Development and validation of a sandwich ELISA for equine IL-1β

Abstract

In this study, we developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) using newly produced monoclonal antibodies (mAbs) for detecting horse/donkey IL-1β in cell culture medium and serum samples. The mAbs were generated via the use of a KLH-conjugated peptide and purified equine IL-1β protein as separate immunogens. Notably, the generated mAbs (3G8 and 5G3) demonstrated no cross-reactivity with other major inflammatory mediators, including IL-1α, IL-1Ra, TNF-α, and SAA. The IL-1β assay, which is based on the screened mAbs, exhibits a detection range of 200–10,000 pg/mL, meeting clinical detection requirements. The coefficients of variation for the repeatability and reproducibility of the assay were both less than 5%, indicating an acceptable level of variation. Subsequently, 84 equine and 24 asinine serum samples were collected, and the IL-1β concentration was measured with both our assay and a commercial kit in parallel. Our results revealed no significant difference between the in-house and commercial ELISA kits for the detection of IL-1β concentrations in horse sera. Moreover, our ELISA method demonstrated superior sensitivity for IL-1β detection in donkey samples compared to existing commercial assays. These findings suggest that the newly developed ELISA provides a reliable analytical method for detecting IL-1β in both equine and asinine samples.

Keywords

IL-1β / Antigen-capture ELISA (acELISA) / Horse / Donkey

References

[1] Abd El-GhaniSE, HamedRMR, EidRA, IbrahimAYM, Abdel-HamidHM, AbdelrahmanW, IbrahimRE, Abdel-AzizMM, MohamedMS. Serum interleukin 1β and sP-selectin as biomarkers of inflammation and thrombosis, could they be predictors of disease severity in COVID 19 Egyptian patients? (a cross-sectional study). Thrombosis Journal, 2022, 20(1): 77

[2] Afify A F, R T Hassanien, R F El Naggar, M A Rohaim, M Munir. 2024. Unmasking the ongoing challenge of equid herpesvirus- 1 (EHV-1): A comprehensive review. Microb Pathog 193106755. https://doi.org/10.1016/j.micpath.2024.106755

[3] Aggeletopoulou I, M Kalafateli, E P Tsounis, C Triantos. 2024. Exploring the role of IL-1β in inflammatory bowel disease pathogenesis. Front Med (Lausanne) 111307394. https://doi.org/10.3389/fmed.2024.1307394

[4] Alfadul H, S Sabico, N M Al-Daghri. 2022. The role of interleukin-1β in type 2 diabetes mellitus: A systematic review and meta-analysis. Front Endocrinol (Lausanne) 13901616. https://doi.org/10.3389/fendo.2022.901616

[5] AllanSM, TyrrellPJ, RothwellNJ. Interleukin-1 and neuronal injury. Nature Reviews Immunology, 2005, 5(8): 629-640

[6] AmaralGA, AlvesJD, Honorio-FrançaAC, FagundesDL, AraujoGG, LobatoNS, LimaVV, GiachiniFR. Interleukin 1-beta is linked to chronic low-grade inflammation and cardiovascular risk factors in overweight adolescents. Endocrine, Metabolic & Immune Disorders: Drug Targets, 2020, 20(6): 887-894

[7] BeaumontRE, SmithEJ, ZhouL, MarrN, ThorpeCT, GuestDJ. Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling. Molecular and Cellular Biochemistry, 2024, 479(5): 1059-1078

[8] Cano-Cano F, L Gómez-Jaramillo, P Ramos-García, A I Arroba, M Aguilar-Diosdado. 2022. IL-1β implications in type 1 diabetes mellitus progression: systematic review and meta-analysis. J Clin Med 11(5). https://doi.org/10.3390/jcm11051303

[9] ChenYT, LohiaGK, ChenS, LiuZ, Wong Fok LungT, WangC, RiquelmeSA. A host- metabolic synchrony that facilitates disease tolerance. Nature Communications, 2025, 16(1): 3729

[10] Chen Y C, Y S Fu, S W Tsai, P K Wu, C M Chen, W M Chen, C F Chen. 2022. IL-1b in the secretomes of MSCs seeded on human decellularized allogeneic bone promotes angiogenesis. Int J Mol Sci 23(23). https://doi.org/10.3390/ijms232315301

[11] Cheng R, Z Wu, M Li, M Shao, T Hu. 2020. Interleukin-1β is a potential therapeutic target for periodontitis: a narrative review. International Journal of Oral Science 12(1). https://doi.org/10.1038/s41368-019-0068-8

[12] ChoiJ, KimSY, KimH, LimBC, HwangH, ChaeJH, KimKJ, OhS, KimEY, ShinJS. Serum α-synuclein and IL-1β are increased and correlated with measures of disease severity in children with epilepsy: Potential prognostic biomarkers?. BMC Neurology, 2020, 20(1): 85

[13] ChurchLD, CookGP, McDermottMF. Primer: Inflammasomes and interleukin 1β in inflammatory disorders. Nature Clinical Practice Rheumatology, 2008, 4(1): 34-42

[14] CummingsKJ, PerkinsGA, KhatibzadehSM, WarnickLD, ApreaVA, AltierC. Antimicrobial resistance trends among Salmonella isolates obtained from horses in the northeastern United States (2001–2013). American Journal of Veterinary Research, 2016, 77(5): 505-513

[15] Dharra R, A Kumar Sharma, S Datta. 2023. Emerging aspects of cytokine storm in COVID-19: The role of proinflammatory cytokines and therapeutic prospects. Cytokine 169156287. https://doi.org/10.1016/j.cyto.2023.156287

[16] DinarelloCA. Blocking interleukin-1β in acute and chronic autoinflammatory diseases. Journal of Internal Medicine, 2011, 269(1): 16-28

[17] DinarelloCA. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunological Reviews, 2018, 281(1): 8-27

[18] DinarelloCA. The IL-1 family of cytokines and receptors in rheumatic diseases. Nature Reviews Rheumatology, 2019, 15(10): 612-632

[19] Dosh R H, N Jordan-Mahy, C Sammon, C Le Maitre. 2019. Interleukin 1 is a key driver of inflammatory bowel disease-demonstration in a murine IL-1Ra knockout model. Oncotarget 10(37): 3559–3575. https://doi.org/10.18632/oncotarget.26894

[20] Dunowska M. 2014. A review of equid herpesvirus 1 for the veterinary practitioner. Part B: pathogenesis and epidemiology. N Z Vet J 62(4): 179–188. https://doi.org/10.1080/00480169.2014.899946

[21] FokET, MoorlagS, NegishiY, GrohLA, Dos SantosJC, GräweC, MongeVV, CraenmehrDDD, van RoosmalenM, da Cunha JolvinoDP, MiglioriniLB, NetoAS, SeverinoP, VermeulenM, JoostenLAB, NeteaMG, FanucchiS, MhlangaMM. A chromatin-regulated biphasic circuit coordinates IL-1β-mediated inflammation. Nature Genetics, 2024, 56(1): 85-99

[22] HarrisonP, PointonJJ, ChapmanK, RoddamA, WordsworthBP. Interleukin-1 promoter region polymorphism role in rheumatoid arthritis: A meta-analysis of IL-1B-511A/G variant reveals association with rheumatoid arthritis. Rheumatology (Oxford), 2008, 47(12): 1768-1770

[23] Hernandez-SantanaYE, GiannoudakiE, LeonG, LucittMB, WalshPT. Current perspectives on the interleukin-1 family as targets for inflammatory disease. European Journal of Immunology, 2019, 49(9): 1306-1320

[24] HinzeCH, FoellD, KesselC. Treatment of systemic juvenile idiopathic arthritis. Nature Reviews Rheumatology, 2023, 19(12): 778-789

[25] IslamuddinM, MustfaSA, UllahS, OmerU, KatoK, ParveenS. Innate Immune Response and Inflammasome Activation During SARS-CoV-2 Infection. Inflammation, 2022, 45(5): 1849-1863

[26] JohnsonKE, ChikotiL, ChandranB. Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and the NLRP3 inflammasome. Journal of Virology, 2013, 87(9): 5005-5018

[27] Katsanos A H, K Kyriakidi, F B Karassa, D Politis, A Skamnelos, D K Christodoulou, K H Katsanos. (2017). Biomarker development in chronic inflammatory diseases. In Biomarkers for Endometriosis (pp. 41–75). https://doi.org/10.1007/978-3-319-59856-7_3

[28] KhannaD, KhannaS, KhannaP, KaharP, PatelBM. Obesity: A Chronic Low-Grade Inflammation and Its Markers. Cureus, 2022, 14(2) e22711

[29] Kim S, M Park, A E Leon, J S Adelman, D M Hawley, R A Dalloul. 2017. Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA system. BMC Veterinary Research 13(1). https://doi.org/10.1186/s12917-017-1199-9

[30] KöhlerG, MilsteinC. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256 5517): 495-497

[31] Laval K, L W Enquist. 2021. The potential role of herpes simplex virus type 1 and neuroinflammation in the pathogenesis of Alzheimer's disease. Front Neurol 12658695. https://doi.org/10.3389/fneur.2021.658695

[32] LiY, QiangR, CaoZ, WuQ, WangJ, LyuW. NLRP3 Inflammasomes: Dual Function in Infectious Diseases. The Journal of Immunology, 2024, 213(4): 407-417

[33] LimWS, EdwardsJF, BoydNK, PayneSL, BallJM. Simultaneous quantitation of equine cytokine mRNAs using a multiprobe ribonuclease protection assay. Veterinary Immunology and Immunopathology, 2003, 91(1): 45-51

[34] LinYZ, CaoXZ, LiL, LiL, JiangCG, WangXF, MaJ, ZhouJH. The pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages. Virus Research, 2011, 160(1–2): 274-282

[35] Liu Q, J Ma, X F Wang, F Xiao, L J Li, J E Zhang, Y Z Lin, C Du, X J He, X Wang, J H Zhou. 2016. Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response. Vet Immunol Immunopathol 17030–40. https://doi.org/10.1016/j.vetimm.2016.01.006

[36] MakaremiS, AsgarzadehA, KianfarH, MohammadniaA, AsghariazarV, SafarzadehE. The role of IL-1 family of cytokines and receptors in pathogenesis of COVID-19. Inflammation Research, 2022, 71(7–8): 923-947

[37] ManganMSJ, OlhavaEJ, RoushWR, SeidelHM, GlickGD, LatzE. Targeting the NLRP3 inflammasome in inflammatory diseases. Nature Reviews. Drug Discovery, 2018, 17(8): 588-606

[38] MantovaniA, DinarelloCA, MolgoraM, GarlandaC. Interleukin-1 and Related Cytokines in the Regulation of Inflammation and Immunity. Immunity, 2019, 50(4): 778-795

[39] MariathasanS, WeissDS, NewtonK, McBrideJ, O'RourkeK, Roose-GirmaM, LeeWP, WeinrauchY, MonackDM, DixitVM. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature, 2006, 440(7081): 228-232

[40] Martinon F, A Mayor, J Tschopp. 2009. The inflammasomes: guardians of the body. Annu Rev Immunol 27229–265. https://doi.org/10.1146/annurev.immunol.021908.132715

[41] MateraMG, CalzettaL, PeliA, ScagliariniA, MateraC, CazzolaM. Immune sensitization of equine bronchus: Glutathione, IL-1beta expression and tissue responsiveness. Respiratory Research, 2005, 6(1): 104

[42] MendiolaAS, CardonaAE. The IL-1β phenomena in neuroinflammatory diseases. Journal of Neural Transmission (Vienna), 2018, 125(5): 781-795

[43] Menzel A, H Samouda, F Dohet, S Loap, M S Ellulu, T Bohn. 2021. Common and novel markers for measuring inflammation and oxidative stress Ex vivo in research and clinical practice-which to use regarding disease outcomes? Antioxidants (Basel) 10(3). https://doi.org/10.3390/antiox10030414

[44] MiglioriniP, ItalianiP, PratesiF, PuxedduI, BoraschiD. The IL-1 family cytokines and receptors in autoimmune diseases. Autoimmunity Reviews, 2020, 19(9) ArticleID: 102617

[45] NeteaMG, Nold-PetryCA, NoldMF, JoostenLA, OpitzB, van der MeerJH, van de VeerdonkFL, FerwerdaG, HeinhuisB, DevesaI, FunkCJ, MasonRJ, KullbergBJ, RubartelliA, van der MeerJW, DinarelloCA. Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages. Blood, 2009, 113 10): 2324-2335

[46] Patrycy M, M Chodkowski, M Krzyzowska. 2022. Role of microglia in herpesvirus-related neuroinflammation and neurodegeneration. Pathogens 11(7). https://doi.org/10.3390/pathogens11070809

[47] Potere N, M G Del Buono, R Caricchio, P C Cremer, A Vecchié, E Porreca, D Dalla Gasperina, F Dentali, A Abbate, A Bonaventura. 2022. Interleukin-1 and the NLRP3 inflammasome in COVID-19: Pathogenetic and therapeutic implications. EBioMedicine 85104299. https://doi.org/10.1016/j.ebiom.2022.104299

[48] Ranson N, M Veldhuis, B Mitchell, S Fanning, A L Cook, D Kunde, R Eri. 2018. NLRP3-dependent and -independent processing of interleukin (IL)-1β in active ulcerative colitis. Int J Mol Sci 20(1). https://doi.org/10.3390/ijms20010057

[49] Ren W, H Li, C Guo, Y Shang, W Wang, X Zhang, S Li, Y Pang. 2023. Serum cytokine biomarkers for Use in diagnosing pulmonary tuberculosis versus chronic pulmonary aspergillosis. Infect Drug Resist 162217–2226. https://doi.org/10.2147/idr.S403401

[50] S M B, L Scherer, J Schaefer, J P Cooney, L Mackiewicz, M Dayton, S R Georgy, K C Davidson, C C Allison, M J Herold, A Strasser, M Pellegrini, M Doerflinger. . IL-1β drives SARS-CoV-2-induced disease independently of the inflammasome and pyroptosis signaling. Cell Death and Differentiation, 2025

[51] She Y X, Q Y Yu, X X Tang. 2021. Role of interleukins in the pathogenesis of pulmonary fibrosis. Cell Death Discovery 7(1). https://doi.org/10.1038/s41420-021-00437-9

[52] SmithEJ, BeaumontRE, McClellanA, SzeC, Palomino LagoE, HazelgroveL, DudhiaJ, SmithRKW, GuestDJ. Tumor necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors. Cell and Tissue Research, 2023, 391(3): 523-544

[53] Sorić Hosman I, I Kos, L Lamot. 2020. Serum amyloid A in inflammatory rheumatic diseases: A compendious review of a renowned biomarker. Front Immunol 11631299. https://doi.org/10.3389/fimmu.2020.631299

[54] Taha N M, M A Salem, M A El-Saied, F F Mohammed, M Kamel, M M El-Bahy, R M Ramadan. 2025. Multifaceted analysis of equine cystic echinococcosis: genotyping, immunopathology, and screening of repurposed drugs against E. equinus protoscolices. BMC Vet Res 21(1): 178. https://doi.org/10.1186/s12917-025-04616-z

[55] Teimuri Nobari S, Y Rasmi, M H Khadem Ansari. 2022. Serum levels of interleukin-1β and disease progression in multiple myeloma patients: A case and control study. Asian Pac J Cancer Prev 23(9): 2937–2942. https://doi.org/10.31557/apjcp.2022.23.9.2937

[56] Wang, J., K. Guo, S. Li, D. Liu, X. Chu, Y. Wang, W. Guo, C. Du, X. Wang, and Z. Hu. 2023. Development and application of Real-Time PCR assay for detection of Salmonella Abortusequi. Journal of Clinical Microbiology 61 (3) : e0137522. https://doi.org/10.1128/jcm.01375-22.

[57] YaseenMM, AbuharfeilNM, DarmaniH. The role of IL-1β during human immunodeficiency virus type 1 infection. Reviews in Medical Virology, 2023, 33 1) ArticleID: e2400

[58] Zhu P, C Liu, B Li, C Zhao, T Zhou, X Xue, B Zhang. 2021. Mangiferin attenuates IL-1β-induced chondrocytes apoptosis. Zhong Nan Da Xue Xue Bao Yi Xue Ban 46(1): 25–31. https://doi.org/10.11817/j.issn.1672-7347.2021.190287

Funding

Natural Science Foundation of Heilongjiang Province(LH2022C109)

National Natural Science Foundation of China(32372985)

National Key Research and Development Program of China(2023YFD1802500)

Tianchi Talent Introduction Plan(IWA2023)

RIGHTS & PERMISSIONS

The Author(s)