Ukr.Biochem.J. 2025; Volume 97, Issue 6, Nov-Dec, pp. 79-92

doi: https://doi.org/10.15407/ubj97.06.079

Enzyme-linked immunosorbent assay for the determination of total prostate-specific antigen

16.12.2025   Uncategorized

K. M. Shevchuk1, O. B. Besarab1*, Yu. V. Gorshunov1, O. Yu. Galkin1,2

1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: besarab@lll.kpi.ua

Received: 26 August 2025; Revised: 08 October 2025;
Accepted: 28 November 2025; Available on-line:  23 December 2025

Prostate-specific antigen (PSA) remains the most widely used biomarker for prostate cancer diagnostics and monitoring. The development of highly informative, sensitive, and reproducible immunoassays for PSA determination is essential for improving diagnostic accuracy. The aim of the study was to develop and optimize a non-competitive “sandwich” ELISA for the determination of total PSA, using a panel of monoclonal antibodies (mAbs) of different specificity groups and epitopes. “Sandwich” ELISA configurations were designed using different capture and detecting antibody pairs. Antibody sorption conditions, reagents working concentrations, incubation parameters and buffer composition were optimized. Analytical performance was evaluated using PSA preparations standardized against the WHO International Standard (96/670). The most effective antibody combinations were found among mAbs targeting epitopes P2 (capture: 21B7, 11G5, 26B9) and P3 (detection: 21F4, 23B4, 27C10), with the high-affinity pair 26B9-27C10 showing the best sorption–detection properties. The use of mixed conjugates did not improve sensitivity in the range of 1-10 ng/ml PSA. Hydrophilization of polystyrene plates surface increased the ELISA signal up to 1.39-fold, depending on the antibody isotype and origin. The developed optimized “sandwich” ELISA demonstrates high specificity and sensitivity for total PSA determination, with analytical characteristics suitable for potential clinical diagnostic applications.

Keywords: , , , , , , , ,

References:

  1. Hollenberg MD. Hypothetical structure of prostate-specific antigen. Clin Chem. 2014;60(4):702. PubMed, CrossRef
  2. Galkin OYu, Komar AG, Besarab OB. Different mice inbred strains humoral immune response against human prostate-specific antigen. Ukr Biochem J. 2019;91(1):30-37. CrossRef
  3. Yasnikova M, Kudriavtsev Y, Ponyrko A, Malyuk A, Riabenko T, Romaniuk A. Comparative analysis of MRI, morphological and biochemical features of malignant neoplasm of the prostate gland. East Ukr Med J. 2024;12(2):369-378. CrossRef
  4. Tkachenko NO, Protsenko OS, Remnyova NO, Chumak LI, Smolienko NP, Bielkina ІO, Marakhovskyi ІO, Korenieva YM, Bondarenko VO. Morphometric features of the prostate gland in rats with sulpiride-induced hyperplasia after combined administration of vitamin D and saw palmetto fruit extract. J V N Karazin Kharkiv Natl Univ Ser Med. 2025;33(1(52)):19-32. CrossRef
  5. Komar A, Kozerecka O, Besarab O, Galkin A. Development and validation of a highly informative immuno-enzymatic analysis for the determination of free prostat-specific antigen. Innov Biosyst Bioeng. 2019;3(4):220-231. CrossRef
  6. Hladkykh FV, Liadova TI. Analgesic potential of cryoextracts of biological tissues and conditioned media of mesenchymal stem cells in the treatment of experimental autoimmune arthritis. Odesa Med J. 2024;(1):35-41. CrossRef
  7. Kulaiets NM. A differentiated approach to the assessment of biomarkers depending on the phenotype of heart failure. Ukr J Cardiovasc Surg. 2025;33(1):39-47. CrossRef
  8. Tytov EV, Yakovtsova II, Ivakhno IV, Nehoduiko VV, Makarov VV, Panasenko SI. Prognostic value of a panel of immunohistochemical markers for determining the risk of recurrence and progression of non-invasive bladder cancer. Ukr J Radiol Oncol. 2023;31(4):391-403. CrossRef
  9. Shevchuk K, Baranovska A, Chernetskyi A, Shchotkina N, Besarab A. Biosafety aspects of hybridoma technology: nature of risks and approaches to their management. Innov Biosyst Bioeng. 2025;9(2):29-41. CrossRef
  10. Dmytrenko O, Golembiovska O, Pashuk V, Zgonnyk S. Problems of classification, safety assessment and risk management of medical devices with biologically active substances. Sci Rise Pharm Sci. 2025;(2(54)):86-104. CrossRef
  11. Practice and theory of enzyme immunoassays. In: Laboratory techniques in biochemistry and molecular biology. Ed. P. Tijssen. Elsevier, 1985. 549 p. CrossRef
  12. Gorsuch TT. The destruction of organic matter. Pergamon, Oxford, 1970. CrossRef
  13. Prydatko AV, Myroniuk AV, Svyderskyi VA. Analysis of approaches to mathematical description of the characteristics of materials with high hydrophobicity. East Eur J Enterp Technol. 2015;5(5(77)):30. CrossRef
  14. Galkin A, Komar A, Gorshunov Y, Besarab A, Soloviova V. New monoclonal antibodies to the prostate-specific antigen: obtaining and studying biological properties. J Microbiol Biotechnol Sci. 2019;9(3):573-577.  CrossRef
  15. Israelachvili JN. Intermolecular and Surface Forces (3rd ed.). Academic Press, 2011. CrossRef
  16. Lin Z, Lo A, Simeone DM, Ruffin MT, Lubman DM. An N-glycosylation Analysis of Human Alpha-2-Macroglobulin Using an Integrated Approach. J Proteomics Bioinform. 2012;5:127-134. PubMed, PubMedCentral, CrossRef
  17.  Wang T, Liu L, Voglmeir J. mAbs N-glycosylation: Implications for biotechnology and analytics. Carbohydr Res. 2022;514:108541. PubMed, CrossRef
  18. van de Bovenkamp FS, Hafkenscheid L, Rispens T, Rombouts Y. The Emerging Importance of IgG Fab Glycosylation in Immunity. J Immunol. 2016;196(4):1435-1441. PubMed, CrossRef
  19. Schneider D, Dühren-von Minden M, Alkhatib A, Setz C, van Bergen CA, Benkißer-Petersen M, Wilhelm I, Villringer S, Krysov S, Packham G, Zirlik K, Römer W, Buske C, Stevenson FK, Veelken H, Jumaa H. Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma. Blood. 2015;125(21):3287-3296. PubMed, PubMedCentral, CrossRef
  20. Courtois F, Agrawal NJ, Lauer TM, Trout BL. Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. MAbs. 2016;8(1):99-112. PubMed, PubMedCentral, CrossRef
  21. de Haan N, Reiding KR, Krištić J, Hipgrave Ederveen AL, Lauc G, Wuhrer M. The N- Glycosylation of Mouse Immunoglobulin G (IgG)-Fragment Crystallizable Differs Between IgG Subclasses and Strains. Front Immunol. 2017;8:608. PubMed, PubMedCentral, CrossRef
  22. Butler M, Spearman M. The choice of mammalian cell host and possibilities for glycosylation engineering. Curr Opin Biotechnol. 2014;30:107-112. PubMed, CrossRef
  23. Kaverinska A, Shevchenko N, Osetsky A, Sukhodub L, Lazurenko V, Zhelezniakov O, Prokopiuk V. The effect of lyophilized and frozen umbilical cord cryoextract on L929 cell culture. Innov Biosyst Bioeng. 2025;9(1):3-12. CrossRef
  24. Bila G, Vovk V, Utka V, Grytsko R, Havrylyuk A, Chopyak V, Bilyy R. NET-inducing diamond nanoparticles with adsorbed hydrophobic SARS-CoV-2 antigens serving as vaccine candidate. Ukr Biochem J. 2024;96(4):95-105. CrossRef
  25. Esser P. Effects of enlarged surface/volume ratio in solid phase assays. Technical Bulletin: 12a. Thermo Fisher Scientific Inc. 2010. Available from: https://www.thermofisher.co.nz/Uploads/file/Scientific/Applications/Lab-Plasticware-Glassware-Supplies/Effects-of-Enlarged-Surface-Volume-Ratio-in-Solid-Phase-Assays-Documented-on-the-basis-of-Nunc-StarWell-Modules.pdf.
  26. Galkin OYu, Savchenko AA, Nikitina KI, Dugan OM. Obtaining and study of properties of new monoclonal antibodies against human IgE. Ukr Biochem J. 2013;85(5):81-87. CrossRef
  27. Galkin OuY, Besarab AB, Lutsenko TN. Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60). Ukr Biochem J. 2017;89(1):22-30. CrossRef
  28. Acevedo B, Perera Y, Ruiz M, Rojas G, Benítez J, Ayala M, Gavilondo J. Development and validation of a quantitative ELISA for the measurement of PSA concentration. Clin Chim Acta. 2002;317(1-2):55-63. PubMed, CrossRef
  29. Gutiérrez-Zúñiga GG, Hernández-López JL. Sandwich-type ELISA impedimetric immunosensor for early detection of prostate-specific antigen (PSA) in human serum. Procedia Chem. 2014;12:47-54. CrossRef
  30. Mashkoor FC, Al-Asadi JN, Al-Naama LM. Serum level of prostate-specific antigen (PSA) in women with breast cancer. Cancer Epidemiol. 2013;37(5):613-618. PubMed, CrossRef
  31. Khudetskyy I, Antonova-Rafi J. Human influence and changes in nature on biological security (overview of the problem). Phytotherapy J. 2023;(2):26-34. CrossRef
  32. Labunets I, Toporova O, Panteleymonova T, Dovbynchuk T, Kyryk V, Kashchuk O, Kordium V. Comparative effects of human umbilical cord-derived mesenchymal stromal cells and their extracellular vesicles in a mouse model of parkinsonism. Cell Organ Transpl. 2025;13(1):46-53. CrossRef
Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.