Introduction
Immunological studies in swine are often limited by the insufficient availability of highly specific immunological reagents. Among the cytokines that play critical roles in porcine immune regulation, interleukin-17A (IL-17A) and interferon-gamma (IFN-γ) are of major scientific and veterinary importance. These cytokines are deeply involved in inflammatory responses, host defense mechanisms, immune signaling pathways, and disease pathogenesis.
The IL-17 cytokine family consists of six identified members, ranging from IL-17A to IL-17F. Among them, IL-17A is considered one of the most biologically significant due to its strong involvement in inflammatory regulation and immune defense. Excessive or dysregulated IL-17A production has been associated with autoimmune disorders, chronic inflammation, and several forms of cancer. IL-17A stimulates the secretion of pro-inflammatory mediators in multiple cell types and is mainly produced by T helper 17 (Th17) lymphocytes and gamma delta (γδ) T cells, although additional immune populations may also contribute to its production.
Interferon-gamma (IFN-γ), the only member of the type II interferon family, is another essential immune cytokine. It is produced by several immune cell populations including CD4+ Th1 lymphocytes, CD8+ cytotoxic T cells, natural killer (NK) cells, NKT cells, dendritic cells, macrophages, and B lymphocytes. IFN-γ plays a central role in antiviral immunity, intracellular bacterial defense, tumor surveillance, and immune regulation. In pigs, IFN-γ has also been implicated in reproductive biology, particularly in uterine remodeling and embryo implantation processes.
This study focused on the generation and detailed characterization of new monoclonal antibodies (mAbs) directed against porcine IL-17A and porcine IFN-γ. The newly developed antibodies were evaluated for antigen specificity, determinant recognition, cross-species reactivity, intracellular staining performance, and their utility in cytokine detection assays such as sandwich ELISA and flow cytometry.
Production of Anti-Porcine IL-17A and IFN-γ Monoclonal Antibodies
Recombinant porcine IL-17A and IFN-γ proteins were produced using the yeast expression system Pichia pastoris. BALB/c mice were immunized subcutaneously with purified recombinant cytokines at multiple time intervals to induce strong antibody responses. Following confirmation of serum antibody production, mice received an additional intravenous booster injection before hybridoma fusion procedures were performed.
Hybridoma supernatants were initially screened using ELISA assays to identify clones producing antibodies specific for porcine IL-17A or IFN-γ. Clones showing undesired carbohydrate reactivity were excluded to ensure high antigen specificity. Selected hybridomas were expanded for monoclonal antibody purification and additional characterization studies.
A total of:
- 10 anti-porcine IL-17A monoclonal antibodies
- 9 anti-porcine IFN-γ monoclonal antibodies
were selected for further validation and immunological analyses.
ELISA Screening and Antibody Specificity Analysis
The specificity of hybridoma-derived antibodies was assessed using enzyme-linked immunosorbent assay (ELISA). Recombinant cytokines were immobilized onto ELISA plates, followed by incubation with hybridoma supernatants. Antibody binding was detected using enzyme-conjugated secondary antibodies and colorimetric substrate reactions.
Only antibodies demonstrating:
- strong binding to the target cytokine,
- absence of non-specific carbohydrate recognition,
- and low background signal
were retained for advanced characterization.
This screening process ensured the development of highly specific monoclonal antibodies suitable for immunological applications in swine research.
Antigenic Determinant Mapping and Cross-Inhibition Studies
To identify antigenic determinants recognized by each monoclonal antibody, purified antibodies were biotinylated and tested in competitive ELISA assays. Cross-inhibition experiments measured the ability of excess unlabeled antibodies to block the binding of labeled antibodies to recombinant cytokines.
The analyses revealed multiple distinct antigenic determinant groups for both IL-17A and IFN-γ antibodies. Some monoclonal antibodies strongly inhibited each other, indicating recognition of overlapping epitopes, while others recognized independent antigenic regions.
For porcine IL-17A, eight determinant groups (A–H) were identified based on:
- inhibition profiles,
- binding patterns,
- and ortholog recognition.
Similarly, anti-IFN-γ antibodies were classified into several determinant groups according to their competitive binding characteristics.
These findings demonstrated substantial epitope diversity among the developed monoclonal antibodies.
Cross-Species Reactivity of the Monoclonal Antibodies
An important aspect of antibody characterization involved evaluating cross-species recognition using recombinant cytokine orthologs from multiple animal species.
IL-17A Cross-Reactivity
Anti-porcine IL-17A antibodies were tested against recombinant IL-17A proteins from:
- bovine,
- ovine,
- caprine,
- equine,
- feline,
- canine,
- monkey,
- human,
- dolphin,
- rabbit,
- zebrafish,
- guinea pig,
- and mouse species.
Several monoclonal antibodies demonstrated broad mammalian cross-reactivity, particularly with human, bovine, ovine, equine, and dolphin IL-17A proteins. However, no reactivity was detected with canine, murine, or zebrafish orthologs for most antibodies.
Interestingly, some antibodies exhibited extremely selective recognition patterns, while others showed broad interspecies binding capacity.
IFN-γ Cross-Reactivity
Anti-porcine IFN-γ monoclonal antibodies also demonstrated varying levels of cross-species reactivity. Strong recognition was observed for:
- bovine,
- ovine,
- caprine,
- canine,
- dolphin,
- and equine IFN-γ proteins.
Limited or no binding was detected with murine, rabbit, and avian IFN-γ orthologs.
One antibody displayed unexpected recognition of zebrafish IFN-γ despite low sequence similarity, highlighting the complexity of epitope recognition mechanisms.
Sequence Alignment and Structural Correlation Analysis
Protein BLAST and multiple sequence alignment analyses were conducted to investigate whether amino acid similarity correlated with antibody cross-reactivity patterns.
For porcine IL-17A, the highest sequence homology was observed with bovine, ovine, caprine, and dolphin orthologs. However, antibody binding intensity did not always directly correlate with sequence identity percentages, suggesting that three-dimensional epitope structure may be more important than overall sequence similarity.
A similar phenomenon was observed for porcine IFN-γ, where some antibodies strongly recognized distant orthologs despite relatively low amino acid identity.
These observations support the idea that monoclonal antibodies often target conformational epitopes rather than simple linear amino acid sequences.
Development of a Sandwich ELISA for Porcine IL-17A Detection
One major objective of this work was the establishment of a sensitive sandwich ELISA for quantitative measurement of porcine IL-17A.
Several antibody combinations were tested to identify the optimal capture and detection pair. The best performance was achieved using:
- anti-IL-17A-2.6 as capture antibody
- anti-IL-17A-1.1 as detection antibody
This antibody pair demonstrated:
- high specificity,
- low background noise,
- and strong sensitivity.
The assay successfully detected recombinant porcine IL-17A within a concentration range of approximately 100–3000 pg/mL.
The system also effectively quantified native IL-17A secreted by stimulated porcine peripheral blood mononuclear cells (PBMCs), confirming its utility for biological sample analysis.
However, increasing pig serum concentrations generated elevated background signals, indicating that serum content above 20% may reduce assay sensitivity.
PBMC Isolation and Cytokine Production Assays
Peripheral blood mononuclear cells were isolated from porcine blood samples using density gradient centrifugation techniques. PBMCs were cultured under stimulatory conditions using:
- phytohemagglutinin (PHA),
- phorbol myristate acetate (PMA),
- and ionomycin.
These stimulants promoted cytokine secretion and activation of immune cells.
Cell culture supernatants were collected after 24 and 48 hours for ELISA analysis. The results showed significantly increased IL-17A production following stimulation, especially after prolonged incubation periods.
PHA stimulation induced stronger IL-17A secretion compared to PMA/Ionomycin treatment.
Flow Cytometry and Intracellular Cytokine Detection
The newly generated monoclonal antibodies were further evaluated for intracellular cytokine staining using flow cytometry.
Purified antibodies were conjugated with Alexa Fluor 647 and tested on activated porcine PBMCs. Several anti-IL-17A antibodies successfully identified IL-17A-producing CD3+ T lymphocytes following PMA/Ionomycin stimulation.
Among the tested antibodies:
- aPoIL-17A-2.5
- aPoIL-17A-2.6
- aPoIL-17A-2.10
showed the strongest and most reliable staining performance.
These antibodies outperformed certain previously available cross-reactive human IL-17A antibodies, suggesting improved sensitivity for porcine immune monitoring.
In contrast, the anti-IFN-γ antibodies showed limited success in intracellular staining applications, likely due to staining artifacts or epitope accessibility limitations.
Scientific Importance and Applications
The development of these novel monoclonal antibodies provides valuable tools for veterinary immunology and translational biomedical research.
These antibodies can support:
- cytokine quantification assays,
- immune cell phenotyping,
- vaccine evaluation studies,
- infectious disease research,
- inflammation studies,
- and therapeutic antibody development.
Because several antibodies also recognize cytokine orthologs from other species, they may have broader applications in comparative immunology research.
The availability of highly specific anti-porcine IL-17A and IFN-γ reagents is expected to significantly improve the study of immune responses in swine models and facilitate the development of advanced immunodiagnostic technologies.
Conclusion
This study successfully established and characterized new monoclonal antibody panels targeting porcine IL-17A and interferon-gamma. Extensive validation demonstrated strong antigen specificity, diverse epitope recognition, cross-species reactivity, and suitability for ELISA and flow cytometry applications.
The newly developed antibodies represent important additions to the limited pool of porcine immunological reagents currently available. These tools will support future investigations into cytokine biology, host immune responses, vaccine development, inflammatory diseases, and translational biomedical studies involving swine models.