Clinical and Experimental Vaccine Research

The Korean Vaccine Society (대한백신학회)
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Development and validation of enzyme-linked immunosorbent assay for anti-mouse pertussis immunoglobulin G using international reference anti-Bordetella pertussis mouse serum NIBSC 97/642

  • Kyu-Ri Kang (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Yi-Hyeon Kwon (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Gyu-Won Cho (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Gi-Sub Choi (CLIPS BnC, Research Center) ;
  • Joon-Hwan Ji (CLIPS BnC, Research Center) ;
  • Hyun-Mi Kang (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Soo-Young Lee (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea) ;
  • Jin-Han Kang (The Vaccine Bio Research Institute, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea)
  • Received : 2024.05.27
  • Accepted : 2024.06.26
  • Published : 2024.07.31
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Abstract

Purpose: In this study, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated. The titer of ELISA was calculated using the reference line (RFL) method based on the standard curve drawn using the international reference anti-mouse serum NIBSC (National Institute for Biological Standards and Control) 97/642. Materials and Methods: In the development step, signal to noise was depicted to select the buffers that showed the most appropriate ratio. In the validation step, standard range, precision, dilution linearity, and specificity were confirmed, and RFL and parallel line (PLL) methods were compared in precision and dilution linearity. Results: Coating concentration for plate was achieved at 0.1 ㎍/mL for pertussis toxin (PT), 0.15 ㎍/mL for filamentous hemagglutinin antigen (FHA), and 0.25 ㎍/mL for pertactin (PRN). The signal to noise ratio was 22.02 for PT, 14.93 for FHA, and 8.02 for PRN with 0.25% goat serum in phosphate-buffered saline (PBS) as a dilution buffer, and 2% skim milk in PBS as a blocking buffer. Based on the precision results, we assessed the lower limit of quantification by 1, 0.2, and 1.5 EU/mL concentration for PT, FHA, and PRN which met the ICH (International Council for Harmonization) M10 criteria of a 25% accuracy and total error of 40%. In specificity, homologous serum was spiked into heterologous serum and the accuracy met the criteria. There was no difference in the results between RFL and PLL calculations (p-value=0.3207 for PT, 0.7394 for FHA, 0.2109 for PRN). Conclusion: ELISA validated with RFL calculation method in this study is a relatively accurate assay for mouse humoral immunogenicity test.

Keywords

Acknowledgement

This research was supported by grant from the Korea Health Industry Development Institute through Ministry of Health and Welfare, Republic of Korea (grant number: HV22C0246).

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