PCR-amplified Immunoassay (Immuno-PCR): Principle of the Method, Variants of Execution, Possibilities and Prospects of Use for the Detection of Pathogenic Biological Agents

Enzyme immunoassay and polymerase chain reaction have become the «gold standard» for the detection of biological pathogens. The method of amplified immunoassay – immuno-PCR allows to combine both methods into a single platform to preserve their advantages and to achieve high sensitivity of the analysis. The purpose of this work is to consider the possibilities and prospects of using PCR-amplified immunoassay for the detection of pathogenic biological agents. Immuno-PCR makes it possible to detect various non-nucleic antigenic determinants in PCR by amplifying a DNA tag conjugated with a specific antibody. The registration of the results is also possible in real time as in the real-time PCR test systems. The main methodological issues in the immuno-PCR technology are: the choice of a carrier of biomolecule complexes, the choice of a method for conjugation of detection antibodies and a reporter nucleic acid, optimization of methods for amplifying signal DNA and accounting for results, and development of methods for reducing background indicators. We consider it necessary to carry out research and development work on the development and the creation of diagnostic kits based on immuno-PCR. With regard to the task of detecting small and trace amounts of antigens of pathogenic biological agents, the most likely diagnostic «niche» of the immuno-PCR method will be the detection of toxins of microbial and non-microbial origin, the minimum clinically significant dose for which is less than the sensitivity of the corresponding immunochemical test systems. Taking into account the prospects for the development of the method, in future it is possible to develop such test systems for the detection of hapten analytes, for example, some toxicants of non-biological origin.

1. Kishkun A.A. Clinical laboratory diagnostics: textbook / 2nd ed., Rev. and add. M.: GEOTAR-Media, 2019. 832 p. (in Russian).

2. Ryazantsev D.Yu., Voronina D.V., Zavriev S.K. Immuno-PCR: Achievements and Prospects // Advances in Biological chemistry. 2016. V. 56. P. 377–410 (in Russian).

3. Chang L., Li J., Wang L. Immuno-PCR: An ultrasensitive immunoassay for biomolecular detection // Anal. Chim. Acta. 2016. V. 910. P. 12–24. https://doi.org/10.1016/j.aca.2015.12.039

4. Sano T., Smith C.L., Cantor Ch.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates // Science. 1992. V. 258. P. 120–122. https://doi.org/10.1126/science.1439758

5. Deng M., Long L., Xiao X. et al. ImmunoPCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers // Vet. Immunol. Immunopathol. 2011. V. 141. № 3–4. P. 183–189. https://doi.org/10.1016/j.vetimm.2011.02.018.

6. Kulbachinsky A.V. Methods for the selection of aptamers for protein targets // Advances in Biological Chemistry. 2006. V. 46. P. 193–224 (in Russian).

7. Morin I., Askin S.P., Schaeffer P.M. IgG-detection devices for the Tus-Ter-lock immuno-PCR diagnostic platform // Analyst. 2011. V. 136. № 22. P. 4815–4821. https://doi.org/10.1039/c1an15731k

8. Johnston E.B., Kamath S.D., Lopata A.L. et al. Tus-Ter-lock immuno-PCR assays for the sensitive detection of tropomyosin-specic IgE antibodies // Bioanalysis. 2014. V. 6. № 4. P. 465–476. https://doi.org/10.4155/bio.13.315

9. Zhou H., Fisher R.J., Papas T.S. Universal immuno-PCR for ultra-sensitive target protein detection // Nucleic Acids Res. 1993. V. 21. P. 6038–6039. https://doi.org/10.1093/nar/21.25.6038

10. Neylon C., Kralicek A.V., Hill T. M., Dixon N.E. Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex // Microbiol. Mol. Biol. Rev. 2005. V. 69. № 3. P. 501–526. https://doi.org/10.1128/MMBR.69.3.501-526.2005

11. Gottlieb P.A., Wu S., Zhang X. et al. Equilibrium, kinetic, and footprinting studies of the Tus-Ter proteinDNA interaction // J. Biol Chem. 1992. V. 267. № 11. P. 7434–7443. https://doi.org/10.3390/molecules24081572

12. Lakhin A.V., Tarantul V.Z., Gening L.V. Aptamers: problems, ways of solving them and prospects // Acta Naturae. 2013. V. 5. № 4. P. 28–39 (in Russian).

13. Sullivan R., Adams M.C., Naik R.R., Milam V.T. Analyzing secondary structure patterns in DNA aptamers identified via CompELS // Molecules. 2019. V. 24. № 8. P. 1572. https://doi.org/10.3390/molecules24081572

14. Chumakov A.M., Yukhina E.S., Frolova E.I. et al. Expanding the possibilities of using DNA aptamers by their functionalization / // Bioorganic chemistry. 2016. V. 42. № 1. P. 3–17 (in Russian).

15. Anzai H., Terai T., Jayathilake C. et al. A novel immuno-PCR method using cDNA display // Anal. Biochem. 2019. V. 578. P. 1–6. https://doi.org/10.1016/j.ab.2019.04.017

16. Graner M., Pointon T., Manton S. et al. Oligoclonal IgG antibodies in multiple sclerosis target patient-specific peptides // PLoS One. 2020. V. 15. № 2. e0228883. https://doi.org/10.1371/journal.pone.0228883

17. Rezaei Z.S., Shahangian S.S., Hasannia S., Sajedi R.H. Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor // Anal. Bioanal. Chem. 2020. V. 412. № 27. P. 7639–7648. https://doi.org/10.1007/s00216-020-02901-4

18. Zhao L., Zhou H., Sun T. et al. Complete antigen-bridged DNA strand displacement amplification immuno-PCR assay for ultrasensitive detection of salbutamol // Sci. Total Environ. 2020. V. 748. P. 142330. https://doi.org/10.1016/j.scitotenv.2020.142330

19. Mehta P.K., Dahiya B., Sharma S. et al. Immuno-PCR, a new technique for the serodiagnosis of tuberculosis // J. Microbiol. Methods. 2017. V. 139. P. 218–229. https://doi.org/10.1016/j.mimet.2017.05.009

20. Sharma S., Sheoran A., Gupta K.B. et al. Quantitative detection of a cocktail of mycobacterial MPT64 and PstS1 in tuberculosis patients by real-time immuno-PCR // Future Microbiol. 2019. V. 14. P. 223– 233. https://doi.org/10.2217/fmb-2018-0284

21. Maerle A.V., Ryazantsev D.Yu., Dmitrenko O.A. et al. Determination of Staphylococcus aureus toxins by immuno-PCR // Bioorganic chemistry. 2014. V. 40. № 5. P. 571 (in Russian).

22. Kirchner M., Sayers E., Cawthraw S. et al. A sensitive method for the recovery of Escherichia coli serogroup O55 including Shiga toxin-producing variants for potential use in outbreaks // J. Appl. Microbiol. 2019. V. 127. № 3. P. 889–896. https://doi.org/10.1111/jam.14345

23. Kolesnikov A.V., Kozyr A.V., Ryabko A.K., Shemyakin I.G. Ultrasensitive detection of protease activity of anthrax and botulinum toxins by a new PCRbased assay // Pathog. Dis. 2016. V. 74. № 1. P.112. https://doi.org/10.1093/femspd/ftv112

24. Das S., Majumder S., Nag M., Kingston J.J. A sandwich duplex immuno PCR for rapid and sensitive identification of Clostridium perfringens alpha and enterotoxin // Anaerobe. 2019. V. 57. P. 63–74. https://doi.org/10.1016/j.anaerobe.2019.03.015

25. Ren X., Zhang Q., Wu W. et al. Anti-idiotypic nanobody-phage display-mediated real-time immunoPCR for sensitive, simultaneous and quantitative detection of total aflatoxins and zearalenone in grains // Food Chem. 2019. V. 297. P. 124912. https://doi.org/10.1016/j.foodchem.2019.05.186

26. Huang W., Tu Z., Ning Z. et al. Development of real-time immuno-PCR based on phage displayed an anti-idiotypic nanobody for quantitative determination of citrinin in monascus // Toxins (Basel). 2019. V. 11. № 10. P. 572. https://doi.org/10.3390/toxins11100572

27. Guan N., Li Y., Yang H., Hu P. et al. Dualfunctionalized gold nanoparticles probe based biobarcode immuno-PCR for the detection of glyphosate // Food Chem. 2021. V. 338. P. 128133. https://doi.org/10.1016/j.foodchem.2020.128133

28. He X., McMahon S., McKeon T.A., Brandon D.L. Development of a novel immuno-PCR assay for detection of ricin in ground beef, liquid chicken egg, and milk // J. Food Prot. 2010. V. 73. № 4. P. 695–700. https://doi.org/10.4315/0362-028x-73.4.695

29. Barkova I.A., Barkov A.M., Viktorov D.N. The method of immuno-PCR in the diagnosis of bacterial and viral infections // J. Microbiol. 2019. № 3. P. 110–117 (in Russian).