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Analytical and Bioanalytical Chemistry

, Volume 409, Issue 15, pp 3831–3842 | Cite as

Dot immunoassay for the simultaneous determination of postvaccination immunity against pertussis, diphtheria, and tetanus

  • Pavel Khramtsov
  • Maria Bochkova
  • Valeria Timganova
  • Svetlana Zamorina
  • Mikhail RayevEmail author
Research Paper

Abstract

A dot immunoassay for simultaneous semiquantitative detection of IgG against tetanus toxoid (Ttx) and diphtheria toxoid (Dtx) and qualitative detection of anti-Bordetella pertussis IgGs in human blood serum using carbon nanoparticles functionalized with streptococcal protein G was developed. Inactivated B. pertussis cells in suspension form were used as an antigen in the immunoassay. Pertussis, tetanus, and diphtheria antigens were separately spotted onto nitrocellulose strips, and then the immunostrips were successively incubated with blood sera and a suspension of carbon nanoparticles. The immunostrips were then scanned with a flatbed scanner, and the images obtained were processed with ImageJ. One hundred fifty-five venous blood serum samples from children vaccinated with diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine were tested in comparison with a conventional ELISA and agglutination test. The total time required for analysis of 32 serum samples was less than 3 h. Comparison between the results of the dot immunoassay and the corresponding ELISA/agglutination test revealed a high level of agreement (Cohen’s kappa between 0.765 and 0.813). The lower limit of quantification was 0.06 IU/ml for anti-Ttx and anti-Dtx. The intra-assay coefficients of variation were less than 15% for anti-Ttx and anti-Dtx and less than 10% for anti-pertussis. The diagnostic sensitivity of detection of the antibody protection level was 93.5% for anti-Ttx [95% confidence interval (CI) 83.5–97.9%], 92.4% for anti-Dtx (95% CI 80.9297.5%), and 90.2% for anti-pertussis (95% CI 75.9–96.8%). The diagnostic specificity was 90.9% for anti-Ttx (95% CI 57.1–99.5%), 85% for anti-Dtx (95% CI 61.1–96.0%), and 89.3% for anti-pertussis (95%CI 80.8–94.5%). The dot immunoassay developed does not require expensive reading equipment, and allows detection of antibodies against three antigens in a single analysis. The immunostrips can be stored for a long time without changes in the coloration of the spots.

Graphical Abstract

The assay procedure. BC Bordetella pertussis cell suspension, CNP carbon nanoparticle, Dtx diphtheria toxoid, Ttx tetanus toxoid

Keywords

Pertussis Tetanus Diphtheria Dot immunoassay Antibody Carbon nanoparticles 

Abbreviations

AU

Arbitrary unit

BSA

Bovine serum albumin

CNP

Carbon nanoparticle

CV

Coefficient of variation

DTaP

Diphtheria, tetanus, and acellular pertussis

DTwP

Diphtheria, tetanus, and whole-cell pertussis

Dtx

Diphtheria toxoid

ELISA

Enzyme linked immunosorbent assay

PBS

Phosphate-buffered saline

PBST

Phosphate-buffered saline with Tween 20

ROC

Receiver operating characteristic

S/P value

Intensity of the sample minus the intensity of the negative control divided by the intensity of positive control minus the intensity of the negative control

Ttx

Tetanus toxoid

Notes

Acknowledgements

The authors thank Nina B. Shemyakina for assistance in blood serum collection. We also express our gratitude to the participating nurses and parents for their contribution. The work was supported by the program for improving the competitiveness of the Ural Federal University (decree no. 211 of the Government of the Russian Federation, contract no. 02.A03.21.0006).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

All procedures performed in studies involving human participants were in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The research was approved by the Review Board of the Institute of Ecology and Genetics of Microorganisms UB RAS (IRB00010009).

Informed consent

Written informed consent was obtained from the children's parents/guardians.

Supplementary material

216_2017_327_MOESM1_ESM.pdf (598 kb)
ESM 1 (PDF 597 kb)

(MP4 19420 kb)

References

  1. 1.
    Viljanen M, Ruuskanen O, Granberg C, Salmi T. Serological diagnosis of pertussis: IgM, IgA and IgG antibodies against bordetella pertussis measured by enzyme-linked immunosorbent assay (ELISA). Scand J Infect Dis. 1982;14:117–22.CrossRefGoogle Scholar
  2. 2.
    Hendriksen C, vd Gun J, Nagel J, Kreeftenberg J. The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera. J Biol Stand. 1988;16:287–97.CrossRefGoogle Scholar
  3. 3.
    Miller J, Silverberg R, Saito T, Humber J. An agglutinative reaction for hemophilus pertussis. J Pediatr. 1943;22:644–51.CrossRefGoogle Scholar
  4. 4.
    Schubert J, Cornell R. Determination of diphtheria and tetanus antitoxin by the hemagglutination test in comparison with tests in vivo. J Lab Clin Med. 1958;52:737–43.Google Scholar
  5. 5.
    Mastroeni P, Leonardi M, Gazzara D, Bizzini B. Rapid assessment of the antitetanus immune status of a subject using dot-ELISA. Eur J Epidemiol. 1989;5:97–100.CrossRefGoogle Scholar
  6. 6.
    Valentini D, Ferrara G, Advani R, Hallander H, Maeurer M. Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis. BMC Immunol. 2015. doi: 10.1186/s12865-015-0090-3.Google Scholar
  7. 7.
    Tighe P, Ryder R, Todd I, Fairclough L. ELISA in the multiplex era: potentials and pitfalls. Proteomics Clin Appl. 2015;9:406–22.CrossRefGoogle Scholar
  8. 8.
    Pickering J, Martins T, Schroder M, Hill H. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for quantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae type b. Clin Diagn Lab Immunol. 2002;9:872–6.Google Scholar
  9. 9.
    Prince H, Lape-Nixon M, Matud J. Evaluation of a tetraplex microsphere assay for Bordetella pertussis antibodies. Clin Vaccine Immunol. 2006;13:266–70.CrossRefGoogle Scholar
  10. 10.
    van Gageldonk P, van Schaijk F, van der Klis F, Berbers G. Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus. J Immunol Methods. 2008;335:79–89.CrossRefGoogle Scholar
  11. 11.
    Reder S, Riffelmann M, Becker C, Wirsing von Konig C. Measuring immunoglobulin G antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay. Clin Vaccine Immunol. 2008;15:744–9.CrossRefGoogle Scholar
  12. 12.
    van Gageldonk P, von Hunolstein C, van der Klis F, Berbers G. Improved specificity of a multiplex immunoassay for quantitation of anti-diphtheria toxin antibodies with the use of diphtheria toxoid. Clin Vaccine Immunol. 2011;18:1183–6.CrossRefGoogle Scholar
  13. 13.
    Stenger R, Smits M, Kuipers B, Kessen S, Boog C, van Els C. Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens. Clin Vaccine Immunol. 2011;18:595–603.CrossRefGoogle Scholar
  14. 14.
    Cabore N, Pierard D, Huygen K. A Belgian serosurveillance/seroprevalence study of diphtheria, tetanus and pertussis using a Luminex xMAP technology-based pentaplex. Vaccine. 2016;4(2):16.CrossRefGoogle Scholar
  15. 15.
    Gorocs Z, Ozcan A. Biomedical imaging and sensing using flatbed scanners. Lab Chip. 2014;14:3248–57.CrossRefGoogle Scholar
  16. 16.
    Petryayeva E, Algar W. Toward point-of-care diagnostics with consumer electronic devices: the expanding role of nanoparticles. RSC Adv. 2015;5:22256–82.CrossRefGoogle Scholar
  17. 17.
    Sabbe M, Vandermeulen C. The resurgence of mumps and pertussis. Hum Vaccin Immunother. 2016;12:955–9.CrossRefGoogle Scholar
  18. 18.
    World Health Organization. Pertussis vaccines: WHO position paper – August 2015. Wkly Epidemiol Rec. 2015;90(35):433–58.Google Scholar
  19. 19.
    van den Brink G, Wishaupt J, Douma J, Hartwig N, Versteegh F. Bordetella pertussis: an underreported pathogen in pediatric respiratory infections, a prospective cohort study. BMC Infect Dis. 2014. doi: 10.1186/1471-2334-14-526.Google Scholar
  20. 20.
    Posthuma-Trumpie G, Wichers J, Koets M, Berendsen L, van Amerongen A. Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays. Anal Bioanal Chem. 2012;402:593–600.CrossRefGoogle Scholar
  21. 21.
    Linares E, Kubota L, Michaelis J, Thalhammer S. Enhancement of the detection limit for lateral flow immunoassays: evaluation and comparison of bioconjugates. J Immunol Methods. 2012;375:264–70.CrossRefGoogle Scholar
  22. 22.
    Liu B, Wang L, Tong B, Zhang Y, Sheng W, Pan M, et al. Development and comparison of immunochromatographic strips with three nanomaterial labels: colloidal gold, nanogold-polyaniline-nanogold microspheres (GPGs) and colloidal carbon for visual detection of salbutamol. Biosens Bioelectron. 2016;85:337–42.CrossRefGoogle Scholar
  23. 23.
    van Amerongen A, Wichers J, Berendsen L, Timmermans A, Keizer G, van Doorn A, et al. Colloidal carbon particles as a new label for rapid immunochemical test methods: quantitative computer image analysis of results. J Biotechnol. 1993;30:185–95.CrossRefGoogle Scholar
  24. 24.
    Mujawar L, Moers A, Norde W, van Amerongen A. Rapid mastitis detection assay on porous nitrocellulose membrane slides. Anal Bioanal Chem. 2013;405:7469–76.CrossRefGoogle Scholar
  25. 25.
    Noguera P, Posthuma-Trumpie G, van Tuil M, van der Wal F, de Boer A, Moers A, et al. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Anal Bioanal Chem. 2011;399:831–8.CrossRefGoogle Scholar
  26. 26.
    Suarez-Pantaleon C, Wichers J, Abad-Somovilla A, van Amerongen A, Abad-Fuentes A. Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron. Biosens Bioelectron. 2013;42:170–6.CrossRefGoogle Scholar
  27. 27.
    Oliveira S, Bisker G, Bakh N, Gibbs S, Landry M, Strano M. Protein functionalized carbon nanomaterials for biomedical applications. Carbon. 2015;95:767–79.CrossRefGoogle Scholar
  28. 28.
    Raev M, Khramtsov P, Bochkova M. Investigation into size distribution of carbon nanoparticles covalently functionalized with proteins. Nanotechnol Russ. 2015;10:140–8.CrossRefGoogle Scholar
  29. 29.
    Crowther J. The ELISA guidebook. 2nd ed. New York: Humana; 2009.CrossRefGoogle Scholar
  30. 30.
    Wild D, editor. The immunoassay handbook. 4th ed. Oxford: Elsevier; 2013.Google Scholar
  31. 31.
    Plikaytis B, Holder P, Pais L, Maslanka S, Gheesling L, Carlone G. Determination of parallelism and nonparallelism in bioassay dilution curves. J Clin Microbiol. 1994;32:2441–7.Google Scholar
  32. 32.
    Borrow R, Balmer P, Roper M. Immunological basis for immunization: module 3: tetanus - update 2006. Geneva: World Health Organization; 2007.Google Scholar
  33. 33.
    Scheifele D, Ochnio J. Immunological basis for immunization: module 2: diphtheria - update 2009. Geneva: World Health Organization; 2007.Google Scholar
  34. 34.
    Wirsing von König C. Immunological basis for immunization: module 4: pertussis - update 2009. Geneva: World Health Organization; 2010.Google Scholar
  35. 35.
    Scheibel I, Bentzon M, Tulinius S, Bojlen K. Duration of immunity to diphtheria and tetanus after active immunization. Acta Pathol Microbiol Scand. 1962;55:483–95.CrossRefGoogle Scholar
  36. 36.
    Schröder J, Kuhlmann W, Trendelenburg C. Knowledge-based approach to clinical decision-support system, with an application in tetanus serology. Clin Chim Acta. 1993;222:79–83.CrossRefGoogle Scholar
  37. 37.
    Xu H, Lohr J, Greiner M. The selection of ELISA cut-off points for testing antibody to Newcastle disease by two-graph receiver operating characteristic (TG-ROC) analysis. J Immunol Methods. 1997;208:61–4.CrossRefGoogle Scholar
  38. 38.
    Youden W. Index for rating diagnostic tests. Cancer. 1950;3:32–5.CrossRefGoogle Scholar
  39. 39.
    Lonnberg M, Carlsson J. Quantitative detection in the attomole range for immunochromatographic tests by means of a flatbed scanner. Anal Biochem. 2001;293:224–31.CrossRefGoogle Scholar
  40. 40.
    Barnes G, Kristiansen P, Caugant D, Naess L. Development and evaluation of a multiplex microsphere assay for quantitation of IgG and IgA antibodies against Neisseria meningitidis serogroup A, C, W, and Y polysaccharides. Clin Vaccine Immunol. 2015;22:697–705.CrossRefGoogle Scholar
  41. 41.
    Smits G, van Gageldonk P, Schouls L, van der Klis F, Berbers G. Development of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG Serum antibodies against measles, mumps, rubella, and varicella-zoster virus. Clin Vaccine Immunol. 2012;19:396–400.CrossRefGoogle Scholar
  42. 42.
    Jol-Van der Zijde C, Labadie J, Vlug A, Radl J, Vossen J, Van Tol M. Dot-immunobinding assay as an accurate and versatile technique for the quantification of human IgG subclasses. J Immunol Methods. 1988;108:195–203.CrossRefGoogle Scholar
  43. 43.
    Edwards K, Meade B, Decker M, Reed G, Rennels M, Steinhoff M, et al. Comparison of 13 acellular pertussis vaccines: overview and serologic response. Pediatrics. 1995;96:548–57.Google Scholar
  44. 44.
    Greco D, Salmaso S, Mastrantonio P, Giuliano M, Tozzi A, Anemona A, et al. Controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. N Engl J Med. 1996;334:341–9.CrossRefGoogle Scholar
  45. 45.
    Gustafsson L, Hallander H, Olin P, Reizenstein E, Storsaeter J. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med. 1996;334:349–56.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Perm State National Research UniversityPermRussia
  2. 2.Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of SciencesPermRussia
  3. 3.Ural Federal University named after the first President of Russia B.N.YeltsinEkaterinburgRussia

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