Preview

Russian Journal of Biotherapy

Advanced search

Development of the test system based on enzyme immunoassay for the detection of recombinant Streptococcus pneumoniae pneumolysin

https://doi.org/10.17650/1726-9784-2023-22-4-52-59

Abstract

Backgraund. Pneumolysin (Ply) is a hemolytic toxin of Streptococcus pneumoniae (S. pneumoniae) expressed by all strains of pneumococci. The use of sandwich enzyme-linked immunosorbent assay (ELISA) can be a simple, fast and effective way of its qualitative and quantitative determination in biological fluids.

Aim. To develop and evaluate the specificity of sandwich ELISA test system for qualitative and quantitative determination of recombinant Ply (rPly) of S. pneumoniae.

Materials and methods. Immobilized on the solid phase rabbit’s polyclonal antibodies (pAbs) to rPly were used as recognition antibodies in sandwich ELISA. The studied antigens were added to the pAbs (rPly). The reaction was manifested by using detecting mouse monoclonal IgG1 (rPly) – antibodies conjugated with horseradish root peroxidase. The specificity of the test system was evaluated when using recombinant α-hemolysin (rα-Hly) and water-soluble S. aureus antigens as reference preparations.

Results. Using sandwich ELISA, rPly was detected at a concentration of 0.15 µ / ml. The test system was characterized by specificity, which was confirmed by the absence of reaction with recombinant rα-Hly of Staphylococcus aureus (S. aureus). Reference preparations of water-soluble surface antigens of S. aureus strains No 209, 1986,1991 and Cowan I gave a false positive reaction due to the presence of protein A (SpA) in their composition, a thermostable surface protein expressed by many strains of staphylococci capable of binding immunoglobulins via Fc-fragment or Fab fragments of the V3H domain of the B cells receptor. A negative reaction was obtained with antigens from the S. aureus wood 46 strain, which does not have the spa gene encoding SpA expression. The presence of protein A in preparations of water-soluble S. aureus antigens was confirmed in the ELISA inhibition assay.

Conclusion. Sandwich ELISA has been developed for qualitative and quantitative determination of S. pneumoniae Ply. The conducted studies have confirmed the specificity of the test system.

About the Authors

E. A. Kurbatova
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



I. V. Yakovleva
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



N. F. Gavrilova
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



D. S. Vorobyev
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



E. S. Petukhova
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



I. B. Semenova
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



A. E. Zaitsev
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



Yu. V. Volokh
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



A. Yu. Leonova
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



A. V. Poddubikov
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



A. A. Kaloshin
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



I. M. Gruber
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

5A Maly Kazenny Ln., Moscow 105064



References

1. Grousd J.A., Rich H.E., Alcorn J.F. Host-pathogen interactions in gram-positive bacterial pneumonia. Clin Microbiol Rev 2019; 32:e00107-18. DOI: 10.1128/CMR.00107-18

2. Hageman J.C, Uyeki T.M, Francis J.S. et al. Severe communityacquired pneumonia due to Staphylococcus aureus, 2003-04 influenza season. Emerg Infect Dis 2006;12:894–9. DOI: 10.3201/eid1206.051141

3. Jahn K., Kohler T.P., Hammerschmidt S. et al. Bacterial adhesins and the pneumococcus. Cells 2022;11:1121. DOI: 10.3390/cells11071121

4. Cockeran R., Anderson R., Feldman C. The role of pneumolysin in the pathogenesis of Streptococcus pneumoniae infection. Curr Opin Infect Dis 2022;15(3):235–9. DOI: 10.1097/00001432-200206000-00004

5. Bubeck Wardenburg J., Bae T., Otto M. et al. Poring over pores: Alpha-hemolysin and panton-valentine leukocidin in Staphylococcus aureus pneumonia. Nat Med 2022;13(12): 1405–6. DOI: 10.1038/nm1207-1405

6. Kebaier C., Chamberland R.R., Allen I.C. et al. Staphylococcus aureus alpha-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome. J Infect Dis 2012;205(5):807–17. DOI: 10.1093/infdis/jir846

7. Cohen T.S., Jones-Nelson O., Hotz M. et al. S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin. Sci Rep 2016;6:35466. DOI: 10.1038/srep35466

8. Kitur K., Parker D., Nieto P. et al. Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage. PLoS Pathog 2015;11(4):e1004820. DOI: 10.1371/journal.ppat.1004820

9. Mitchell T.J., Dalziel C.E. The biology of pneumolysin. Subcell Biochem 2014;80:145–60. DOI: 10.1007/978-94-017-8881-6_8

10. Gingerich A.D., Mousa J.J. Diverse mechanisms of protective anti-pneumococcal antibodies. Front Cell Infect Microbiol 2022;12:824788. DOI: 10.3389/fcimb.2022.824788

11. Wheeler J., Freeman R., Steward M. et al. Detection of pneumolysin in sputum. J Med Microbiol 1999;48(9):863–6. DOI: 10.1099/00222615-48-9-863.8

12. Rajalakshmi B., Kanungo R., Srinivasan S., Badrinath S. Pneumolysin in urine: A rapid antigen detection method to diagnose pneumococcal pneumonia in children. Indian J Med Microbiol 2002;20(4):183–6. PMID: 17657067

13. Matos J.A., Madureira D.J., Rebelo М.С. et al. Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin. Mem Inst Oswaldo Cruz 2006;101(5):559–63. DOI: 10.1590/s0074-02762006000500014

14. Lahti E., Mertsola J., Kontiokari T. et al. Pneumolysin polymerase chain reaction for diagnosis of pneumococcal pneumonia and empyema in children. Eur J Clin Microbiol Infect Dis 2006;25(12):783–9. DOI: 10.1007/s10096-006-0225-9

15. Becker R.E., Berube B.J., Sampedro G.R. Tissue-specific patterning of host innate immune responses by Staphylococcus aureus alpha-toxin. J Innate Immun 2014;6(5):619–631. DOI: 10.1159/000360006

16. Song L., Hobaugh M.R., Shustak C. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 1996;274(5294):1859–66. DOI: 10.1126/science.274.5294.1859

17. Vorobyev D.S., Sidorov A.V., Kaloshin A.A. et al. Preparation a recombinant form of pneumolysin protein from Streptococcus pneumoniae. Byulleten ‘Eksperimental’noi Biologii i Meditsini = Bulletin of Experimental Biology and Medicine 2022;174(12):723–7. (In Russ.) DOI: 10.47056/0365-9615-2022-174-12-723-727

18. Vorobyev D.S., Sidorov A.V., Kaloshin A.A. et al. Recombinant plasmid DNA pSp-raPLY encoding the synthesis of recombinant atoxic pneumolysin protein Streptococcus pneumoniae, Escherichia coli strain M15/pSp-raPLY is a producer of recombinant atoxic form of pneumolysin and obtaining this protein for the development of a vaccine preparation for the prevention of pneumococcal infections. RU 2 782 598C1. (In Russ.)

19. Broecker F., Anish C., Seeberger P.H. Generation of monoclonal antibodies against defined oligosaccharide antigens. Methods Mol Biol 2015;1331:57–80. DOI: 10.1007/978-1-4939-2874-3_5

20. Nakane P.K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 1974;22(12):1084–91. DOI: 10.1177/22.12.1084

21. Segel I.H. Biochemical calculations: How to solve mathematical problems in general biochemistry. New-York, London, Sydney, Toronto: John Wiley & Sons Inc., 1976; 441 p.

22. Levinson A.I., Tar L., Carafa C., Haidar M. Potent stimulus of immunoglobulin M rheumatoid factor production. J Clin Invest 1986;78(3):612–7. DOI: 10.1172/JCI112617

23. Falugi F., Kim H.K., Missiakas D.M., Schneewind O. The role of protein A in the evasion of host adaptive immune responses by Staphylococcus aureus. mBio 2013; 4(5):e00575-13. DOI: 10.1128/mBio.00575-13

24. Rigi G., Ghaedmohammadi S., Ahmadian G.A. Сomprehensive review on staphylococcal protein A (SpA): Its production and applications. Biotechnol Appl Biochem 2019;66(3):454–64. DOI: 10.1002/bab.1742

25. Balachandran M., Giannone R.J., Bemis D.A., Kania S.A. Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46. PLoS One 2017;12(8):e0183913. DOI: 10.1371/journal.pone.0183913


Review

For citations:


Kurbatova E.A., Yakovleva I.V., Gavrilova N.F., Vorobyev D.S., Petukhova E.S., Semenova I.B., Zaitsev A.E., Volokh Yu.V., Leonova A.Yu., Poddubikov A.V., Kaloshin A.A., Gruber I.M. Development of the test system based on enzyme immunoassay for the detection of recombinant Streptococcus pneumoniae pneumolysin. Russian Journal of Biotherapy. 2023;22(4):52-59. (In Russ.) https://doi.org/10.17650/1726-9784-2023-22-4-52-59

Views: 349


Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.


ISSN 1726-9784 (Print)
ISSN 1726-9792 (Online)