A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes

• Published in: Journal of microbiological methods Vol. 79; no. 3; pp. 289 - 294
• Main Authors: Yang, Jae Seung, Choi, Seulggie, Kim, David D., Kang, Seok-Seong, Yun, Cheol-Heui, Lee, Kangseok, Han, Seung Hyun
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.12.2009; Elsevier
• Subjects: Antibodies, Bacterial - blood; Antibodies, Bacterial - immunology; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cholera vaccine; Cholera Vaccines - immunology; Cytotoxicity Tests, Immunologic - methods; Fundamental and applied biological sciences. Psychology; Humans; Microbial Viability - immunology; Microbiology; Regression Analysis; Vibrio cholerae; Vibrio cholerae O1 - immunology; Vibriocidal assay; Vibrio cholerae; Cholera vaccine; Vibriocidal assay; Human; Antibody; Method; Infection; Bacteriosis; Bacteria; Vibrionaceae; Serum; Cholera; Serotype; Quantitative analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2009.09.016

Currently available cholera vaccines are formulated with killed-whole cells of Vibrio cholerae O1 Inaba and Ogawa serotypes. A serum vibriocidal assay has been widely used to evaluate the immunogenicity of cholera vaccines in clinical trials. In this study, we developed a duplex vibriocidal assay to obtain vibriocidal antibody titers against both serotypes simultaneously. Initially, serial dilutions of serum from vaccinees were incubated with guinea pig complements along with both streptomycin-resistant Inaba and ampicillin-resistant Ogawa strains for 1 h. The mixture was then inoculated on separate agar plates containing each antibiotic to selectively culture each corresponding serotype and incubated at 37 °C for 16 to 20 h. Bacterial colonies were enumerated using an automated colony counting system to obtain the vibriocidal antibody titers defined by the reciprocal of serum dilution inhibiting bacterial growth by 50%. Performance of the duplex vibriocidal assay was examined by comparison with a single serotype vibriocidal assay using 20 clinical sera consisting of ten-paired sera prepared at pre- and post-vaccination. Both assays showed a good correlation for vibriocidal titers against the two serotypes, respectively, as determined by Pearson correlation coefficient ( r) and regression coefficient ( β) analyses; r = 0.998 , β = 1.003 for Inaba and r = 0.997, β = 0.999 for Ogawa, respectively. The duplex vibriocidal assay can diminish the amount of sera required for the assay and enhance assay efficiency in terms of time, labor intensity, and expenditure.