Ethical imperatives, regulatory reforms, and scientific advancements have propelled the development and implementation of non-animal testing strategies. The introduction of the 3Rs principle has guided global efforts to minimize animal use in research and testing. In parallel, the wide array of in vitro and in silico methods has offered more human-relevant, cost-effective, and reproducible alternatives for toxicity assessment (Jin et al., 2020; Huang et al., 2021).

Regulatory agencies, such as the OECD, have also endorsed validated alternative test guidelines for endpoints, including skin sensitization, ocular irritation, and systemic toxicity (Corvaro et al., 2017). In the United States, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has spearheaded the promotion of the regulatory acceptance of assays, such as the Bovine Corneal Opacity and Permeability (BCOP) and Isolated Chicken Eye (ICE) tests, recognized by the FDA, EPA, and CPSC, among other agencies. Legislation, such as the Lautenberg Chemical Safety Act in the U.S. and the REACH regulation in the European Union (EU), has mandated alternative methods where feasible.

More recently, the FDA Modernization Act 2.0 authorized the use of non-animal data for drug safety and efficacy evaluations, reinforcing the shift toward ‘New Approach Methodologies’ (NAMs) (Busquet et al., 2020). The EU has promoted the use of non-animal-derived antibodies due to their reproducibility and ethical advantages (Gray et al., 2020). The COVID-19 pandemic highlighted the limitations of traditional animal models in infectious disease research, accelerating global interest in alternatives. In this context, small organism models, including zebrafish (Danio rerio) and Caenorhabditis elegans, have gained attention (Gö ethel et al., 2022), and advanced techniques, such as microphysiological systems (MPSs), have not been widely adopted.

Despite substantial progress, regulatory integration of alternative methods remains challenging. Differences in national validation standards, lack of internationally harmonized protocols, and limited cross-species translation have hindered broader implementation. Therefore, continued global collaboration and investment in validation and standardization remain essential to achieving widespread regulatory acceptance of non-animal approaches.

While non-animal alternatives continue to evolve, animal models retain a critical role in specific aspects of drug development. Rodents, particularly mice, have made significant contributions to understanding disease mechanisms and identifying therapeutic targets through evolutionarily conserved gene networks and regulatory pathways (Powell et al., 2022; Sullivan et al., 2023). Comparative genomic studies involving primate species have improved the interpretation of human disease variants (Gao et al., 2023). Integration of mouse phenotyping data with machine learning has enhanced the prediction of disease-relevant genes (Alghamdi et al., 2022).

Animal models are indispensable in pharmacokinetics (PK) research for evaluating absorption, distribution, metabolism, and excretion (ADME) properties. Rodent-based PK studies have helped define exposure–efficacy and exposure–toxicity relationships in early-phase development. Advances, such as microsampling, dried blood spot analysis, and physiologically based pharmacokinetic (PBPK) modeling, have enhanced efficiency and reduced animal use (Leblanc et al., 2018; Yuan et al., 2022). For instance, chimeric mice with humanized livers offer a better predictive system for human drug metabolism (Naritomi et al., 2019), and in vivo datasets continue to inform machine learning-based PK prediction models (Obrezanova et al., 2022).

Nonetheless, concerns persist regarding the limited capacity of animal models to replicate the complexity of human diseases. Methodological inconsistencies and interspecies differences frequently hinder effective clinical translation. Models for chronic conditions, such as Alzheimer’s disease or diabetic cardiomyopathy, have been criticized for failing to capture key pathological features, including aging or blood–brain barrier dynamics (Lezoualc’h et al., 2023; Padmanabhan and Götz, 2023). Similar limitations exist in cancer, pulmonary hypertension, and other disease models (Long et al., 2022; Wu X. H. et al., 2022; Sahara and Yanai, 2023), necessitating human-relevant, mechanism-reflective alternatives.

Despite these limitations, animal models remain indispensable at specific stages of drug development. Particularly in late-stage preclinical testing, they are crucial tools for assessing systemic toxicity, immune response, and overall pharmacodynamic behavior under complex physiological conditions. As regulatory authorities often require in vivo validation before clinical entry, animal models function as a transitional bridge between in vitro data and first-in-human studies. Therefore, they are not easily replaceable by current alternatives, highlighting the complementary nature of animal and non-animal methods.

Several well-established in vitro and ex vivo test methods have been internationally recognized as replacements for traditional animal-based assays. For example, BCOP and ICE tests, using excised bovine or chicken eyes to assess corneal opacity and permeability, are OECD-approved alternatives for ocular irritation testing widely applied in regulatory safety evaluations, particularly in cosmetics and ophthalmic formulations. Although reliant on animal-derived tissues, they significantly reduce the need for live animal use and provide reproducible mechanistic data. Their limitations include reduced sensitivity to reversible or mild irritants, necessitating the integration of these tests into tiered testing strategies.

Skin sensitization assessment has advanced with the development of assays targeting key molecular events in the adverse outcome pathway (AOP) and is now supported by methods endorsed in the OECD TG 442 series. Representative examples include the Direct Peptide Reactivity Assay (DPRA), which evaluates the chemical reactivity of test substances through covalent binding to model peptides, KeratinoSens™, which measures transcriptional activation of the antioxidant response element (ARE) pathway in keratinocytes, and the human Cell Line Activation Test (h-CLAT), which assesses immune cell activation via the expression of CD86 and CD54 in dendritic cell-like lines. Though not antibody-based, these methods have contributed to substantial reductions in animal testing and are routinely used in defined approaches for hazard identification.

Receptor binding assays (RBAs), performed to quantify ligand-receptor interactions, are alternatives to in vivo toxicity assays that have replaced mouse bioassays in the detection of marine biotoxins, such as ciguatoxins and paralytic shellfish poisoning toxins (Ruberu et al., 2003; Hardison et al., 2016). Indeed, RBAs are extensively used in pharmacology, particularly in studies on GPCRs and nuclear receptors. While not inherently antibody-dependent, certain RBA platforms utilize antibody capture strategies; nonetheless, limitations such as poor mimicry of dynamic in vivo conditions and susceptibility to artifacts, such as non-specific binding and fluorescence quenching, remain to be addressed (Botana et al., 2010; Hulme and Trevethick, 2010; Breen et al., 2016).

Enzyme-linked immunosorbent assay (ELISA) remains a widely used method for evaluating immune responses and measuring antigen-specific antibody titers, especially in vaccine potency testing for diphtheria and tetanus (Jariyapan et al., 2017; De-Simone et al., 2023). ELISA’s potential for standardization and high-throughput capacity has made it broadly applicable in research and regulatory settings, with enhanced formats (e.g., biotin–streptavidin systems and antigen adsorption), improving sensitivity and reproducibility (Maple et al., 2001). However, ELISA has limitations in directly assessing neutralization efficacy and may show inter-laboratory variability and cross-reactivity (van Hoeven et al., 2008; Hifumi et al., 2014). In toxicology and pharmacology, classical alternatives, including organ-based assays, cell activation models, receptor binding assays, and ELISA, have reduced the reliance on animal testing. Nevertheless, classical methods may fall short in evaluating toxin potency or antitoxin activity with sufficient precision. Thus, advancing specialized, mechanism-based in vitro assays capable of replacing in vivo models in toxin and antitoxin evaluation remains necessary. In the following section, we discuss this need by exploring key toxin categories and emerging antibody-driven strategies for accurate detection and efficacy testing.

Most alternative approaches described herein are primarily utilized during the early stages of drug development, such as discovery, screening, or pilot studies. While these methods are effective in reducing animal use during initial phases, their application in later-stage confirmatory toxicology remains limited. In particular, animal studies are still required in the final stages of preclinical development to evaluate systemic toxicity, recovery from adverse effects, and whole-organism pharmacokinetics, all of which are critical aspects that have yet to be fully replicated by in vitro or in silico methods. Therefore, despite ongoing advancements, animal models remain indispensable for ensuring the safety of drug candidates prior to first-in-human trials.

Toxins produced by various organisms can broadly be classified based on their biological origin, including bacterial, fungal, or animal-derived. Microorganisms in diverse environments produce a wide range of toxins (Rajkovic, 2014; Gupta et al., 2016; Nwaji et al., 2022). Bacterial toxins can be classified into endotoxins, which are released during bacterial lysis, and exotoxins, which are secreted into the external environment (Cavaillon, 2018). Clostridium botulinum, Corynebacterium diphtheriae, and Staphylococcus aureus produce potent exotoxins that severely impact human health (Lindsay and Griffiths, 2013; Cenciarelli et al., 2019; Wenzel et al., 2020). Botulinum neurotoxin (BoNT) from Clostridium species blocks neurotransmitter release, causing botulism and muscle paralysis. Their production and use are strictly regulated in many countries owing to the potential for bioterrorism (Cenciarelli et al., 2019; Janik et al., 2019). Staphylococcal enterotoxins (SEs), particularly SEB, are superantigens that bind to MHC class II molecules on antigen-presenting cells and T cell receptors, resulting in the release of high levels of cytokines and severe inflammation (Lindsay and Griffiths, 2013). Shiga toxin, produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli, comprises two subunits: the A subunit binds to ribosomes, inhibiting protein synthesis and causing cell death, while the B subunit binds to the globotriaosylceramide receptor on the cell membrane to facilitate cellular entry (Gyles, 2007; Melton-Celsa, 2014). Exposure to Shiga toxin can lead to diarrhea, fever, and vomiting, and, in severe cases, hemolytic uremic syndrome (Obrig, 2010; Keir et al., 2012).

Fungi produce mycotoxins, secondary metabolites synthesized by genera such as Aspergillus, Penicillium, and Fusarium (Abraham et al., 2022). These toxins include aflatoxins, ochratoxins, and zearalenone, which are associated with liver and kidney toxicity and, in some cases, carcinogenicity (Reddy et al., 2010; Claeys et al., 2020). Aflatoxin B1, produced by Aspergillus flavus and A. parasiticus, is among the most potent carcinogens commonly found in contaminated grains and nuts (Awuchi et al., 2020). It is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (Wogan et al., 2012). Consequently, many regulatory agencies have established strict detection standards to mitigate potential public health risks (Robens and Cardwell, 2003; Wu and Guclu, 2012). Zootoxins, produced by insects, arachnids, reptiles, and marine organisms, pose significant health risks (Nwaji et al., 2022). These toxins act through various mechanisms, including inflammation, neurotoxicity, coagulopathy, and cytolysis (Guido-Patiño and Plisson, 2022). Common toxin-producing animals include ants, wasps, scorpions, spiders, snakes, algae, and shellfish (Garthwaite, 2000; Kularatne and Senanayake, 2014).

Most antitoxins and antivenoms are polyclonal antibodies derived from the sera of toxin- or venom-immunized animals. While effective, these treatments may induce acute immunogenic responses and lack cross-reactivity to different toxin variants (Dixit et al., 2016). Furthermore, their production and potency testing depend on animal use, further raising ethical and technical challenges. The development of in vitro assays and antibody-based detection platforms to replace animal-based systems for antitoxin production and evaluation has gained traction (Mukherjee et al., 2012; Doke and Dhawale, 2015; Akkermans et al., 2020).

Accurate and sensitive detection of toxins is essential for effective diagnosis, surveillance, and therapeutic evaluation. Although PCR enables the identification of pathogenic bacteria by targeting specific DNA sequences (Persson et al., 2008; Alahi and Mukhopadhyay, 2017), it cannot distinguish whether the detected nucleic acids originate from live or dead organisms, which can lead to false positives. Additionally, factors like low-quality samples or nonspecific primer binding can further compromise accuracy (Fevraleva et al., 2022). Moreover, PCR does not provide direct information on the presence or activity of toxic proteins, thereby limiting its utility in assessing actual toxicity.

Complementary methods have been developed to overcome these challenges. Techniques, such as high-performance liquid chromatography or mass spectrometry, provide high-resolution chemical analysis but require costly equipment, trained personnel, and lengthy procedures (Banerjee and Bhunia, 2010; Hickert et al., 2015; Féraudet Tarisse et al., 2021). In contrast, immunoassays using antibodies provide an efficient and sensitive platform for detecting toxins and evaluating antitoxin efficacy.

Antibody-based immunoassays are relatively simple to perform, require minimal sample preparation, and can be standardized across laboratories. ELISAs, for instance, are highly reproducible and allow high-throughput screening of samples. Miniaturized formats, such as LFAs, enable rapid and on-site testing. Immunoassays can be adapted for use with optical and electrochemical biosensors, providing enhanced sensitivity through signal amplification and device integration (Weng et al., 2016; Li et al., 2021). Hence, antibody-based immunoassays are considered suitable alternatives to animal-based assays for detecting toxins and assessing the neutralizing efficacy of antitoxins. Regulatory authorities, including the US Food and Drug Administration (FDA), European Centre for Disease Prevention and Control (ECDC), and World Health Organization (WHO), have incorporated immunoassays into their approved detection frameworks. For example, the U.S. has approved PCR-based detection of the Shiga toxin genes (Stx1 and Stx2) and commercial ELISA kits for detecting Shiga toxin-producing E. coli (STEC) (Gould et al., 2022; He et al., 2013). Meanwhile, the ECDC permits the use of cultures, PCR, and STEC-specific ELISA for confirmatory diagnostics. Similarly, Clostridioides difficile can be diagnosed using PCR and ELISA following fecal culture (McDonald et al., 2018).

Although significant progress has been made, toxins such as BoNTs are routinely tested using animal models, with the mouse bioassay considered the benchmark for BoNT detection (Hobbs et al., 2019). Similarly, evaluating the antivenom potency of venom toxins, particularly those derived from snakes, relies heavily on in vivo lethality testing due to their complex and variable composition. These assays necessitate a large number of animals and present challenges related to reproducibility and mechanistic insights. Consequently, continued efforts are needed to develop and validate antibody-based in vitro alternatives that offer equivalent or superior sensitivity, reproducibility, and functional relevance for bacterial- and animal-derived toxins.

Since many toxins exist in multiple isomeric forms, detection antibodies must recognize all clinically or environmentally relevant forms to avoid underestimating toxin levels and misinterpreting associated risks. Accordingly, selecting and validating antibodies requires comprehensive cross-reactivity testing with all relevant toxin isomers to ensure accurate measurement.

Many natural toxins can cause significant biological damage even at extremely low concentrations, necessitating the development of highly sensitive detection systems. Among the available technologies, antibody-based assays, such as ELISA, are widely used to identify and quantify bacterial and venom toxins. These assays offer a rapid and specific means of detection, often replacing or complementing traditional culture methods or in vivo bioassays (Peng et al., 2022). Table 1 summarizes representative antibody-based detection assays and their applications in toxin titer measurement.

Antitoxin potency for bacterial diseases, such as botulism, tetanus, and diphtheria, has traditionally been assessed using mouse lethality assays. Animal-free alternatives, including ELISA-based platforms and cell-based functional assays, have been developed as ethically compliant and mechanistically relevant options (Bak et al., 2017; Torgeman et al., 2017). For instance, neuronal cell lines are utilized to evaluate botulinum antitoxins by quantifying cleaved SNAP-25, a BoNT/A-specific intracellular target. This method demonstrates strong reproducibility and alignment with in vivo data, highlighting its suitability for replacing animal tests in potency assessment. Similarly, antitoxin potency for tetanus and diphtheria has historically been evaluated in toxin-challenged animal models. However, ongoing efforts aim to replace such assays with in vitro methods, including immunoassays targeting specific epitopes or functional domains of the toxins (Manghi et al., 1994; Zhuang et al., 2021; Chen et al., 2022). These strategies result in reduced variability, decreased costs, and greater ethical acceptability.

Animal models have traditionally been utilized to determine the neutralization efficacy of antivenoms. However, these methods present substantial ethical and logistical challenges, including the substantial number of animals required and the variability of venom composition across species and regions (Pucca et al., 2019). To address these concerns, ELISA platforms have been developed to evaluate the binding and neutralization potential of antivenoms using purified venom components. One such system employs β-bungarotoxin from B. multicinctus as the antigen to test 55 equine plasma samples with established in vivo efficacy, yielding 97% sensitivity and 93% specificity even in the presence of competing toxic PLA2 enzymes (Liu et al., 2024). These results indicate that ELISA-based approaches can serve as effective surrogates for animal testing in antivenom evaluation.

In addition to ELISA, alternative in vitro methodologies—including enzymatic activity assays and PPI platforms—are being investigated to capture broader aspects of venom neutralization (Gutiérrez et al., 2017; Liu et al., 2024). The complex composition of snake venoms, featuring diverse proteins and enzymes, such as phospholipase A2 (PLA2), snake venom metalloproteinases (SVMPs), and matrix metalloproteinases (MMPs), presents significant challenges for developing standardized universal in vitro assays (Warrell, 2010; Calvete, 2011). Consequently, single-antigen assays often fail to capture the full spectrum of toxic activities. To achieve an accurate assessment of antivenom efficacy, an integrated approach employing cell-based models, receptor-binding studies, and multiplexed antigen panels may be required.

An emerging promising strategy involves the use of multivalent or multispecific antibodies capable of simultaneously targeting multiple toxin epitopes or components. This methodology has been shown to increase therapeutic efficacy in both in vitro and in vivo models. For instance, a bispecific antibody targeting two distinct pertussis toxin epitopes showed more than a two-fold increase in neutralizing activity compared to its monospecific counterparts (Wagner et al., 2016). Similarly, a bispecific antibody that binds to the surface exopolysaccharide Psl and the PcrV proteins of the type III secretion system sis currently being evaluated in clinical trials for Pseudomonas aeruginosa (DiGiandomenico et al., 2014). These developments suggest that advances in antibody engineering—particularly the development of bispecific or multispecific formats—are likely to improve the accuracy and standardization of animal-free platforms for assessing antitoxins and antivenoms.

CIVMs, including organoids and MPSs, enable the assessment of human-specific physiological responses under controlled experimental conditions. These models replicate certain features of tissue architecture, perfusion, and immune interactions that are often lacking in conventional 2D cell cultures. When coupled with antibodies that bind to specific toxins for quantification, CIVMs serve as an effective platform for toxin detection, mechanistic studies, and neutralization screening.

Lung- and liver-on-a-chip models can be used to evaluate drug-induced toxicity and immunological responses, while brain-on-a-chip systems are increasingly utilized to model neurotoxicity and infectious challenges (Baran et al., 2022). These systems facilitate precise kinetic measurements and multiplex immune or signaling pathway readouts, which are valuable for assessing toxin-antibody interactions in biologically relevant contexts without relying on animals.

Snake venoms pose unique challenges due to their complex composition and limited genetic characterization. Most venom-encoding genes and toxin-producing cell types remain poorly understood, which has limited progress in recombinant antivenom production and non-animal test development. To address this issue, a high-quality reference genome for the Indian cobra has been published, identifying 139 toxin genes across 33 families (Suryamohan et al., 2020). This genomic resource enables the design of recombinant antigens and antibody targets.

Additionally, venom gland organoids derived from adult stem cells can secrete functionally active toxins in vitro (Post et al., 2020; Alafeef et al., 2021). These long-term expandable cultures offer a human-relevant, scalable alternative to venom extraction from live animals, serving as a novel tool for studying venom biology and testing neutralizing antibodies under controlled laboratory conditions.

Advances in AI and in silico modeling now allow rapid simulation of toxicological effects, antibody optimization, and reduced reliance on animal testing. Leveraging extensive biological datasets and machine learning algorithms, these methods can predict toxicity, immune responses, and antigen–antibody interactions. AI systems integrated with in vitro platforms can rapidly screen potential antibody candidates and rank their binding affinities or neutralization potential. For instance, patient-on-a-chip systems combined with AI algorithms have been used to profile drug responses and develop predictive models of immunotoxicity (Barrett et al., 2023; Chopra et al., 2023). In venom research, these tools may enable virtual screening of antibody libraries against toxin epitope databases derived from genomic and proteomic studies. As these computational pipelines mature, they promise a scalable, cost-efficient framework for advancing antibody-based alternatives to animal testing.

Antibodies enable the precise detection and neutralization of a wide range of toxins due to their high specificity and selectivity. Diagnostic immunoassays, such as ELISA, are used to identify bacterial toxins (e.g., C. botulinum, Clostridium difficile, S. aureus, and C. diphtheriae), fungal toxins (e.g., aflatoxins), and animal-derived venoms (Reverté et al., 2014; Zhu et al., 2014; Pöhlmann and Elßner, 2020).

Therapeutic-neutralizing antibodies are being developed against toxins such as botulinum toxin, diphtheria toxin, and snake venoms, given their high lethality and bioterrorism risks (Cenciarelli et al., 2019; Rasetti-Escargueil and Popoff, 2019). Accurate detection and timely neutralization with these therapeutic antibodies are crucial for public health preparedness and treatment. Antibody-based methods now enable animal-free assays for potency and toxicity, including ELISA-based systems, skin sensitization tests, and SNAP-25 cleavage-based assays for botulinum toxin (Nepal and Jeong, 2020; Gądarowska et al., 2022). While immunoassays offer faster, simpler, and more cost-effective alternatives to mouse lethality assays, they often lack the ability to measure the biological activity or toxic effects of functional proteins (Koh et al., 2015; El-Sayed et al., 2022).

High-quality antibodies optimized for quantification and assessment of biological activity enable broader application of antibody-based alternatives. Key considerations include selecting antigen regions critical for biological function, designing recombinant or peptide antigens that maintain immunogenicity, and developing detection formats suitable for specific applications (Dormitzer et al., 2008; Caradonna and Schmidt, 2021; Guarra and Colombo, 2023). Increasing attention is focused on animal-free antibody discovery technologies due to ethical considerations. These alternatives to traditional animal immunization comprise phage, yeast, ribosome, and microbial display platforms (Jones et al., 2016; Zhang, 2023), direct B cell isolation from peripheral blood (Pedrioli and Oxenius, 2021), and epitope-directed selection methods (Chen et al., 2020; Zhu et al., 2023). Developments in AI have enabled in silico antibody discovery, design, and optimization through the integration of protein structure prediction and de novo sequence generation (Jeong et al., 2022; Hie et al., 2024).

High-throughput, label-free platforms, such as BLI, SPR, flow cytometry, lab-on-a-chip, and PPI assays, enable precise and reproducible antibody characterization, surpassing traditional animal-based methods (Picollet-D’hahan et al., 2021; Chun et al., 2024; Vacca et al., 2024). Standardizing these technologies may accelerate the transition to animal-free testing paradigms that meet scientifically robust and regulatory standards.

To gain regulatory and industrial acceptance, antibody-based alternatives to animal testing require methodological standardization and rigorous validation. Although immunoassays with antibodies are widely used for assessing drug and vaccine quality, they must undergo rigorous comparative studies with traditional animal-based protocols (Nepal and Jeong, 2020; Bas et al., 2021). Despite their high specificity and sensitivity, immunoassays often fail to fully capture toxin functions or the in vivo neutralization capacity of antitoxins. Therefore, complementary cell-based or biochemical methods are needed to better reflect in vivo efficacy (Guideline, 2005; Koh et al., 2015). Ultimately, new approaches must demonstrate comparable or superior accuracy, reproducibility, and biological relevance to qualify as a true alternative to animal testing (Griesinger et al., 2016).

A central requirement during standardization is alignment with international guidelines, such as those established by the OECD, ICH, or WHO. These frameworks articulate best practices for validation and outline criteria for assay acceptance, including robustness, transferability, and inter-laboratory reproducibility (Kandárová and Letašiová, 2011; GIVIMP, 2018). Peer-reviewed validation and assessment by recognized accreditation bodies are critical in legitimizing novel methodologies intended for regulatory implementation (Bruner et al., 1996; Hartung, 2007). The key stages of standardization for antibody-based alternatives, along with corresponding guidelines and authorities, are summarized in Table 2. Furthermore, institutional and economic barriers require careful consideration. The pharmaceutical sector has traditionally depended on established animal testing paradigms due to their historical regulatory precedence, cost-efficiency, and familiarity. Consequently, even scientifically valid alternatives may face resistance unless they demonstrate scalability, cost-effectiveneess, and ease of use (Doke and Dhawale, 2015; Kanďárová and Pôbiš, 2024).

Antibody-based assay development should prioritize scientific rigor, practical implementation, and regulatory compatibility. By creating methods that satisfy market requirements and international validation standards, researchers can accelerate the adoption of animal replacement strategies in public health and commercial domains (Bas et al., 2021; Kahrass et al., 2024).

In toxin-related research, antibodies are commonly employed for diagnostic and therapeutic purposes. In diagnostics, immunoassays with specific antibodies are used to identify harmful agents, such as food allergens, bacterial exotoxins, and fungal toxins (Janik et al., 2019; Féraudet Tarisse et al., 2021; Avril et al., 2022). For example, ELISA kits containing targeted antibodies are frequently utilized to monitor aflatoxins, BoNTs, and SEs in food and clinical samples (Zhu et al., 2014; Janik et al., 2019).

Neutralizing antitoxins are included in the WHO Model List of Essential Medicines for treating potentially lethal intoxications, such as botulism, diphtheria, and tetanus. However, conventional antitoxin therapies, often derived from animal plasma, carry a significant risk of adverse effects, including serum sickness, hypersensitivity reactions, and nephritis (Mayers et al., 2003; Pirazzini and Rossetto, 2017). The development of monoclonal antibodies as therapeutics offers a more targeted and safer alternative. Examples include raxibacumab (approved for anthrax) and bezlotoxumab (approved for Clostridioides difficile infection), both approved by the US FDA and representing milestones in antibody-based antitoxin therapies (Tsai and Morris, 2015; Kufel et al., 2017). Beyond these examples, a broader range of antibody-based therapeutics for toxin neutralization at different development stages is summarized in Table 3.

In the context of animal replacement, antibodies serve as tools for detecting toxins and are pivotal in developing non-animal-based potency assays for antitoxins. For example, neutralizing antibodies specific to BoNTs have been applied in ELISA-based assays to replace mouse lethality tests, demonstrating accuracy and reproducibility (Nepal and Jeong, 2020; Gdoura et al., 2022). Therapeutic antibodies targeting snake venoms are being investigated to substitute conventional equine-derived antivenoms, with promising efficacy and reduced immunogenicity (Pucca et al., 2019).

Although therapeutic antibody production has traditionally relied on animal immunization, ethical considerations have prompted the adoption of non-animal alternatives, such as phage display, yeast and ribosome display, and single B cell cloning from human peripheral blood mononuclear cells (Jones et al., 2016; Uchański et al., 2019; Pedrioli and Oxenius, 2021; Zhang, 2023; Porebski et al., 2024). The use of AI-assisted in silico antibody design and structural optimization is demonstrating potential to accelerate development while reducing reliance on animal models (Watson et al., 2023; Hie et al., 2024). Additionally, antibody-based analyses can be performed using high-throughput technologies such as SPR, BLI, flow cytometry, and lab-on-a-chip systems. These platforms facilitate rapid, quantitative, and reproducible data generation, contributing to the standardization of assays and minimizing operator-dependent variability (Picollet-D’hahan et al., 2021; Chun et al., 2024). As these tools become increasingly integrated with MPSs and machine learning algorithms, the capacity to functionally evaluate toxin activity and antitoxin efficacy without the need for animal testing will be substantially enhanced.

Antibodies are utilized as analytical and therapeutic tools in toxin research and are foundational in the development of animal-free testing strategies. Incorporating antibodies into high-precision immunoassays and computational workflows may influence approaches to toxin detection, characterization, and neutralization in preclinical research and public health preparedness.