title: Dot immunoassay for the simultaneous determination of postvaccination immunity against pertussis, diphtheria, and tetanus.
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.
authors.affiliation: Perm State National Research University, 15 Bukireva str., 614000, Perm, Russia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Khramtsov
authors.affiliation: Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences, 13 Goleva str., 614081, Perm, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Bochkova
authors.affiliation: Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences, 13 Goleva str., 614081, Perm, Russia.
authors.forename: Valeria
authors.initials: V
authors.lastname: Timganova
authors.affiliation: Perm State National Research University, 15 Bukireva str., 614000, Perm, Russia.|Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences, 13 Goleva str., 614081, Perm, Russia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Zamorina
authors.affiliation: Institute of Ecology and Genetics of Microorganisms of the Ural Branch of the Russian Academy of Sciences, 13 Goleva str., 614081, Perm, Russia. mraev@iegm.ru.|Ural Federal University named after the first President of Russia B.N.Yeltsin, 620002, Ekaterinburg, Russia. mraev@iegm.ru.
authors.forename: Mikhail
authors.identifier: http://orcid.org/0000-0001-6882-4928
authors.initials: M
authors.lastname: Rayev
mesh_terms: D000293:Adolescent; D000907:Antibodies, Bacterial / Q000097:blood* / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry; D001886:Bordetella pertussis / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D004165:Diphtheria / Q000097:blood / Q000276:immunology / Q000517:prevention & control*; D004168:Diphtheria Toxoid / Q000276:immunology / Q000627:therapeutic use; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007109:Immunity; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D007223:Infant; D053758:Nanoparticles / Q000737:chemistry; D010567:Pertussis Vaccine / Q000276:immunology / Q000627:therapeutic use; D013742:Tetanus / Q000097:blood / Q000276:immunology / Q000517:prevention & control*; D013745:Tetanus Toxoid / Q000276:immunology / Q000627:therapeutic use; D014611:Vaccination; D014917:Whooping Cough / Q000097:blood / Q000276:immunology / Q000517:prevention & control*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004168:Diphtheria Toxoid; C034284:IgG Fc-binding protein, Streptococcus; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D010567:Pertussis Vaccine; D013745:Tetanus Toxoid
keywords: Antibody; Carbon nanoparticles; Diphtheria; Dot immunoassay; Pertussis; Tetanus