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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 528-533, Vol. 8, No. 3
Departments of Pediatrics and Pathology,
University of Rochester, Rochester, New York
Received 25 September 2000/Returned for modification 28 November
2000/Accepted 31 January 2001
Assays measuring opsonophagocytic killing capacity of immune sera
are good surrogate assays for assessing pneumococcal vaccine responses,
but they are tedious to perform primarily because the enumeration of
surviving bacteria requires the counting of individual bacterial
colonies. To overcome this limitation, we have developed a simple and
rapid chromogenic assay for estimating the number of surviving
bacteria. In this method, the conventional opsonophagocytic killing
assays were performed in microtiter wells with differentiated HL-60
cells as phagocytes. At the end of the assay the reaction mixture was
cultured for an additional 4.5 h to increase the number of
bacteria. After the short culture, XTT
(3,3'-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) and coenzyme Q were added to the wells
and the optical density at 450 nm was measured. Our study shows that
changes in the optical density were proportional to the number of CFU
of live bacteria in the wells. Also, the number of bacteria at the end
of the 4.5-h culture was found to be proportional to the original
number of bacteria in the wells. When the performance of the
chromogenic assay was evaluated by measuring the opsonizing titers of
Streptococcus pneumoniae serotypes 6B and 19F, the
sensitivity and precision of the new method were similar to those of
the conventional opsonization assay employing the colony counting
method. Furthermore, the results of this chromogenic assay obtained
with 33 human sera correlate well with those obtained with the
conventional colony counting method (R > 0.90) for
the two serotypes (6B and 19F). Thus, this simple chromogenic assay
would be useful in rapidly measuring the capacities of antisera to
opsonize pneumococci.
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.528-533.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chromogenic Assay Measuring Opsonophagocytic
Killing Capacities of Antipneumococcal Antisera
and
*
Corresponding author. Mailing address: Departments of
Pediatrics and Pathology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 608, Rochester, NY 14642. Phone:
(716) 273-4157. Fax: (716) 273-1101. E-mail:
moon_nahm{at}urmc.rochester.edu.
Present address: Faculty of Biological Sciences, College of Natural
Sciences, Chonbuk National University, Chonju 561-756, Korea.
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