The Production of Antivenom and Implementing the 3Rs

By Mariah Bowman | February 26th, 2026

The Guide for the Care and Use of Laboratory Animals (the Guide) provides well-accepted definitions of replacement, reduction, and refinement (the 3Rs), which is a nearly universal approach applied “when deciding to use animals in research and in designing humane animal research studies.” Each has goals related to the care of animals and the procedures performed on them; however, the Guide explains that these ethical considerations “should serve as a starting point,” and researchers “are encouraged to go beyond these provisions.” Replacement “refers to methods that avoid using animals” or the complete replacement of animals; reduction “refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate pain and distress;” and, refinement “involves strategies for obtaining comparable levels of information from the use of fewer animals or for maximizing the information obtained from a given number of animals (without increasing pain or distress) so that in the long run fewer animals are needed to acquire the same scientific information.”

Despite these directives from the Guide, the production of antivenom has remained fundamentally unchanged since its inception. Lack of innovation means that the 3Rs are not being fully considered. Therefore, this process needs to be advanced, which will then produce better results and also consider the animals used in its production. The process begins with venom collection. Venomous snakes are milked for their venom by forcing them to bite into a mesh-covered vial. Pressure is then applied to the snake’s venom glands to extract as much venom as possible. The snakes are meticulously selected; accordingly, most antivenom producers house their own snakes for venom milking and use species native to their area.

The collected venom, which may be a cocktail of several different species, is then diluted so it can be injected into animals, usually horses or sheep. Monovalent antivenoms are created with venom from a single snake species, whereas polyvalent antivenoms use the venom of several snake species. When a venomous snake bites someone, it may be difficult to identify the species involved. In these cases, polyvalent antivenoms are most helpful when a healthcare provider is aware of the different species native to the area but does not know the exact species that caused the bite.

Horses are known to produce large quantities of antibodies when injected with small doses of venom. These antibodies are part of the animal’s immune response and are tailored to the venom or venoms that are introduced. Several blood samples are collected and separated into plasma, erythrocytes, and platelets. The erythrocytes, or red blood cells, are then returned to the animal. The plasma is the key component of this procedure because it is where antibodies are found. After removing impurities and processing the antibodies, the final product (the antivenom) is then tested, typically injecting mice with lethal doses of venoms mixed with the processed antibodies.

However, this process has clear drawbacks due to its use of non-human animals. Although plasma is separated from the other cells, trace amounts of animal-derived erythrocytes may remain, potentially causing life-threatening allergic reactions in people injected with the final product. To combat this issue, monoclonal antibodies should be considered as an alternative because they are more compatible with the human immune system. Fully human monoclonal antibodies are proteins produced in a lab that function like human antibodies and can be replicated much faster than the months required for antibody production in horses. Refining this approach may alleviate any adverse effects caused by allergies and conform to the 3Rs since animals will be replaced with other technologies.

Despite this alternative approach to antibody production, the final product will likely be tested on mice. Since monoclonal antibodies are more compatible with humans, testing on mice may enhance the venom’s toxicity. This is called antibody-dependent enhancement of toxicity (ADET). There is not a clear reason as to why this is occurring, but the researchers who have identified this issue explain there are limitations of preincubation assays, “which are currently used as the gold standard for assessing the preclinical efficacy of toxin-neutralizing agents in the field of antivenom research.” Consequently, this reaction would likely call for multiple tests due to the increased chances of inaccurate results, which would lead to the death of more mice. Preclinical alternatives are not only needed to improve the efficacy of antivenom, but also to replace the use of mice and other animals in testing. The last part of this blog will focus on two alternatives that conform to the 3Rs: toxicovenomics and antivenomics. Employing these approaches has the potential to advance antivenom development in several important ways.

Venomics uses the genetic material of venom to create a comprehensive map of genes and proteins, after which bioassays can be utilized to examine the biological effects of venoms. Together, this approach is known as toxicovenomics, which occurs before the process of making antivenom. The same venomous species can vary in the types of toxins it produces, usually depending on environmental factors or diet. This is why toxicovenomics is important – identifying the type of venom and the proteins involved helps produce antivenoms that are ethical, consistent with the 3Rs, and potentially more effective. Lethality tests with mice are replaced with proteomic analyses, enzymatic activity assays, and cell-based toxicity tests. Additionally, immunization mixtures are refined to include only medically relevant toxins, reducing stress and the number of procedures. Consequently, the venom component is perfected before the antivenom process begins.

The key difference between toxicovenomics and antivenomics is that the former focuses on the impact of toxins rather than on their composition or response to antivenom. Improving and utilizing antivenomics will help expand the range of species that a single antivenom can treat. Antivenomics is the process of investigating venom proteins that elicit immune responses, as well as venom proteins that do not elicit these responses. In this technique, snake venom is incubated with antivenom, the toxins are diluted, and additional antibodies are introduced to trigger the less-potent venoms. This helps produce an antivenom that targets all toxins within a venom, not just the most potent ones, and reduces the need for procedures done on animals. Venom proteins and antibody binding can then be measured in a laboratory, thereby replacing lethality tests traditionally conducted on mice. Therefore, antivenomics is an important 3Rs advancement because it helps reduce the number of animals used, both for testing antivenoms and for antibody production.

There is a substantial need to replace and refine the process of testing antivenoms, as well as a need to reduce and refine the production of antibodies used to create antivenoms. Advancing the science of toxicovenomics and antivenomics, and supporting adoption of better science and technology, is a positive move in the right direction and represents important steps toward implementing the 3Rs.

The views expressed do not necessarily reflect the official policy or position of Johns Hopkins University or Johns Hopkins Bloomberg School of Public Health.