from 2-500 µg/mL. Each antivenom preparation was pre-incubated with a fixed dose of crude N. kaouthia venom equivalent to the 2xLD100 at a 1:1 (v/v) ratio at 37 °C for 30 min. Mice were injected intravenously with 0.5 mL of the venom-antivenom mixture via the caudal vein. The dilution range was selected to yield a mortality curve between 20% and 80%. Survival and mortality were recorded over 48 h. ED₅₀ values were calculated by Probit analysis.

Survival rate assessment

To assess the neutralization potency, the venom (2x LD100) was mixed with VHH-Fc preparation at 1x, 2x, and 5x ED50 concentrations. Mice were divided into four groups (n = 6 per group) including VHH-αNTX-Fc, bispecific VHH-Fc, the cocktail of VHH-αNTX-Fc and VHH-PLA2-Fc (1:1 molar ratio) and polyclonal antibody (positive control). The venom and antibody mixtures were prepared at a 1:1 volume ratio and incubated at 37 °C for 30 min. Each mouse received 0.5 mL of the mixture via the caudal vein injection. This volume was consistent with animal ethics approval and in accordance with the Thai Pharmacopoeia. Morbidity and mortality were recorded at 1, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h post-injection. Survival rates were calculated and expressed as percentages.

Statistical analysis

Kaplan–Meier survival analysis was performed to evaluate survival over time following antivenom treatment, and differences between survival curves among groups were assessed using the Log-rank (Mantel–Cox) test. In addition, survival outcomes at the 48-hour endpoint were analyzed using a two-way analysis of variance (Two-way ANOVA), with antibody type and ED₅₀ concentration as independent variables. The ANOVA outputs (effect size and variance) were further applied for power analysis to confirm that the sample size (n = 6 per group) was sufficient to achieve adequate statistical power, in line with the Thai Pharmacopoeia guideline. A post-hoc power analysis demonstrtated that the achieved statistical power exceeded 80% at 0.05, supporting the adequacy of the sample size beyond reliance on regulatory guidelines. A p-value of less than 0.05 was considered statistically significant. All animal experiments were approved by the Queen Saovabha Memorial Institute Animal Care and Use Committee.

Results

Production of VHH fused with human IgG1 Fc domain in ExpiCHO-s cells

To generate bispecific VHH-Fc fusion proteins, two single-domain antibodies (VHHs) recognizing the main components of N. kaouthia venom including α-neurotoxin (αNTX) and phospholipase A₂ (PLA₂) were genetically linked via a flexible glycine-serine linker ((G₄S)₃) as illustrated in Fig 1A. The linker enables each VHH domain to fold properly and maintains its function. Incoporating both VHHs within the same polypeptide arm is advantageous, as it eliminates the need to co-transfect two separate genes. The bispecific VHH cassette was cloned in-frame upstream of the human IgG1 Fc domain in the pFUSE-hIgG1-Fc2 expression vector. This results in the correct translation and dimerization of the fusion protein. A secretion signal peptide at the N-terminus enabled secretion of proteins into the culture supernatant. The expressed bispecific VHH-Fc adopts the architecture shown in Fig 1B. For comparison, monospecific nanobody-Fc constructs were also generated by cloning individual VHHs targeting either αNTX or PLA2 into the same expression vector. These served as controls to evaluate the functional advantage of the bispecific format. Monospecific constructs are shown in Fig 1A and the dimerization orientation are shown in Fig 1B.

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Fig 1. Design of Monospecific-Fc and Bispecific VHH-Fc Fusion proteins.

(A) Genetic design of VHH-Fc constructs including monospecific and bispecific antivenom. (B) Schematic representation of Fc dimerization and fusion protein configuration.

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The recombinant constructs whose sequences were verified by DNA sequencing were expressed in ExpiCHO-S cells to ensure the correct protein folding and human-like glycosylation. Each construct contained IL-2 signal sequences to facilitate secretion of the fusion proteins into the culture medium. Transiently transfected cells were cultured for 5–6 days while maintaining cell viability between 65–75%. Preliminary optimization experiments indicated that monospecific constructs showed optimal yield at 37 °C with harvesting on day 5, whereas the bispecific construct exhibited improved stability and higher expression at 34.5 °C with harvesting on day 6. After cultivation, culture supernatants containing the secreted VHH-Fc fusion proteins were harvested. The supernatants were purified using a recombinant protein A FF column, which selectively binds to the Fc region of the fusion proteins. Chromatographic elution profiles revealed distinct peaks corresponding to VHH-αNTX-Fc (Fig 2A), VHH-PLA₂-Fc (Fig 2B), and bispecific VHH-Fc (Fig 2C). Expression optimization experiments were conducted to identify conditions yielding the highest protein levels. VHH-αNTX-Fc (63.814 mg/L) and VHH-PLA₂-Fc (95.680 mg/L) achieved peak expression on day 5 at 37 °C using a DNA:PEI-Max ratio of 1:5. In contrast, the bispecific VHH-Fc fusion reached its highest yield (71.472 mg/L) on day 6 under the same transfection ratio but at a reduced temperature of 34.5°C.

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Fig 2. Purification of VHH-Fc Proteins by rProtein A FF column.

Elution profile of (A)VHH-αNTX-Fc, (B) VHH-PLA2-Fc, and (C) Bispecific VHH-Fc fusion proteins. Green, Blue and Purple lines represent absorbance at 280 nm (A280) while red lines indicate the percentage of Buffer B (%Conc B) in each chromatogram.

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Characterization of VHH-Fc fusion proteins

To determine the biochemical properties of VHH-αNTX-Fc, VHH-PLA2-Fc and Bispecific VHH-Fc fusion proteins, samples were analyzed by SDS-PAGE under both reducing and non-reducing conditions using 10% polyacrylamide gels. Under reducing conditions, the expected bands of VHH-αNTX-Fc, VHH-PLA2-Fc and bispecific VHH-Fc showed at ~39 kDa, ~ 39 kDa and ~53 kDa, respectively. Similarly, under non-reducing conditions, the expected molecular weights of VHH-αNTX-Fc, VHH-PLA2-Fc and bispecific VHH-Fc under non-reducing condition migrated at ~78 kDa, ~ 78 kDa and ~106 kDa, respectively (Fig 3A). This is consistent with Fc-mediated dimerization. Protein identity was confirmed by Western blot using an anti-human IgG1 Fc domain-specific antibody. All fusion proteins were observed under both reducing and non-reducing conditions. This confirmed the presence of the human Fc region and successful expression in the mammalian system (Fig 3B) These results verify that VHH-αNTX-Fc, VHH-PLA₂-Fc, and bispecific VHH-Fc were correctly expressed, folded, and secreted by the ExpiCHO-S cell system. Notably, additional high molecular weight bands ranging from ~150–250 kDa were observed in all samples. These likely represent minor amounts of protein aggregates. However, the abundance of these aggregates was low relative to the dominant bands corresponding to the correctly folded target proteins.

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Fig 3. SDS-PAGE and Western blot analyzes of purified VHH-Fc Fusion Proteins.

(A) Coomassie blue-stained SDS-PAGE showing protein migration under non-reducing (-) and reducing (+) conditions. (B) Immunoblotting using anti-human IgG1 Fc specific antibodies under non-reducing (-) and reducing (+) conditions.

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To evaluate the N-linked glycosylation on VHH-αNTX-Fc, VHH-PLA2-Fc and bispecific VHH-Fc, enzymatic deglycosylation was performed using PNGase F and Endo H. PNGase F cleave all N-linked glycoforms including high mannose, hybrid, and complex types while Endo H specifically cleave high mamnose glycans by targeting the bond between two N-acetylglucosamine (GlcNAc) residues. After treatment with PNGase F, the fusion proteins exhibited increased electrophoretic mobility compared to the untreated controls, indicating successful deglycosylation and suggesting the presence of N-linked glycans. In contrast, Endo H treatment did not alter the migration patterns of the proteins, suggesting that the N-linked glycans are of the complex type rather than high-mannose (Fig 4). These results imply that the VHH-Fc fusion proteins contain complex N-glycans. Additionally, protein bands observed at ~36 kDa and ~25 kDa in the PNGase F- and Endo H-treated samples, respectively, likely correspond to the enzymes themselves. This shift confirms the removal of N-linked glycans. Importantly, deglycosylation was performed solely for analytical validation of glycan type and was not applied to antibodies used in any subsequent in vitro binding assays or in vivo neutralization experiments.

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Fig 4. Deglycosylation analysis of purified target proteins using PNGase F and Endo H enzymes.

SDS-PAGE analysis followed by Coomassie-blue staining under reducing conditions (with DTT). (A) VHH-αNTX-Fc, (B) VHH-PLA2-Fc, and (C) Bispecific VHH-Fc.

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In vitro binding activity by Enzyme-linked immunosorbent assay (ELISA)

The antigen-binding ability of the VHH-Fc fusion proteins including VHH-αNTX-Fc, VHH-PLA2-Fc, and bispecific VHH-Fc was evaluated using an ELISA-based assay against crude N. kaouthia (cobra) venom. Microtiter plates were coated with crude cobra venom and incubated with serial dilutions of the VHH-Fc fusion proteins. Receptor binding domain of SARS-CoV-2 fused with hIgG1 Fc, RBD-Fc, served as a negative control. As a commercial anti-cobra serum containing an human Fc domain was not available, no positive control was included in this study. The initial concentrations were 0.093 µM (5 µg/mL) for bispecific VHH-Fc, 0.13 µM (5 µg/mL) for VHH-αNTX-Fc, 3.6 µM (140 µg/mL) for VHH-PLA2-Fc, and 0.103 µM (5 µg/mL) for RBD-Fc, with final concentrations of 4 × 10 ⁻ ⁶ µM (8.5 × 10 ⁻ ⁵ µg/mL), 6.6 × 10 ⁻ ⁶ µM (8.5 × 10 ⁻ ⁵ µg/mL), 7.03 × 10 ⁻ ³ µM (1.4 × 10 ⁻ 1 µg/mL), and 4.02 × 10 ⁻ ⁴ µM (8.5 × 10 ⁻ ⁵ µg/mL), respectively, using 3-fold serial dilutions for VHH-αNTX-Fc, bispecific VHH-Fc, and RBD-Fc, and 2-fold serial dilutions for VHH-PLA2-Fc. Following incubation, binding was detected using an anti-human IgG (Fc-specific) peroxidase-conjugated secondary antibody. The results demonstrated that all VHH-Fc fusion proteins were capable of binding to the immobilized crude cobra venom, whereas the RBD-Fc negative control showed no detectable binding (Fig 5). Among the tested proteins, VHH-αNTX-Fc and the bispecific VHH-Fc showed stronger binding activity compared to VHH-PLA2-Fc, which exhibited the weakest binding. VHH-PLA2-Fc was subjected to a 2-fold serial dilution instead of 3-fold to improve resolution in the mid-slope region of the binding curve, given its weaker binding activity observed in preliminary experiments. Under the tested concentration range, the binding curve did not reach a full saturation plateau, consistent with its relatively low affinity toward the venom.

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Fig 5. In vitro binding activity of VHH-Fc fusion proteins to crude N. kaouthia venom by ELISA.

Binding curves are shown for VHH-αNTX-Fc (blue), VHH-PLA2-Fc (green), bispecific VHH-Fc (red), and RBD-Fc (pink, negative control). Data are presented as mean ∆OD450 ± SD from a triplicate assay for each protein concentration.

https://doi.org/10.1371/journal.pntd.0014119.g005

In vivo neutralization of VHH-Fc fusion proteins against crude N. kaouthia venom

The neutralization ability of the VHH-Fc fusion proteins against crude N. kaouthia venom was evaluated using a mouse model. The intravenous injection volume of 0.5 mL used in this study was conducted in strict accordance with the Thai Pharmacopoeia, which permits intravenous injection volumes up to this level in mice of this weight range under controlled experimental conditions. This practice is standard in venom–antivenom neutralization assays in Thailand, has been formally approved under our institutional animal ethics protocol, and no signs of cardiac overload or abnormal physiology were observed during the experiments. Initially, the in vivo toxicity of crude venom in mice was determined. Probit analysis established the median lethal dose (LD50) at 6.55 µg/mouse (0.34 µg/g) with a 95% confidence interval (CI) of 5.27-7.83 µg/mouse, calculated as the mean of three independent experiments. The absolute lethal dose (LD₁₀₀) was determined to be 7.205 µg/mouse (S1 and S2 Tables). Subsequently, the median effective dose (ED₅₀) for each VHH-Fc were assessed by observing the percentage survival rate of mice within 48 hours after receiving the mixture of venom and VHH-Fc. An equine-derived anti-N. kaouthia venom was used as a positive control, while normal saline solution (NSS) served as a negative control. The ED₅₀ values of the antibody are shown in Fig 6 and Table 1. Raw survival data used for probit-based ED50 determination are provided in S3 and S4 Tables. The ED₅₀ value for VHH-PLA2-Fc could not be determined due to its low affinity compared to the other VHH-Fc constructs. The ED₅₀ values of the bispecific VHH-Fc and VHH-αNTX-Fc were highly comparable, indicating no meaningful difference in median neutralization potency among these recombinant antibody formats under the defined experimental conditions. In contrast, the cocktail and equine-derived antivenom exhibited a higher ED₅₀ value, suggesting lower neutralization potency relative to the recombinant VHH-Fc constructs.

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Table 1. Median effective doses (ED₅₀) of antibody formats against crude cobra venom.

https://doi.org/10.1371/journal.pntd.0014119.t001

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Fig 6. Dose–response curves showing the neutralization efficacy of various antibody formats against crude cobra venom.

Antibody constructs tested include bispecific VHH-Fc, a cocktail of VHH-αNTX-Fc and VHH-PLA2-Fc, VHH-αNTX-Fc, and an equine-derived anti-N. kaouthia venom. Median effective doses (ED₅₀) were calculated using nonlinear regression (four-parameter logistic model). The ED₅₀ values were 3.215 µg for the bispecific VHH-Fc, 4.533 µg for the cocktail, 3.163 µg for VHH-αNTX-Fc, and 4.360 µg for the equine-derived antivenom (positive control).

https://doi.org/10.1371/journal.pntd.0014119.g006

The neutralization efficacy of the antibodies was assessed at 1x, 2x, and 5x their respective ED₅₀ values against the 2xLD₁₀₀ dose of crude N. kaouthia venom. as this challenge dose established a stringent and reproducible lethality endpoint. This approach minimizes variability in survival outcomes and facilitates accurate ED₅₀ estimation. The use of 2xLD₁₀₀ in venom–antivenom neutralization assays was reviewed and approved under our institutional ethics protocol (QSMI-ACUC-05–2024). Antibody–toxin mixtures were administered to mice via caudal vein injection. The observation interval for survival monitoring is shown in Fig 7A. The challenge test (Fig 7B) revealed that at 1x ED₅₀, VHH-αNTX-Fc achieved the highest survival rate (62%), while both the bispecific VHH-Fc and cocktail VHH-Fc resulted in a 50% survival rate. The equine-derived antivenom from the Thai Red Cross provided the lowest survival rate at this dose (33%). At 2x ED₅₀, both VHH-αNTX-Fc and bispecific VHH-Fc achieved 100% survival, whereas the horse anti-venom and cocktail VHH-Fc resulted in survival rates of 83% and 66%, respectively. Complete protection was observed in all groups when the crude N. kaouthia venom (2xLD₁₀₀) was preincubated with 5x ED₅₀ of any VHH-Fc construct or horse anti-venom, and the mixture was administered via caudal vein injection. In contrast, all mice that received crude N. kaouthia venom mixed with NSS died within 60 minutes post-injection. Kaplan–Meier survival analysis with the Log-rank test confirmed statistically significant differences among treatment groups (p < 0.05). Two-way ANOVA further indicated significant effects of both antibody type and ED₅₀ concentration on survival outcomes, consistent with the dose-dependent neutralization trends observed. The analyses were applied to evaluate dose- and antibody format–dependent effects on survival outcomes and were not intended to establish definitive therapeutic superiority among antibody formats. The statistical findings support that the sample size (n = 6 per group) was sufficient to ensure robust conclusions in line with the Thai Pharmacopoeia guideline.

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Fig 7. In vivo neutralization of crude N. kaouthia venom-induced lethality (2xLD100) in mice using different VHH-Fc constructs.

(A) Observation intervals following injection of antibody-toxin mixtures. (B) Survival rates of mice receiving crude N. kaouthia venom with 1x, 2x and 5x ED50 of VHH-αNTX-Fc, bispecific VHH-Fc, cocktail VHH-Fc and equine-derived anti-N. kaouthia venom as a positive control. Illustration from NIAID NIH BioArt Source (bioart.niaid.nih.gov/bioart/283 and bioart.niaid.nih.gov/bioart/505).

https://doi.org/10.1371/journal.pntd.0014119.g007

Discussion

The Monocled Cobra (N. kaouthia) is a significant venomous snake found in tropical areas, especially in Southeast Asia, and is a leading cause of snakebit-related morbidity and mortality [29,30]. Current standard treatment involves the administration of antivenoms derived from the plasma of immunized animals, typically horses or sheep. Even though these antivenoms have been lifesaving, there are several critical limitations including high immunogenicity, risk of severe allergic reaction, batch-to-batch variability and the presence of non-neutralizing antibodies that do not contribute to venom neutralization [21,31,32]. Therefore, there is an urgent need for antivenoms that are more effective, safer and consistent in targeting the diverse components of snake venom.

Recombinant antibody-based therapeutics represent a promising alternative to conventional antivenoms. In particular, camelid-derived single-domain antibodies have several appealing physiochemical properties including high affinity, high soluble, high stability and the ability to penetrate tissues effectively [26]. These properties make them attractive candidates for venom neutralization. Previous studies have shown that VHH- αNTX fused with Fc was expressed in plant-based, and it can enhance the neutralizing activity [21]. However, this protein expression in plants has drawbacks, especially post-translational modification and a tendency for protein misfolding, which can limit their suitability for human therapeutics [33,34]. To overcome these issues, we produced both monospecific (VHH-αNTX, VHH-PLA2) and bispecific (targeting both toxins) fused with the human IgG1 Fc domain using a mammalian expression system, ExpiCHO-S cells. This system was chosen for its capacity to support high-yield protein expression with human-like complex-type glycosylation and proper protein folding [35,36]. In our PEI-Max-based transient expression system, we performed transfections at 1.0 × 10⁶ viable cells/ml, corresponding to the early to mid-exponential phase, where cells remain metabolically active and receptive to DNA uptake. Preliminary trials indicated that higher densities, such as ≥ 3–4 × 10⁶ cells/ml, led to a decline in transfection efficiency and reduced protein yield, likely due to physiological shifts as cells progress further into the log phase [37]. In contrast, VHH-Fc constructs produced in tobacco plants predominantly carry high-mannose glycans [38], which are associated with rapid clearance from the circulation and reduced serum half-life [39,40]. In ExpiCHO-S cells, the glycosylation profile more closely resembles native human antibodies, potentially improving pharmacokinetic properties and therapeutic performance.

Further binding analysis using ELISA confirmed the functional activity of the VHH-Fc constructs against crude N. kaouthia venom. Among the three constructs, the bispecific VHH-Fc exhibited the strongest antigen-binding response, followed by VHH-αNTX-Fc and VHH-PLA2-Fc. While these results indicate high antigen recognition in vitro, they did not correlate with neutralization efficacy in vivo. In the murine model, VHH-αNTX-Fc conferred the greatest protection at an 2xLD₁₀₀ dose corresponding to 2.2xLD₅₀, yielding the highest survival rates, whereas the bispecific VHH-Fc showed only moderate protective efficacy. This discrepancy underscores that strong in vitro binding is not necessarily predictive of therapeutic performance in vivo. However, it is important to note that the venom challenge conditions used in this study deviate from standardized antivenom efficacy protocols, which typically utilize higher multiples of LD₅₀ (e.g., 3-6xLD₅₀) to ensure uniformly lethal challenge conditions [41,42]. In contrast, we employed an empirically determined 2xLD100 equivalent to 2.2xLD₅₀. The application of a defined 2xLD₁₀₀ challenge provides a consistent lethality endpoint for ED₅₀ determination and enables direct comparison of neutralization efficacy among antibody formats under controlled conditions. Previous studies evaluating scFv- and VHH-based antibodies have similarly used relatively low multiples of LD50 in murine neutralization assays. Kulkaew et al. defined LD₁₀₀ of N. Kaouthia long α-neurotoxin as 1.5 × LD₅₀ for murine neutralization studies while Richard et al. employed an LD100 α-cobratoxin challenge to assess antibody-mediated protection [21,43]. Additionally, Liu et al. utilized an LD100 crude N. Kaouthia venom challenge model equivalent to 1.4x LD₅₀ for neutralization assessment [11]. Following these approaches, the present study applied an empirically determined LD₁₀₀ of crude N. kaouthia venom in order to assess antibody performance within a multi-component envenoming context. This approach is particularly relevant given the dual-targeting design of the bispecific VHH-Fc construct directed against both α-neurotoxin and PLA₂. Nevertheless, the limitations of this study should be taken into account including i) the lower multiples of LD₅₀ may reduce the stringency of the challenge and limit the ability to discriminate antibody performance under higher toxin burdens and ii) batch-to-batch variability in venom toxicity, as well as geographic variation in venom composition and toxic potency among snakes from different regions, may affect the accuracy of the LD₁₀₀ determination [29]; in some cases, 2.2 × LD₅₀ may be insufficient to consistently achieve uniform lethality compared with higher challenge doses (e.g., 3–6 × LD₅₀). Furthermore, rescue experiments in which antivenom is administered after venom exposure were not performed, which limits direct extrapolation to clinical treatment scenarios. Accordingly, the in vivo findings presented in this study should be interpreted as proof-of-concept within the defined experimental framework rather than as definitive evidence of therapeutic superiority over existing antivenom products.

The bispecific construct, despite its dual-targeting design, may suffer from reduced functional affinity due to structural constraints or steric hindrance between its two antigen-binding domains. Such interdomain interference has been reported in various bispecific antibody formats and can impair simultaneous or effective binding to multiple targets [44]. Additionally, the increased molecular complexity of the bispecific VHH-Fc may render it more prone to partial misfolding, aggregation, or proteolytic degradation in vivo, thereby reducing the pool of bioactive antibody available at the site of envenoming. These issues may be exacerbated by the increased molecular complexity of the bispecific construct compared to its monospecific counterparts, particularly under physiological conditions [45]. The limited binding activity of VHH-PLA₂-Fc observed in ELISA could be attributed to the origin of the nanobody sequence itself. This construct was derived from a previously published VHH selected from a naïve phage display library rather than from an immunized camelid. Nanobodies from naïve libraries often display lower intrinsic affinities due to the absence of in vivo affinity maturation. Although Fc fusion can improve apparent binding via avidity effects, this enhancement may not fully compensate for inherently weak affinity. Moreover, epitope accessibility may also play a role, since PLA₂ epitopes within whole crude venom can be conformationally shielded or structurally less exposed compared to isolated toxin preparations. Together, these factors likely contribute to the reduced binding observed for VHH-PLA₂-Fc in vitro. It is important to clarify that PLA2 toxins are unlikely to be the primary drivers of lethality at 2xLD₁₀₀ venom challenge, where α-neurotoxins are expected to dominate. Nonetheless, PLA2 is biologically relevant due to its abundance in N. kaouthia venom (~27%) and its reported contribution to local myotoxicity as well as potentiation of αNTX-induced neurotoxicity. Thus, while PLA2 may not independently determine survival outcomes at low venom doses, its inclusion as a target remains rational when considering broader pathophysiological consequences of envenoming. These structural and conformational limitations likely contribute to the observed disconnect between its in vitro binding potency and in vivo protective efficacy.

Although several studies have demonstrated the potential of recombinant antibody formats in neutralizing cobra venom components, direct comparisons are limited. Richard G et al., 2013 reported the successful use of high-affinity llama-derived single-domain antibodies (VHHs) and VHH-Fc constructs to neutralize α-cobratoxin in vivo, achieving full protection in mice following toxin challenge [21]. Ledsgaard L et al., 2022 utilized a phage display to isolate a human monoclonal IgG capable of neutralizing whole N. kaouthia venom in a murine model, underscoring the potential of fully human antibodies for therapeutic use [46]. A broadly neutralizing human monoclonal antibody was developed from a synthetic library to target conserved α-neurotoxins across multiple snake species, demonstrating high-affinity binding, in vitro receptor blockade, and in vivo protection [10]. These studies support the efficacy of single-target antibody constructs but do not explore bispecific designs.

In this context, previous studies from Thailand and Southeast Asia have provided important insights into the composition and neutralization of N. kaouthia venom. Proteomic analyses have shown that Thai N. kaouthia venom is dominated by three-finger toxins, particularly α-neurotoxins, which constitute the principal lethal components, whilephospholipase A₂ representing the second most abundant toxin family [42,47]. Detailed venomics and immunoproteomic studies have characterized these profiles and demonstrated its relevance to venom lethality and antivenom immunorecactivity. Comparative venomics by Tan et al. have revealed significant geographic variation in the venom proteomes of N. kaouthia populations across Thailand, Malaysia and Vietnam [42]. Similarly, proteomics-informed analyses in India and China also demonstrated the variation of toxin profiles [48,49]. These studies highlight how regional differences in toxin abundance, specially the ratio of three-finger toxins to PLA2, contribute to variations in LD50 values and the neutralization efficacy of QSMI antivenom [29]. Collectively, these primary and comparative studies provide essential context for interpreting the present findings and underscore the necessity of evaluating antivenom efficacy, including Thai QSMI-derived antivenom, within a framework specific to venom composition and geographical source. Methodological evaluations of antivenom efficacy have further emphasized that challenge dose selection and experimental design critically influence outcome interpretation and limit direct cross-study comparisons [14]. In parallel, toxin-specific studies targeting individual α-neurotoxins and PLA₂s have demonstrated the biological relevance of these toxin families in mediating lethality and local tissue damage [20,21,23–26]. Together, these studies provide essential context for interpreting the present findings and support the rationale for focusing on α-neurotoxin- and PLA₂-directed antibody formats, while acknowledging the inherent limitations of comparative assessment across different experimental frameworks. Our study extends this work by introducing a bispecific VHH-Fc targeting both α-neurotoxin and phospholipase A2 from N. kaouthia. Although this construct exhibited the highest antigen-binding signal in vitro, its in vivo efficacy was lower than that of the monospecific VHH-αNTX-Fc. This contrasts with the expectation that targeting multiple venom components would yield enhanced neutralization. One possible explanation lies in structural constraints inherent to bispecific formats, such as steric hindrance between domains or reduced conformational stability, as previously discussed.

Importantly, this outcome advances our understanding of antivenom development by demonstrating that high antigen-binding activity in vitro does not always correlate with protective efficacy in vivo, underscoring the need to integrate both binding and functional assays when evaluating antibody performance and to address the structural and mechanistic barriers in the design of next-generation recombinant antivenoms.

To build upon these findings, future research should focus on structural characterization of bispecific constructs to assess folding, stability, and potential domain interference using biophysical and imaging tools such as cryo-EM, SEC-MALS, and thermal stability assays. Second, pharmacokinetic and biodistribution studies to evaluate serum half-life, tissue targeting, and degradation under physiological conditions. Third, functional epitope mapping to determine whether both antigen-binding sites can engage their targets simultaneously and effectively. Fourth, engineering optimized bispecific formats, such as alternative domain orientations or linker designs, to reduce steric hindrance and improve functional synergy. Finally, in vivo efficacy testing should be extended to include not only different geographical variants of N.kaouthia venom but also related snake species containing αNTX and PLA2. Such evaluations would clarify whether the constructs can provide cross-protection beyond a single venom source, thereby demonstrating their generalizablility across venom variants and their robustness against interspecific and intraspecific venom variation.

Supporting information

S1 Table. Determination of the median lethal dose (LD₅₀) of crude Naja kaouthia venom in ICR mice.

https://doi.org/10.1371/journal.pntd.0014119.s001

(DOCX)

S2 Table. Determination of the 100% lethal dose (LD₁₀₀) of crude Naja kaouthia venom based on multiples of the LD₅₀.

https://doi.org/10.1371/journal.pntd.0014119.s002

(DOCX)

S3 Table. Raw survival data used for probit analysis of crude Naja kaouthia venom neutralization in mice.

https://doi.org/10.1371/journal.pntd.0014119.s003

(DOCX)

S4 Table. Probit-based median effective dose (ED₅₀) estimates and model fit for Naja kaouthia venom neutralization assays.

https://doi.org/10.1371/journal.pntd.0014119.s004

(DOCX)

Acknowledgments

The author gratefully acknowledges the support provided by the Department of Biochemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, and the Queen Saovabha Memorial Institute for providing laboratory facilities.

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