Khramtsov Pavel, Bochkova Maria, Timganova Valeria, Zamorina Svetlana, Rayev Mikhail
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.
Anal Bioanal Chem. 2017 Jun;409(15):3831-3842. doi: 10.1007/s00216-017-0327-5. Epub 2017 Apr 3.
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.
开发了一种斑点免疫测定法，用于同时半定量检测人血清中抗破伤风类毒素（Ttx）和抗白喉类毒素（Dtx）的IgG，并定性检测抗百日咳博德特氏菌IgG，该方法使用经链球菌蛋白G功能化的碳纳米颗粒。免疫测定中使用悬浮形式的灭活百日咳博德特氏菌细胞作为抗原。将百日咳、破伤风和白喉抗原分别点样到硝酸纤维素条上，然后将免疫条依次与血清和碳纳米颗粒悬浮液孵育。然后用平板扫描仪扫描免疫条，并使用ImageJ对获得的图像进行处理。对155份接种白喉、破伤风和全细胞百日咳（DTwP）疫苗的儿童静脉血清样本进行检测，并与传统酶联免疫吸附测定（ELISA）和凝集试验进行比较。分析32份血清样本所需的总时间少于3小时。斑点免疫测定结果与相应的ELISA/凝集试验结果之间的比较显示出高度一致性（科恩kappa系数在0.765至0.813之间）。抗Ttx和抗Dtx的定量下限为0.06 IU/ml。抗Ttx和抗Dtx的批内变异系数小于15%，抗百日咳的批内变异系数小于10%。检测抗体保护水平的诊断敏感性对于抗Ttx为93.5%[95%置信区间（CI）83.5 - 97.9%]，对于抗Dtx为92.4%（95%CI 80.9 - 97.5%），对于抗百日咳为90.2%（95%CI 75.9 - 96.8%）。诊断特异性对于抗Ttx为90.9%（95%CI 57.1 - 99.5%），对于抗Dtx为85%（95%CI 61.1 - 96.0%），对于抗百日咳为89.3%（95%CI 80.8 - 94.5%）。所开发的斑点免疫测定法不需要昂贵的读数设备，并且能够在单次分析中检测针对三种抗原的抗体。免疫条可以长时间保存，斑点颜色无变化。图形摘要 测定程序。BC百日咳博德特氏菌细胞悬液，CNP碳纳米颗粒，Dtx白喉类毒素，Ttx破伤风类毒素。
