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


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


Pertussis Tetanus Diphtheria Dot immunoassay Antibody Carbon nanoparticles 



Arbitrary unit


Bovine serum albumin


Carbon nanoparticle


Coefficient of variation


Diphtheria, tetanus, and acellular pertussis


Diphtheria, tetanus, and whole-cell pertussis


Diphtheria toxoid


Enzyme linked immunosorbent assay


Phosphate-buffered saline


Phosphate-buffered saline with Tween 20


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


Tetanus toxoid



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)


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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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