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.

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