Development and Validation of a Highly Informative Immuno-Enzymatic Analysis for the Determination of Free Prostat-Specific Antigen


  • Anatolii Komar SE “Ukrainian Medical Certification Center”, the Ministry of Health of Ukraine; Igor Sikorsky Kyiv Polytechnic Institute, Ukraine
  • Oksana Kozerecka Hema LLC, Ukraine
  • Olexandr Besarab Igor Sikorsky Kyiv Polytechnic Institute, Ukraine
  • Alexander Galkin Igor Sikorsky Kyiv Polytechnic Institute; Palladin Institute of Biochemistry, NAS of Ukraine, Ukraine



Free prostate-specific antigen, Enzyme immunoassay, Validation


Background. Prostate-specific antigen (PSA) is a key marker used in the monitoring of patients with prostate hyperplasia, risk of deve­lopment and cancer of prostate. Clinical laboratory diagnostics for such patients are implemented using appropriate serological tests. Enzyme immunoassay kits are bioanalytical products that are subject to the requirements for medical devices for in vitro diagnostics. Thus, the urgent task of modern analytical biotechnology is improving bioanalytical, technical, and economic indicators of diagnostic kits, as well as their validation, as a prerequisite for the admission of such products to the market.

Objective. Scientific substantiation of the composition and technology of highly informative enzyme-linked immunosorbent assay (ELISA) test-kit for the quantitative determination of free PSA in human serum (blood), as well as its bioanalytical validation at the time of manufacture and expiry date.

Methods. A previously prepared and characterized set of monoclonal antibodies (mAb) to different PSA epitopes was used to construct ELISA. Peroxidase conjugates of mAbs were synthesized by periodate oxidation. We used the WHO Prostate Specific Antigen International Standard and the human serum with different PSA amount. The validation of the developed ELISA was performed according to the conventional scheme used for quantitative bioanalytical methods.

Results. At the first stage of the work, the determination of the optimal mAbs configuration was carried out for the development of a non-competitive sandwich ELISA. The mAbs studied were characterized by the following indicators: activity in ELISA (relative to PSA, its complex with α1-antihymotrypsin (α1-ACT) and related protein kallikrein-2), isotype, titer, affinity constant, comparative epitope specificity, degree of PSA enzymatic activity inhibition, source of origin (Balb/c or NZB mouse). Based on these characteristics, the principle of using mAbs to construct ELISA for the determination of various forms of PSA (free molecule and one associated with α1-ACT) was formed. For different ranges of PSA concentrations, it was assessed the feasibility of sharing different mAbs to determine free PSA. At the second stage of the work, a protocol for the quantitative ELISA was formulated (determination of the number of reagent components, optimal time, and temperature indicators for all stages of the analysis). At the final stage of the study, we carried out validation of the developed ELISA, de­termining the following indicators: diagnostic specificity, linearity, limit of detection and quantification (analytical sensitivity), precision, correctness (accuracy).

Conclusions. The antibodies of the epitopes P2 and P4 (and different specificity groups) are the most optimal combination for the sandwich ELISA to determine free PSA. The sorption-detection ability of different mAb pairs correlates with their affinity. Antibodies 26B9, 21B7, and 11G5 of the P2 epitope (group II specificity) were the best antibodies for use in solid phase sorption, and antibodies 14C8 and 21D7 of the epitope P4 (group I specificity) were the best ones for detections. The pair of high-affinity mAbs 26B9 and 14C8 possessed the most pronounced sorption-detection properties. The combined use of the mAbs 14C8 and 21D7 enzyme conjugates leads to increased sensitivity in the study of PSA concentrations in the range of 1–10 ng/ml. Validation characteristics were determined at the time of kit release and at the end of the shelf life (1 year). The average value of diagnostic specificity is 100.6%. The technique is linear in the range of 0.1–30 ng/ml, the uncertainty of the calibration graph is insignificant. The estimated detection limit is 0.001782 ng/ml, and the limit of quantification (analytical sensitivity) coincides with the reproducibility limit and is 0.0054 ng/ml. The correctness (systematic error) is 0.03 ng/ml and is statistically insignificant.


Fletcher RH. Guideline: Experts recommend against prostate cancer screening with prostate-specific antigen test. Ann Intern Med. 2019;170(2):1. DOI: 10.7326/acpjc-2019-170-2-002

Duskova K, Vesely S. Prostate specific antigen. Current clinical application and future prospects. Biomedical Papers. 2015;159(1):18-26. DOI: 10.5507/bp.2014.046

Haythorn MR, Ablin RJ. Prostate-specific antigen testing across the spectrum of prostate cancer. Biomark Med. 2011;5(4):515-26. DOI: 10.2217/bmm.11.53

Sydyakina YV, Sivakova AA, Komar AG, Galkin AY. Prostat-specific antigen: biochemical, molecular-biological, and analytical aspects. Innov Biosyst Bioeng, 2019;3(2):86-95. DOI: 10.20535/ibb.2019.3.2.164790

Galkin OY, Komar AG, Pys'menna MO. Specificity of manifacturing process validation for diagnostic serological devices. Biotechnologia Acta. 2018;11(1):25-38. DOI: 10.15407/biotech11.01.025.

Galkin OY, Komar AG, Grigorenko AA. Bioanalytical standardizing for serological diagnostic medical devices. Biotechnologia Acta. 2015;8(2):112-9. DOI: 10.15407/biotech8.02.112

Practice and theory of enzyme immunoassays. In: Tijssen P, editor. Laboratory techniques in biochemistry and molecular biology. 1st ed. Elsevier; 1985. 674 p.

Gryzodub OI, editor. State pharmacopoeia of Ukraine. 1st ed. Kharkiv: RIREG; 2008. 617 p.

Urbakh VY. Mathematical statistics for biologists and physicians. Moscow: AS USSR; 1963. 321 p.

Standards Russian Federation. State system for ensuring uniformity of measurements. Indicators of accuracy, correctness, precision of methods of quantitative chemical analysis. Evaluation methods. Мoscow: Standardinform. RMG 61–2010.

Galkin OY, Besarab OB, Gorshunov YV, Dugan OM. Bioanalytical validation of immunoenzymatic test-kit for quantitative determination of total human immunoglobulin E. Scientific Issue Ternopil Volodymyr Hnatiuk National Pedagogical University Series Biology. 2014;4:7-18.

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 Food Sci. 2019;9(3):573-7. DOI: 10.15414/jmbfs.2019/

Galkin OY, Savchenko AA, Nikitina KI, Dugan OM. Obtaining and study of properties of new monoclonal antibodies against human IgE. Ukr Biochem J. 2013;85(5):81-7. DOI: 10.15407/ubj85.05.081

Galkin A, Besarab A, Gorshunov Y, Soloviova V, Dugan O. Mouse monoclonal antibodies to horseradish peroxidase isoenzyme C: obtaining and study of biological properties. Izvestiya Vuzov Prikladnaya Khimiya i Biotekhnologiya. 2014;5:47-59.

International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use, validation of analytical procedures: methodology, ICH Q2B. Geneva; 1996. 11 р.

W.H.O. Expert Committee on Biological Standardization. World Health Organization Technical Report Series. 1981;658:21.

Technical regulation on medical devices for in vitro diagnostics, approved by the Resolution of the Cabinet of Ministers of Ukraine No. 754 of 21.10.2013. Ukrainian Official Gazette. 2013;82:3047.

Standards Ukraine. Medical products. Quality management system. Regulatory requirements. Kyiv: State Committee for Technical Regulation and Consumer Policy; 2007. ISO 13485:2005.

Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Official Journal of the European Communities. 1998;L 331(41):1.

Standards Russian Federation. Clinical laboratory technologies. Quality requirements for clinical laboratory tests. Мoscow: Standardinform, 2009. R 53022.2-2008; Part 2. Assessment of the analytical reliability of research methods (accuracy, sensitivity, specificity).

Parreño V, Romera SA, Makek L, Rodriguez D, Malacari D, Maidana S, et al. Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards. J Virol Methods, 2010;169(1):143-53. DOI: 10.1016/j.jviromet.2010.07.014

Schares G, Basso W, Majzoub M, Rostaher A, Scharr JC, Langenmayer MC, et al. Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti. Vet Parasitol. 2011;175(1-2):52-9. DOI: 10.1016/j.vetpar.2010.09.024

Volkova RA. Medical immunobiological drugs quality control system using chemical and immunochemical methods [dissertation]. Moscow: LA Tarasevich State Research Institute of Standardization and Control of Medical Biological; 2009. 276 p.

Galkin O, Myhalchuk M, Kazmirchuk V, Gurzhenko Y. Development and comparison of different versions of ELISA for detection of specific IgE-antibodies. Bulletin of Taras Shevchenko National University of Kyiv Ser Biology. 2014;1:15-21.



How to Cite

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 [Internet]. 2019Dec.10 [cited 2023May30];3(4):220-31. Available from: