Obtaining High-Quality Listeriosis Antigen for Serological Diagnosis of Animal Listeriosis by Enzyme-Linked Immunosorbent Assay

Authors

  • O.V. Biloivan National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0002-9973-4551
  • V.I. Bolotin National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0001-9875-6639
  • A.P. Paliy National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0002-9193-3548
  • N.V. Marchenko National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0001-5897-3284
  • L.O. Ganova D.K. Zabolotny Institute of Microbiology and Virology, NAS of Ukraine, 154 Akademika Zabolotnoho Str., Kyiv, 03143, Ukraine
  • A.I. Zavgorodnii National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0003-3563-0478
  • Yu.K. Dunaiev National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0001-7378-430X
  • O.V. Pavlichenko National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Hryhoriia Skovorody Str., Kharkiv, 61023, Ukraine https://orcid.org/0000-0002-6577-6577

DOI:

https://doi.org/10.15407/

Keywords:

Listeria monocytogenes, antigen, listeriosis, ELISA, serological studies

Abstract

Listeriosis diagnostics is a key factor in livestock welfare control regarding this infection. Purpose. To develop a technology for obtaining a high-quality listeriosis antigen for the serological diagnosis of animal listeriosis using the enzyme-linked immunosorbent assay (ELISA). Methods. For performing the complement fixation test (CFT) and ELISA, blood serum samples from cattle, pigs, and sheep collected in 2017–2021 were used. Working serum dilutions and ELISA procedures were determined according to OIE recommendations. The optimal antigen concentration was identified by titration, selecting the dilution that provided the best discrimination between negative and positive sera. Control samples were tested in triplicate, and optical density was measured with a Multiscan FC ELISA reader (450 nm). A reaction was considered reliable when discrepancies between control sera ranged from 0.35 to 1.10; in the case of deviations, the assay was repeated. Antigen specificity was assessed in ELISA using sera containing antibodies to Brucella, Campylobacter, and Chlamydia. Reproducibility was evaluated by the difference in optical density values across three replicates. The threshold value was calculated as the mean + standard deviation × 3. The assay’s performance was compared with CFT, AGID, and commercial kits. Statistical analysis was performed using Student’s t-test in Statistica 6.0 and Microsoft Excel. Results. To develop a technology for producing listeriosis antigens for serological diagnosis in animals, five museum strains of Listeria were revived and used to obtain production volumes of bacterial mass. The resulting protein concentration was 50 μg/mL. The activity and specificity of the obtained antigen were evaluated using indirect ELISA with positive and negative sera from cattle, pigs, and sheep, as well as positive sera to other pathogens (Brucella, Campylobacter, Yersinia) previously submitted to the laboratory within 2017–2021. The difference between the mean values of positive and negative control sera ranged from 0.35 to 1.10, confirming the reliability of the results. Antigen specificity was demonstrated in ELISA using sera containing antibodies against Brucella abortus, Brucella ovis, Campylobacter fetus, and Chlamydia spp., as well as sera from intact animals. All tested sera were negative, indicating high specificity of the obtained antigen. Conclusions. The obtained data may be used to improve the diagnosis of animal listeriosis in Ukraine through the development of highly specific antigens.

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Published

2026-08-10

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Vol. 88 No. 2 (2026): Mikrobiolohichnyi Zhurnal

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Research Articles

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How to Cite

Biloivan, O., Bolotin, V., Paliy, A., Marchenko, N., Ganova, L., Zavgorodnii, A., Dunaiev, Y., & Pavlichenko, O. (2026). Obtaining High-Quality Listeriosis Antigen for Serological Diagnosis of Animal Listeriosis by Enzyme-Linked Immunosorbent Assay. Mikrobiolohichnyi Zhurnal, 88(2), 48-57. https://doi.org/10.15407/