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KMID : 1039120190080010064
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Clinical and Experimental Vaccine Research
2019 Volume.8 No. 1 p.64 ~ p.69
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Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse
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Choi Gi-Sub
Huh Dong-Ho
Han Seung-Beom
Ahn Dong-Ho
Kang Kyu-Ri
Kim Ji-Ahn
Choi Bo-Mi
Kim Hea-Ryun
Kang Jin-Han
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Abstract
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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.
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KEYWORD
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Bordetella pertussis, Enzyme-linked immunosorbent assay, Murine, Whooping cough
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