Chromogenic Assay Measuring Opsonophagocytic Killing Capacities of Antipneumococcal Antisera

Abstract

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.

Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae

“…The viability OPA has been successfully implemented by multiple laboratories (12,16,17,23), and it has been recognized as the reference method for the evaluation of other OPA methods. Opsonophagocytosis can be measured by many methods including flow cytometric, chromogenic, and radioisotopic assays (11,12,15,16,23,24). We provide the median OPA titers, as determined by this multilaboratory study, for a set of 12-paired sera (Table 1) to facilitate the evaluation of other OPA methods.…”

Use of HL-60 Cell Line To Measure Opsonic Capacity of Pneumococcal Antibodies

“…Kim et al overcame the colony-counting problem by colony enhancement with dye and automated colony counting (50). Alternatively, a chromogenic modification has been developed for the faster determination of titers (54). Another disadvantage is that the OPKA has many manual steps and requires at least 40 l of serum sample to test a single serotype.…”

Trojan Horse Effect: Phagocyte-Mediated Streptococcus iniae Infection of Fish

“…In this case, S. iniae probably gains access to the bloodstream and maintains a high level of bacteremia, leading to the onset of a generalized disease and meningitis, as described for other diseases (14,27). Similarly to Streptococcus pyogenes (group A streptococcus) infection in humans, where serotype replacement in a population (24) is most likely the result of the immune status of the individuals along with the introduction of a highly virulent organism (8), the propensity of S. iniae to cause an invasive disease in fish is likely related not only to the immune status of the fish but also to the pathogenetic mechanism(s) of virulent strains.…”