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Clinical and Diagnostic Laboratory Immunology, October 2005, p. 1238-1242, Vol. 12, No. 10
1071-412X/05/$08.00+0 doi:10.1128/CDLI.12.10.1238-1242.2005
Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333
Received 16 June 2005/ Returned for modification 11 July 2005/ Accepted 15 August 2005
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In this study, we developed a rapid OPA that uses bacterial cell viability as an endpoint for the measurement of functional pneumococcal antibodies. We compared killing OPA titers obtained by two different viability endpoints (fluorometric versus colony counting) in a single-serotype and multivalent format. We evaluated the fluorescent OPAs using a panel of quality control sera (19). A set of 12 paired reference sera (pre- and postvaccination) from adult volunteers immunized with a 23-valent polysaccharide vaccine were collected, aliquoted, and lyophilized at the Oxford Blood Transfusion Service, Oxford, United Kingdom.
All Pnc strains used in the single-serotype or monovalent fluorescent OPA (fOPA) were as previously described (18). The strains used in the fluorescent multivalent OPA (fmOPA) were as described by Bogaert et al. (2) except for Pnc serotype 14 (optochin resistant, donated by M. Nahm, University of Alabama, Birmingham, AL), and Pnc serotype 19F (DS1235-99, penicillin resistant at 0.5 µg/ml), a clinical isolate from the Streptococcal Reference Laboratory, CDC, Atlanta, GA. Pnc serotypes were confirmed by Quellung reaction using type-specific antiserum. Antibiotic resistance was confirmed for the fmOPA strains. All strains were at least 90% opaque except for the rifampin-resistant 18C strain (50% opaque).
The reference OPA used was performed as published previously (18). This single-serotype OPA has a colony counting endpoint, and it was used for comparison to develop and standardize the fOPA and fmOPA. The reference OPA used HL-60 cells differentiated into polymorphonuclear leukocytes for 5 days with an effector-to-target cell ratio of 400:1 (6, 18). OPA titers were determined as the reciprocal of the serum dilution with 
50% killing of the bacterial load. Rabbit complement was purchased from PelFreez (Brown Deer, WI). The bacterial load was 1,000 CFU per well. The reference preparation was purified immunoglobulin G (Bayer, Elkhart, IN).
The single-serotype fOPA followed exactly the same format and used the same reagents and target strains as the reference OPA (18). Modifications include the following. After the 45-minute incubation period for opsonophagocytosis, the microtiter plate was centrifuged for 5 min at 600 x g to differentially pellet HL-60 cells, while leaving the bacteria in suspension. Using a robotic liquid handler (Precision 2000; Biotek, Winooski, VT) programmed to avoid disturbing the HL-60 cell pellet, 20 µl from each well of the plate was replica plated onto a daughter plate containing 40 µl per well of alamar blue buffer. The alamar blue buffer consisted of 25% Todd-Hewitt broth containing 0.5% yeast extract (THYE), 65% opsonophagocytosis buffer (Hanks balanced salt solution with 0.1% gelatin), and 10% alamar blue indicator (Trek Diagnostic Systems, Cleveland, OH). The daughter plates were incubated at 37°C and 5% CO2 for 5 to 6 h until the complement control wells read 20,000 fluorescent units (FU) in a fluorometer at 530/590 nm for excitation/emission wavelengths (BioTek model FL600; gain, 75). The metabolic activity of the bacteria changed the alamar blue to a lavender color. OPA titers were determined as the reciprocal of the dilution with 
50% of the mean fluorescence detected in the complement control wells in the absence of serum antibodies but with all other assay components present.
The format of the fmOPA was similar to that of the fOPA, but the fmOPA used a mixture of seven Pnc strains, each with a different antibiotic resistance marker. The multivalent assay required additional modifications, such as an effector/target cell ratio of 11:1, 8 x 105 differentiated HL-60 cells per well, additional complement source (20 µl per well), and additional opsonophagocytosis buffer (140 µl per well) to be added to a mother plate to generate seven daughter plates. Each strain was diluted in OPA buffer, and all seven strains were mixed to yield a final suspension containing approximately 10,000 bacteria per strain in a 20-µl volume for a total bacterial suspension of 70,000 bacteria in each well. Lower numbers of bacteria per strain in the mother plate did not affect the titer (1,000 to 10,000); however, it resulted in longer incubation periods for the daughter plates (up to 8 h) to obtain similar fluorescent signals. Each daughter plate contained a different antibiotic at half the MIC for each given strain. OPA titers were determined as described above for the fOPA.
Figure 1 gives the signal-to-noise ratios obtained with five different reaction mixtures containing various THYE concentrations and a constant alamar blue concentration (10%) at various inocula of bacteria. A 10% concentration of alamar blue in the reaction mixture yielded a final concentration of 6.5% to measure metabolic activity by the bacterial inoculum.
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92% matched within 3 twofold dilutions). Agreement was lower (3 dilutions) for serotypes 6B, 14, and 18C, with 75%, 79.2%, and 83.3%, respectively. In addition, the fmOPA titers were compared to the published median values from a multilaboratory study where five laboratories tested the same sera using the reference OPA method (19). OPA titers that were more than 3 twofold dilutions apart from this median titer were repeated, and the median values of all repeats were used for further analysis. Cumulative percentages are shown in Table 1.
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0.09). Only serotype 9V showed a statistically significant difference (P < 0.036) by the ANOVA comparison described above and by Mann-Whitney rank sum analysis of fmOPA titers and in-house reference OPA titers. ANOVA among reference OPA, reference multilaboratory OPA, and fOPA titers for serotype 9V showed no statistically significant difference (P = 0.138), nor did the Mann-Whitney rank sum test comparing fmOPA titers to reference multilaboratory OPA titers (P = 0.256). The logarithmically transformed titers (base 2) were plotted in identity graphs for regression analysis using Microsoft Excel, version 2002 (Fig. 2). Comparison of reference OPA with fOPA titers yielded r values ranging from 0.79 to 0.98 (Table 1), and slopes were between 0.71 and 1.0. When fmOPA titers were compared to in-house reference OPA titers, the r values ranged from 0.66 to 0.92 and the slopes from 0.37 to 0.78. Finally, comparison of the fmOPA titers to the median values from the multilaboratory study gave r values ranging from 0.76 to 0.97 and slopes from 0.47 to 1.19.
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Our alamar blue fOPA yielded a signal-to-noise ratio of 6:1 under optimal buffer conditions (Fig. 1), which is an improvement over the chromogenic indicator OPA developed for two Pnc serotypes by Lin et al. (13) with similar numbers of bacteria. The alamar blue indicator has been successfully incorporated into serum bactericidal activity assays for Haemophilus influenzae type b and Neisseria meningitidis to achieve fluorometric readouts of viability without affecting titer determination (15, 20).
Our fmOPA yielded high agreement (Table 1) with median titers from a previously reported multilaboratory study (19). Although different bacterial strains were used by the methods, the titers were largely conserved. Titers for all serotypes agreed within 3 dilutions (twofold) for at least 75% of the sera. The multilaboratory evaluation of the reference OPA resulted in 80% or greater interlaboratory agreement in OPA titers for the serotypes evaluated (19). Advantages of the fmOPA are semiautomation of a single-day assay, elimination of colony counting, multivalent capacity, and ease in data analysis. In addition, the amount of sera required for testing of seven serotypes is reduced by 86%, and throughput is greatly enhanced in comparison to the reference viability assay.
Comparison of the fmOPA to the reference multilaboratory OPA yielded better agreement than to the in-house reference OPA. Since the multilaboratory titers were the median values from several laboratories, they account better for the equivocal titers given by some sera. The lack of agreement in titer for selected sera may be due to limiting complement concentrations or to individual serum antibody avidities. Increasing the amount of complement in the assay improved the titers for some trouble sera, particularly in serotype 14, but compromised the performance of other serotypes (data not shown). In addition, three trouble sera had low avidity for serotype 9V or 14 (33 to 53% reduction in antibody concentration with 0.5 M thiocyanate elution). It has been suggested that sera with high titers (above 512) be retested at an initial dilution higher than 1:8 (7). This would be a practical approach to confirm the linearity of high-titer sera, as well as ensuring a complement excess in the multivalent reaction mixture.
The antibiotic resistance strain for serotype 6B used in the multivalent assay (fmOPA) exhibited significantly lower growth rates compared to all other strains in the panel. We have identified a replacement strain for serotype 6B with erythromycin resistance for selection. Quality assurance is needed when using antibiotic-resistant Pnc strains. Growth, maintenance, and storage of master and working bacterial stocks must be controlled for strain stability in terms of growth rate, opacity, and antibiotic resistance marker.
In summary, the fmOPA allows for the simultaneous determination of a viability endpoint for seven Pnc serotypes in one mother plate setup, thereby reducing the serum volume required for testing. Our analysis showed high agreement of the fmOPA with the reference viability method: at least 75% of titers matched within 3 dilutions for all serotypes. The assay eliminates colony counting, produces results in a single day, and can be a useful tool for pneumococcal vaccine evaluation.
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ACKNOWLEDGMENTS |
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We thank the Association of Public Health Laboratories (APHL) for providing funding for Kathryn Bieging and Gowrisankar Rajam through the Emerging Infectious Diseases Laboratory Fellowship program.
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FOOTNOTES |
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REFERENCES |
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| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. | Infect. Immun. |
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| J. Clin. Microbiol. | J. Virol. | ALL ASM JOURNALS |
