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

Abstract

PurposeAlthough 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 MethodsBordetella 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).ResultsOptimal 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.ConclusionThe 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.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model

“…The serum IgG, IgG1, and IgG2a titers against the three pertussis antigens, PT, FHA, and PRN, were evaluated using an indirect enzyme-linked immunosorbent assay (ELISA). The assays were conducted according to previous methods [ 40 ], with modifications. Immunoplates were coated with 0.1, 0.15, and 0.25 μg/mL of PT, FHA, and PRN antigens obtained from NIBSC (National Institute for Biological Standards and Control, South Mimms, UK), and the plate was incubated overnight.…”

Immunomodulation as a Novel Strategy for Prevention and Treatment of Bordetella spp. Infections

“…Among the proposed reasons for its resurgence are waning immunity (13), the end of the "honeymoon period" (14), the past vaccination calendar (15), and the failure of the current acellular vaccine to confer sterilizing immunity and long-lasting herd immunity. The increase in the number of cases is associated with more advanced diagnostic tools than ever before, allowing for an increase in the number of identified cases (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), but also with increased morbidity and mortality that creates an unambiguous imperative for improved prevention methods.…”