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Clinical and Diagnostic Laboratory Immunology, January 2004, p. 89-93, Vol. 11, No. 1
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.1.89-93.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Measurement of Serum Bactericidal Activity Specific for Haemophilus influenzae Type b by Using a Chromogenic and Fluorescent Metabolic Indicator

Sandra Romero-Steiner,1* Willie Spear,1,{dagger} Nekeidra Brown,1,{ddagger} Patricia Holder,1 Thomas Hennessy,2 Patricia Gomez de Leon,3 and George M. Carlone1

Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,1 Arctic Investigations Program, Centers for Disease Control and Prevention, Anchorage, Alaska,2 School of Medicine, Universidad Nacional Autónoma de México, Mexico City, Mexico3

Received 15 September 2003/ Returned for modification 3 November 2003/ Accepted 7 November 2003

We evaluated alamarBlue as a metabolic indicator in a standardized assay for the measurement of serum bactericidal activity (SBA) to Haemophilus influenzae type b (Hib) using sera containing natural and vaccine-induced anticapsular (polyribosylribitol phosphate) antibodies. SBA assays with a colorimetric and a fluorometric end point in the presence of alamarBlue were developed and compared to a standard SBA assay, where colony counts are performed to determine the titer (12). A colorimetric end point required a spectrophotometer, whereas a fluorometric end point required a fluorometer. Prevaccination sera (n = 27) and postvaccination sera (n = 13) were tested by all three methodologies, and the SBA titers obtained in the presence of alamarBlue were compared to those from the standard method. Both the colorimetric and the fluorometric SBA titers were significantly correlated (r = 0.87 and r = 0.95, respectively) with those of the standard assay (>=50% killing as the SBA titer end point), and titers were not significantly different when compared to those of the standard assay (P > 0.68). However, the fluorometric end point had superior performance and ease of titer determination compared to the colorimetric end point (95 versus 87% of SBA titers were within 2 dilutions of the standard titer). Hib SBA assays with alamarBlue are reproducible, faster (same-day assay), and easier to perform than the standardized assay, which requires manual or automated colony counts. These semiautomated methodologies result in increased sample throughput and collection of data in digital formats that can be exported to data analysis programs for determination of SBA titers.

* Corresponding author: MS A-36, Respiratory Diseases Immunology Section, Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-2473. Fax: (404) 639-3115. E-mail: SSteiner{at}cdc.gov.

{dagger} Present address: Department of Pulmonary Medicine, Emory University, Atlanta, GA 30322.

{ddagger} Present address: College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX 77288.

Clinical and Diagnostic Laboratory Immunology, January 2004, p. 89-93, Vol. 11, No. 1
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.1.89-93.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bieging, K. T., Rajam, G., Holder, P., Udoff, R., Carlone, G. M., Romero-Steiner, S. (2005). Fluorescent Multivalent Opsonophagocytic Assay for Measurement of Functional Antibodies to Streptococcus pneumoniae. CVI 12: 1238-1242 [Abstract] [Full Text]  
  • Romero-Steiner, S., Holder, P. F., Gomez de Leon, P., Spear, W., Hennessy, T. W., Carlone, G. M. (2005). Avidity Determinations for Haemophilus influenzae Type b Anti-Polyribosylribitol Phosphate Antibodies. CVI 12: 1029-1035 [Abstract] [Full Text]  


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