Development and Validation of a Fourfold Multiplexed Opsonization Assay (MOPA4) for Pneumococcal Antibodies

American Society for Microbiology - Tập 13 Số 9 - Trang 1004-1009 - 2006
Robert L. Burton1,2, Moon H. Nahm1,2
1Departments of Pathology
2Microbiology, University of Alabama at Birmingham, Birmingham, Alabama

Tóm tắt

ABSTRACT Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well ( r 2 > 0.95). Although 22 (out of 390; ∼6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 μg/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.

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Tài liệu tham khảo

10.1128/IAI.66.12.5848-5853.1998

Bogaert, D., M. Sluijter, R. De Groot, and P. W. Hermans. 2004. Multiplex opsonophagocytosis assay (MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine. Vaccine22:4014-4020.

Briles, D. E., M. Nahm, K. Schroer, J. Davie, P. Baker, J. Kearney, and R. Barletta. 1981. Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 Streptococcus pneumoniae. J. Exp. Med.153:694-705.

Fedson, D. S., and D. M. Musher. 1994. Pneumococcal vaccine, p. 517-564. In S. A. Plotkin and E. A. Mortimer (ed.), Vaccines, 2nd ed. W. B. Saunders Co., Philadelphia, Pa.

Fleck, R. A., S. Romero-Steiner, and M. H. Nahm. 2005. Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies. Clin. Diagn. Lab. Immunol.12:19-27.

10.1128/CDLI.12.2.287-295.2005

10.1016/S0264-410X(03)00230-5

10.1128/IAI.67.5.2327-2333.1999

Kim, K. H., J. Yu, and M. H. Nahm. 2003. Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies. Clin. Diagn. Lab. Immunol.10:616-621.

10.1128/CVI.13.4.459-466.2006

Nahm, M. H., D. E. Briles, and X. Yu. 2000. Development of a multi-specificity opsonophagocytic killing assay. Vaccine18:2768-2771.

10.1128/JCM.38.6.2043-2050.2000

10.1128/IAI.72.1.114-122.2004

Rennels, M. B., K. M. Edwards, H. L. Keyserling, K. S. Reisinger, D. A. Hogerman, D. V. Madore, I. Chang, P. R. Paradiso, F. J. Malinoski, and A. Kimura. 1998. Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants. Pediatrics101:604-611.

Robbins, J. B., R. Austrian, C. J. Lee, S. C. Rastogi, G. Schiffman, J. Henrichsen, P. H. Makela, C. V. Broome, R. R. Facklam, R. H. Tiesjema, and J. C. Parke, Jr. 1983. Considerations for formulating the second-generation pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups. J. Infect. Dis.148:1136-1159.

10.1128/CVI.13.2.165-169.2006

10.1128/cdli.4.4.415-422.1997

Romero-Steiner, S., D. M. Musher, M. S. Cetron, L. B. Pais, J. E. Groover, A. F. Fiore, B. D. Plikaytis, and G. M. Carlone. 1999. Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity. Clin. Infect. Dis.29:281-288.

10.1128/IAI.67.11.5979-5984.1999

Shinefield, H. R., and S. Black. 2000. Efficacy of pneumococcal conjugate vaccines in large scale field trials. Pediatr. Infect. Dis. J.19:394-397.

Shinefield, H. R., S. Black, P. Ray, I. Chang, N. Lewis, B. Fireman, J. Hackell, P. R. Paradiso, G. Siber, R. Kohberger, D. V. Madore, F. J. Malinowski, A. Kimura, C. Le, I. Landaw, J. Aguilar, and J. Hansen. 1999. Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers. Pediatr. Infect. Dis. J.18:757-763.

10.1128/CDLI.10.4.514-519.2003

Whitney, C. G., M. M. Farley, J. Hadler, L. H. Harrison, N. M. Bennett, R. Lynfield, A. Reingold, P. R. Cieslak, T. Pilishvili, D. Jackson, R. R. Facklam, J. H. Jorgensen, and A. Schuchat. 2003. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. N. Engl. J. Med.348:1737-1746.

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American Society for Microbiology

Tập 13 Số 9

1004-1009

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