Clin Exp Vaccine Res.  2019 Jan;8(1):64-69. 10.7774/cevr.2019.8.1.64.

Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse

• Choi GS
• Huh DH
• Han SB
• Ahn DH
• Kang KR
• Kim JA
• Choi BM
• Kim HR
• Kang JH

• KMID: 2438967
• DOI: http://doi.org/10.7774/cevr.2019.8.1.64

Abstract

PURPOSE
Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice.
MATERIALS AND METHODS
Bordetella pertussis Tohama phase I was cultured for 24-30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250-1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP).
RESULTS
Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively.
CONCLUSION
The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.

Keyword

Bordetella pertussis; Enzyme-linked immunosorbent assay; Murine; Whooping cough

MeSH Terms

Animals
Antibodies
Bordetella pertussis
Chromatography
Diagnosis
Enzyme-Linked Immunosorbent Assay*
Horseradish Peroxidase
Methods
Mice*
Models, Animal
Pertussis Toxin
Streptavidin
Vaccines
Whooping Cough
Antibodies
Horseradish Peroxidase
Pertussis Toxin
Streptavidin
Vaccines

Figure

• 

  Fig. 1 Determination of optimal antigen concentration. Dilution of positive serum was 1:80,000. Positive, National Institute for Biological Standard and Control serum; Negative, normal mouse serum; S/N, signal to noise.

• 

  Fig. 2 Pre-validation of QC samples to determine proper optical range. QC, quality control; PT, pertussis toxoid; HQC, high-range quality control sample; MQC, middle-range quality control sample; LQC, low-range quality control sample.

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