Antigen and Immunogen: An Investigation into the Heterogeneity of Immunology Terminology in Learning Resources

Immunology is a rapidly evolving discipline (1). In addition to fast-paced discoveries in the field, immunological content is chockfull of discipline-specific jargon, which comes with pedagogical challenges. To understand key concepts, instructors and students must master the ever-growing web of technical language, complex acronyms, and abbreviations in immunology texts and articles. Educators strive to teach key concepts so that students maintain motivation to pursue biology and are well-prepared to tackle advanced content in later courses. Regardless of the level or extent of knowledge intended, educational resources (e.g., textbooks and digital learning resources) are critical in delivering course content, influencing what is taught, and serving as vital resources in the classroom (2, 3). Unlike research articles, textbooks present key concepts in a carefully organized format for students (also known as scientists-in-training) and the general public to understand. Key terms are often bolded and included in glossaries so that the niche language of immunology does not impede students from understanding major concepts. In digital resources, the key terms are often hyperlinked directly to a glossary definition. Consistency in the description of the key terms is essential for novice learners so that misconceptions are avoided. Surprisingly, evidence suggests that inconsistencies in definitions and concepts are still encountered in well-vetted learning resources (2, 4–8). In this article, we analyzed the inconsistencies in the definition of two key immunological terms: antigen and immunogen.

Understanding immunology is critical for training the next generation of scientists and medical practitioners (9), but the point at which students first encounter key terms is likely to be outside of a formal immunology course. For example, vaccine science, which has permeated digital resources (e.g., https://www.cdc.gov), news articles, and social media, as it makes its way into public dialogue and policies, is rooted in immunology. Immunology content may also be introduced in middle and high schools (10) or courses for nonmajors in colleges, such as human biology, microbiology for prenursing students, anatomy and physiology, public health, among others (11, 12). Immunology is not offered as a stand-alone course at most 2-y colleges, with the rare exceptions of medical laboratory technologist programs. Formal education in immunology typically happens in a graduate-level course, a one-semester upper-level undergraduate course, or a module within a microbiology or physiology course. For each of these routes, learners can encounter immunological concepts and terminology that differ, depending on the learning resources (e.g., textbooks, digital resources) used and the disciplinary expertise of educators that serve as a foundation for learning.

Traditionally, immunology textbooks have been an important source of in-depth knowledge. In addition, open educational resources (OERs; e.g., OpenStax textbooks and resources available through Howard Hughes Medical Institute’s Biointeractive) available digitally, under an open license or in the public domain that permits no-cost access, use, adaptation, and redistribution with no or limited restrictions (13), are being increasingly adopted by instructors due to equitable access for students and flexibility for the instructor (14). In addition to standard published textbooks and OERs, a new wave of digital educational resources, such as Khan Academy, has left its mark on the educational landscape. When considering concept definitions, the reach of these digital tools cannot be overlooked. For example, Khan Academy serves more than 100 million users per year (15).

With the use of rapid tests for coronavirus disease 2019 (COVID-19) diagnosis, the word antigen has achieved common usage (16), whereas awareness of the term immunogen lags. For instance, a Google trends database search for “antigen” show a stark increase since May 2020, whereas the term “immunogen” remained constant over the past 5 years (Fig. 1). At the time of performing this analysis, Google’s English Dictionary provided by Oxford Languages defined antigen as “a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies.” Definition of the term immunogen is not provided through Google’s English Dictionary but is included in other digital sources. For example, the Merriam-Webster dictionary defines immunogen as “a substance that produces an immune response” and reports that its first use was in 1959 without providing a citation. The use of the word immunogenic can be dated as far back as 1917 (17).

Unlike the term immunogen, the origin of the term antigen has been well documented. Antigen was coined by Ladislav Deutsch, also known as Lásló Detre, a Hungarian researcher, who used the phrase “substances immunogènes ou antigenes” in a French article in 1899 (18). Although Deutsch described an “antigen” as bacterial products that may turn into Abs (18), an Austrian scientist, E.P. Pick, described “antigens” as proteins in 1912 (19), and Mazumdar described “antigen” as any substance to which an animal can make Abs (20). The work of Bordet (21), Ehrlich (22), and many others showed that antigens could be composed of a variety of biomolecules. The historical context and the discoveries that led to its current definition were reviewed by authors such as Jean Lindenmann (23), Pauline Mazumdar (20), and Klaus Eichmann (24). Like several other immunological concepts, the term antigen has evolved as scientists uncovered the chemical nature of the substances that interact with Abs, TCRs, MHC molecules, and pattern recognition receptors (PRRs), leading to a variety of contexts and subdisciplines in which the term applies. The evolution of the definition of antigen reflects the exploratory nature of experimental science based on emerging knowledge from the bench. Nonetheless, we anticipate that contemporary immunologists and educators would agree that a standard definition of antigen is fundamental, not only to understanding immunobiology but also biotechnology, medicine, medical laboratory science, and many other related applications (Fig. 2).

To formalize an approach to undergraduate immunology education, a group of university educators recently undertook the task of initiating a community discussion on key concepts in immunology (25–27). One thing that became evident through these discussions was that disciplinary experts were interpreting the term antigen differently. We hypothesized that this discrepancy may have originated from variability in the authoritative textbooks and learning resource definitions. This prompted us to explore and analyze the definition of the term antigen in varied educational resources. Several of the resources that were a part of our analysis listed antigen as synonymous with immunogen. Therefore, in this analysis, we compare the definitions of the terms antigen and immunogen provided in the glossaries of widely adopted immunology textbooks and other educational resources that contain immunology-related content. Based on our analysis, we recommend a conceptual framework for these terms in the context of undergraduate education, to foster consistency and to reflect the current state of immunological knowledge.

The coauthors of this article were surveyed to determine the most commonly used educational resources (i.e., textbooks and digital resources), and based on their recommendations, 49 educational resources in four categories were reviewed and analyzed for the definitions of antigen and immunogen: (1) immunology textbooks (n = 13; Supplemental Table I); (2) commonly used digital resources and OERs (n = 12; Supplemental Table II); (3) microbiology textbooks (n = 10; Supplemental Table III); and (4) other nonimmunology/microbiology textbooks that include immunology-related topics, such as biochemistry, biotechnology, cell and molecular biology, genetics, general biology, human anatomy and physiology, medical laboratory practice, medical terminology, and zoology (n = 14; Supplemental Table IV).

Within each textbook’s narrative, the terms antigen and immunogen can be used in varying contexts. For example, if the discussion pertains to Ab titers, then an “antigen” may be referred to as a substance that triggers Ab production, and its T cell interactions may not be mentioned at all. In contrast, glossary definitions are intended to be all-encompassing of the varying contexts in which a term could be used. Thus, to ensure consistency, we compared only the glossary definition of the terms antigen and immunogen or immunogenicity (if immunogen was not included) in the 49 chosen educational resources.

Once the definitions were collected, two of the authors (S.L.E. and S.P.) analyzed the definitions to identify broad themes emerging from these definitions, and they developed a codebook (Figs. 3–7). Next, three authors (S.P., R.L.S.-T., and A.L.) independently coded the data, exchanged notes on themes after the first round of coding, and revisited and finalized the codebook. After this consensus discussion, the three coders independently coded the data once again. The relative frequency of each code category was computed using Microsoft Excel (Microsoft Corporation, Redmond, WA), and graphs were plotted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). The p values were computed using a nonparametric statistical test for categorical data with small sample sizes (Fisher’s exact test) using R (version 3.6.1). Cohen’s κ interrater reliability score was computed using an R (version 3.6.1) using the psych package and was found to be in an acceptable range (>0.8–1) for all categories of data (28).

On analysis of the glossary definition of immunogen/immunogenicity, three categories emerged: (1) glossaries that do not mention (code category DNM) the term immunogen; (2) glossaries that mention immunogen as nonsynonymous with antigen (code category NS); and (3) glossaries that mention immunogen as synonymous with antigen (code category S). Code descriptions and specific examples of glossary definitions representing each of the three code categories are depicted in (Fig. 3A. The Cohen’s κ interrater score for coding this dataset was 1.

The analysis showed that 55% of textbooks surveyed did not mention the term immunogen in the glossary (Fig. 3B). Whereas 33% of the textbooks indicated that antigen is not synonymous with immunogen, 12% of textbooks mentioned that antigen is synonymous with immunogen (Fig. 3B). In immunology-specific textbooks, immunogen is predominantly defined as nonsynonymous with antigen (Fig. 3C). In contrast, microbiology textbooks had an approximately equal split between the three code categories (Fig. 3C). Most digital resources did not mention the term immunogen, and none of the “other biology” textbooks mentioned the term immunogen (Fig. 3C).

The glossary definition of antigen in different learning resources was examined next. Because of the large diversity in how antigen was defined in these texts, four different thematic categories were identified: (1) whether the substance binds to immune cell receptors and/or activates the immune system (Fig. 4); (2) whether the substance interacts with BCRs and Abs, both TCRs and BCRs, or a receptor in the immune system with no specific details noted (Fig. 5); (3) the biochemical nature of the substance itself (protein, polysaccharide, lipid, or nucleic acid) (Fig. 6); and (4) whether antigen is defined exclusively as a substance that is foreign or nonself (Fig. 7).

On analysis of the glossary definition of antigen to determine whether antigen was described based on the molecule’s capability to bind to immune cell receptors and/or activate the immune response, six code categories emerged: (1) a molecule that activates (code category A), stimulates, or triggers an immune response; (2) a molecule that is recognized by, reacts with, or binds to an immune cell receptor (code category ReB); (3) a substance that is recognized by, reacts with, or binds to an immune cell receptor and activates/stimulates/triggers an immune response (code category ReB+A); (4) a substance that is recognized by, reacts with, or binds to an immune cell receptor and may activate/stimulate/trigger an immune response (code category ReB+MA); (5) not applicable (code category NA) for when antigen is not described based on the criteria used for this analysis or is not mentioned in the glossary; and (6) does not mention (code category DNM) the term antigen (Fig. 4A). Code descriptions and specific examples of glossary definitions representing each code category are depicted in (Fig. 4A. The Cohen’s κ interrater score for this dataset was 0.84.

Thirty-nine percent of textbooks (Fig. 4B), primarily immunology specific (Fig. 4C), mention antigen as a substance that is recognized by or reacts with or binds to an immune cell receptor (code category ReB) without mentioning anything about its capability to activate an immune response. Thirty-three percent of textbooks (Fig. 4B), mostly microbiology based (Fig. 4C), describe antigen as a substance that activates immune response (code category A). Whereas 18% of textbooks describe antigen as a substance that is recognized by or reacts with or binds to an immune cell receptor and activates the immune response (code category ReB+A), only 6% describe it as a substance that is recognized by or reacts with or binds to an immune cell receptor and may activate the immune response (code category ReBD+MA). Two percent of resources (n = 1 digital resource/OER) define antigen in a way that does not lend itself to the analysis in this category (code category NA) (Fig. 4A–C), and 2% percent of resources (n = 1 microbiology textbook) do not mention (code category DNM) the term antigen in the glossary (Fig. 4A–C).

On analysis of the glossary definition of antigen, based on a molecule’s capability to interact with BCRs, TCRs and BCRs, or immune cell receptors generally, four categories emerged: (1) glossaries that describe antigen as a molecule that interacts with components of the humoral immune system (code category HIS) that includes BCRs and Abs; (2) glossaries that describe antigen as a molecule that activates or binds to adaptive immune system (code category AIS) components (TCRs and BCRs); (3) glossaries that describe antigen as a substance that interacts with immune system (code category IS) components, and no specific arm of the immune system is mentioned; and (4) glossaries that do not mention the term antigen (code category DNM) (Fig. 5A). Code descriptions and specific examples of glossary definitions representing each code category are depicted in (Fig. 5A. The Cohen’s κ interrater score for this dataset was 0.94.

We determined that 43% of textbooks (Fig. 5B), mostly immunology based (Fig. 5C), mention antigen as a substance that interacts with TCRs and BCRs and stimulates an adaptive immune response. Twenty-nine percent of the textbooks do not distinguish between TCRs and BCRs and instead note a target of the immune system in a general sense (Fig. 5B). Twenty-seven percent of resources (Fig. 5B), mostly digital (Fig. 5C), retain the older definition of antigen: a molecule that interacts only with B lymphocytes or only leads to Ab production (20). One microbiology textbook did not mention the definition of antigen in the glossary (Fig. 5B, 5C).

Further analysis of the definition of antigen, based on its biochemical identity, led to classification into four categories: (1) glossaries that describe antigen as any molecule (code category AM), without specific mention of its biochemical composition; (2) glossaries that describe antigen as a protein-only or protein and polysaccharide molecule (code category PM); (3) glossaries that describe antigen as a protein, polysaccharide, nucleic acid, or lipid molecule (code category PNLM); and (4) glossaries that do not mention the term antigen (code category DNM) (Fig. 6A). Code descriptions and specific examples of glossary definitions representing each code category are depicted in (Fig. 6A. The Cohen’s κ interrater score for this dataset was 0.96.

Most textbooks (76%), in all categories, do not mention the biochemical composition and describe antigen as any molecule (code category AM) (Fig. 6B). Antigen was described as a protein or a polysaccharide molecule (code category PM) in 14% of textbooks. Eight percent of textbooks in all but the “other biology” category mention antigen as a molecule that could be a protein, polysaccharide, nucleic acid, or lipid (code category PNLM) (Fig. 6B, 6C). One microbiology textbook did not mention the definition of antigen in the glossary (code category DNM) (Fig. 6B, 6C).

Lastly, the definition of antigen, based on origin, led to three code categories: (1) glossaries that describe antigen as any molecule (code category ANY), (2) glossaries that describe antigen as nonself or foreign (code category NSel), and (3) glossaries that do not mention the term antigen (DNM) (Fig. 7A). Code descriptions and specific examples of glossary definitions representing each code category are depicted in (Fig. 7A. The Cohen’s κ interrater score for this dataset was 1.

Most textbooks (78%) describe antigen as any molecule (code category ANY), leaving it to the instructors’ interpretation of whether it is a self, nonself, or altered self-antigen (Fig. 7B). Twenty percent of textbooks describe antigen as nonself or foreign (Fig. 7B), and 2%; (n = 1 microbiology textbook) do not include the definition of antigen in the glossary (Fig. 7B). The three code categories were represented in all categories of resources (Fig. 7C).

In this study, we explored the degree of discrepancy in definitions across educational resources for the key immunological terms “antigen” and “immunogen,” to find a common language that could be used to converse with both experts and novices. Antigen is a widely used term in textbooks, scientific literature, and other learning resources, although in different contexts. In cellular and molecular biology, the term antigen is used in the context of lymphocyte development, immune cell activation, and immunological memory. In biotechnology, antigen is used in terms of biomolecule purification and detection and for drug delivery. In diagnostics, antigen is used in terms of blood typing, pregnancy tests, rapid antigen tests, and agglutination. Similarly, antigen is used in different contexts in vaccinology, epidemiology, pathogenesis, and immune system–related medical conditions, such as cancer, autoimmune disorders, and hypersensitivity disorders. Therefore, in our previous study (25), it was not surprising that understanding antigen–Ab interactions was rated as a very important skill for students to learn. Although critically important for understanding immunology, the definition of antigen may also vary, depending on the instructor’s background and the textbook being used.

In contemporary times, definitions can easily be obtained with a few keystrokes and a web browser. However, to master the scientific language, students need to define a term and confidently use the term within the correct context (29). Mastering disciplinary vocabulary is a critical component of comprehension (30, 31) and allows one to fluently communicate the context and ideas related to these terms with peers, colleagues, mentors, and the general public. Scientific communication is a core competency for undergraduate life science education (32). Effective science communication skills predict trainees’ intentions to persist in science and are critical for career success in science, technology, engineering, and mathematics (33). Consequently, difficulty in comprehending the discipline-specific language is likely to impede a learner’s ability to establish a sufficient conceptual understanding (34, 35). Due to their abstract nature, the understanding of molecular terms in biology is perceived by students as particularly difficult (36). Multiple, ambiguous definitions for a particular concept can impede understanding by both educators and students alike. If disciplinary experts encounter difficulties in understanding these key terms, then the difficulties for novices are likely to be amplified.

The majority of resources analyzed in this study (55%) did not include the term immunogen in their glossaries. Immunogen was predominantly found in immunology textbooks, to a certain extent in microbiology textbooks and digital resources, but was absent from other biology textbooks. On the contrary, all educational resources, except for a single microbiology textbook (2% of the total), included the term antigen in the glossary, suggesting that, as compared with antigen, immunogen is not a common term in disciplines other than immunology. This mirrors the trends that we observed on Google search trends, demonstrating that “antigen” is a more widely used term than “immunogen.”

The reason for delving into the definition of immunogen in the first place was prompted by definitions that described antigen and immunogen as synonymous (12% of the total), or those that mentioned antigen as a molecule that activates the immune response (51% of the total). Although the term may be used elsewhere in the texts, the absence of immunogen in glossaries is a missed opportunity to add clarity to the definition of antigen. Specifically, immunogenic is an adjective that describes an antigen’s ability to activate immune responses. Most immunology textbooks described antigen as a molecule that is recognized by, reacts with, or binds to an immune cell receptor. However, a surprisingly large number of resources described antigen as a substance that activates the immune system (∼33% of the total) or a substance that binds and activates (∼18% of the total) the immune system. Using these terms synonymously can be a source of grave misconception.

How scientists describe these terms can critically influence the types of hypotheses generated and experimental questions addressed. For example, knowledge of the viral antigenic sites recognized by Abs does not necessarily indicate which immunogenic structure initiates the production of Abs in the immunized host. Failure to differentiate between antigenicity and immunogenicity may lead to failures in developing synthetic peptide vaccines against viral diseases (37–39). How an immune response is measured has been a matter of great debate (40), and a lack of clarity regarding the difference between the two terms can further impede the understanding of advanced concepts associated with immunological cascades, such as haptens, adjuvants, allergens, tolerogens, etc.

Further analysis showed that immunology textbooks define antigen as a molecule that interacts with adaptive immune components, i.e., lymphocyte antigen receptors. When compared with other resources, inconsistencies were evident. A significant proportion of educational resources (∼27%) retain an older definition of antigen as “Ab generating,” which implies that antigens interact only with B lymphocyte antigen receptors. Another significant proportion of resources (∼29%) identify antigen as a molecule that interacts with the immune system generally—and leaves it to the reader’s interpretation of whether this relates to only the lymphocyte antigen receptors or both the lymphocyte antigen receptors and PRRs.

The nuanced distinction between lymphocyte antigen receptor ligands and pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) is critical to driving home other advanced concepts in immunology, such as the extent of specificity associated with innate and adaptive immune responses. These molecular terms are abstract and not intuitive for novices to grasp (36). This distinction is also foundational for understanding how and why the lymphocyte antigen receptors are more diverse in recognizing a myriad of antigens, whereas the diversity among germline-encoded PRRs is limited, yet highly effective.

The distinction between PRR ligands (specifically called PAMPs and DAMPs) and lymphocyte antigen receptor ligands is critical for researchers interested in comparative immunology and evolutionary biology–related questions. The adaptive immune system is a relatively new branch of the immune system and arose ∼500 million years ago in jawed fish (41). Invertebrates lack the typical adaptive immune components. Nonetheless, invertebrates are being increasingly recognized as important model organisms for ecoimmunology-based studies, which have implications for evolutionary medicine, emerging infectious disease, public health, One Health, conservation, and wildlife management (42–46). The varied use of the term antigen is an example of a barrier to interdisciplinary knowledge, communication, and scientific progress.

As discussed earlier, the definitions of antigen and immunogen are nuanced, yet critical for novice learners to understand. In (Fig. 8, we show a step-by-step approach and contextual map that instructors can use to introduce these terms in a classroom (47, 48). This framework, along with our set of recommendations (Table I), should help educators consistently delineate these terms. To initiate this topic in the classroom, instructors may consider describing the origin of the word antigen as “Ab-generating,” which can help to couch the terms in a story format and expose students to how the terms continue to evolve with newer immunological discoveries (20, 23, 24). As a general paradigm, ligands would be considered a physical entity that specifically binds to a cognate receptor. A subset of ligands (called antigens) bind specifically to lymphocyte antigen receptors, and another subset (called PAMPs and DAMPs) bind to the PRRs. This approach accommodates the principle that both innate immune cells and cells of the adaptive immune response use different types of receptor that bind to ligands to discriminate self from nonself, while at the same time specifying that antigens bind specifically to lymphocyte antigen receptors. Further classification, as described in (Fig. 8, would allow sharing the context associated with these terms.

With the knowledge of γδ-T cells, NK cells, intraepithelial lymphocytes, B1 lymphocytes, etc., we have learned that innate and adaptive immune components are on a spectrum and cannot be separated with a rigid line. There is constant communication between innate and adaptive immune components, and as discussed earlier, invertebrates do not exhibit typical adaptive immune components, and yet can serve as excellent models for comparative and ecoimmunological studies. In addition, most immunology textbooks largely focus on the human/mammalian immune system with innate and adaptive immune mechanisms. This is a missed opportunity to consider terminology and concepts from the perspective of an organism that lacks typical elements of adaptive immunity. Educators may consider pointing out these exceptions and gray areas between innate and adaptive immunity with regard to specific cell types and receptors, in the context of comparative immunology. Lastly, pedagogical studies have shown that before introducing new terms, relating the concept to everyday life scenarios improves student articulation of understanding (49). For this, case studies (e.g., a case centered around the rapid antigen test for COVID-19) or problem-based learning can be highly effective (50) and something to which immunology easily lends itself.

Although we chose 49 educational resources from varied disciplines of biology, the list is far from comprehensive. Nonetheless, the list was sufficient to highlight the issue at hand and initiate a community discussion. Also, this study focuses on antigen and immunogen, which are foundational to understanding advanced immunological concepts. We did not investigate the glossary definitions of related advanced terms such as haptens, adjuvants, and allergens. Lastly, the data analyzed in this study were obtained from educational resources. Moreover, this analysis does not address student misconceptions, which will be elucidated in forthcoming studies.

Writing educational materials is an iterative process, and the authors of this article recognize this fully. This article does not intend to criticize particular authors, publishers, or disciplinary experts who might prefer to use the word antigen in various contexts. Antigen is indeed a very common term, as compared with the other terms noted in (Fig. 8. Instead, the analysis brings forth an observation that differences in the way that specific terms are defined can potentially perpetuate misconceptions or reinforce differences across disciplines on the part of instructors and students alike. The educational resources included in this investigation are listed only as examples, to illustrate the point and not as a comprehensive list of resources.

With this study, we hope that educators and educational resource authors will be mindful of the usage of the terms antigen and immunogen and the varied contexts in which each term applies. Lastly, our analysis and recommendations are based on our current understanding of the immunology literature. Because immunology is a rapidly evolving discipline, these terms must be periodically revisited, to establish consensus among scientists and educators, such that clear terminology can be shared with novice learners, newer instructors, and seasoned instructors. We also hope that immunology-focused societies and disciplinary experts will review emerging disciplinary knowledge to update the key terms and disseminate this knowledge to the broader community through periodicals and scholarly literature (Table I).

Dr. Brian D. Wisenden and Dr. Chris Merkord are duly acknowledged for help with statistical analysis–related questions.

The authors have no financial conflicts of interest.