RESUMO
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 µg/mL for pertussis toxin (PT), 0.15 µg/mL for filamentous hemagglutinin antigen (FHA), and 0.25 µg/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.
RESUMO
Purpose: Pertussis bacteria have many pathogenic and virulent antigens and severe adverse reactions have occurred when using inactivated whole-cell pertussis vaccines. Therefore, inactivated acellular pertussis (aP) vaccines and genetically detoxified recombinant pertussis (rP) vaccines are being developed. The aim of this study was to assess the safety profile of a novel rP vaccine under development in comparison to commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccines. Materials and Methods: The two positive control DTaP vaccines (two- and tri-components aP vaccines) and two experimental recombinant DTaP (rDTaP) vaccine (two- and tri-components aP vaccines adsorbed to either aluminum hydroxide or purified oat beta-glucan) were used. Temperature histamine sensitization test (HIST), indirect Chinese hamster ovary (CHO) cell cluster assay, mouse-weight-gain (MWG) test, leukocytosis promoting (LP) test, and intramuscular inflammatory cytokine assay of the injection site performed for safety assessments. Results: HIST results showed absence of residual pertussis toxin (PTx) in both control and experimental DTaP vaccine groups, whereas in groups immunized with tri-components vaccines, the experimental tri-components rDTaP absorbed to alum showed an ultra-small amount of 0.0066 IU/mL. CHO cell clustering was observed from 4 IU/mL in all groups. LP tests showed that neutrophils and lymphocytes were in the normal range in all groups immunized with the two components vaccine. However, in the tri-components control DTaP vaccine group, as well as two- and tri-components rDTaP with beta-glucan group, a higher monocyte count was observed 3 days after vaccination, although less than 2 times the normal range. In the MWG test, both groups showed changes less than 20% in body temperature and body weight before the after the final immunizations. Inflammatory cytokines within the muscle at the injection site on day 3 after intramuscular injection revealed no significant response in all groups. Conclusion: There were no findings associated with residual PTx, and no significant differences in both local and systemic adverse reactions in the novel rDTaP vaccine compared to existing available DTaP vaccines. The results suggest that the novel rDTaP vaccine is safe.