Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Standardization of
Neisseria meningitidis
Serogroup B
Colorimetric Serum Bactericidal Assay
Tamara Rodrı´guez,* Miriam Lastre, Barbara Cedre´, Judith del Campo, Gustavo Bracho,
Caridad Zayas, Carlos Taboada, Miriam Dı´az, Gustavo Sierra, and Oliver Pe´rez
Departments of Basic and Clinical Immunology and of Enteropathogenic Bacteria, Finlay Institute, Habana City, Cuba Received 18 July 2001/Returned for modification 5 September 2001/Accepted 26 October 2001
The correlate of protection for serogroup B meningococci is not currently known, but for serogroup C it is believed to be the serum bactericidal assay (SBA). The current SBAs are labor intensive and the variations in protocols among different laboratories make interpretation of results difficult. A colorimetric SBA (cSBA), based on the ability ofNeisseria meningitidisserogroup B to consume glucose, leading to acid production, was standardized by using group B strain Cu385-83 as the target. The cSBA results were compared to those obtained for a traditional colony-counting microassay (mSBA). Glucose and bromocresol purple pH indicator were added to the medium in order to estimate growth of cSBA target cell survivors through color change. Different variants of the assay parameters were optimized: growth of target cells (Mueller Hinton agar plates), target cell number (100 CFU/per well), and human complement source used at a final concentration of 25%. After the optimization, three other group B strains (H44/76, 490/91, and 511/91) were used as targets for the cSBA. The selection of the assay parameters and the standardization of cSBA were done with 13 sera from vaccinated volunteers. The titers were determined as the higher serum dilution that totally inhibited the bacterial growth marked by the color invariability of the pH indicator. This was detected visually as well as spectrophotometrically and was closely related to a significant difference in the growth of target cell survivors determined using Student’sttest. Intralaboratory reproducibility wasⴞ1 dilution. The correlation between bactericidal median titers and specific immunoglobulin G serum concentration by enzyme immunoassay was high (rⴝ0.910,P< 0.01). The bactericidal titers generated by the cSBA and the mSBA were nearly identical, and there was a high correlation between the two assays (rⴝ0.974,P< 0.01). The standardized cSBA allows easy, fast, and efficient evaluation of samples.
Serogroup B strains ofNeisseria meningitidis are an impor-tant cause of meningitis, an epidemic disease that is a major health problem in various parts of the world (10, 24). The role of circulating antibody and complement in protection from meningococcal disease was demonstrated in 1918 (14, 16). In the 1960s, classic studies by Goldschneider et al. provided evidence that protection of humans from meningococcal dis-ease correlated with the presence of serum bactericidal activity (12, 14).
The serum bactericidal assay (SBA) is a functional measure of the ability of antibodies in conjunction with complement to kill bacteria and is considered the assay of choice for measure-ment of functional antimeningococcal antibodies in vitro. Dif-ferent protocols have been developed to demonstrate the pres-ence of bactericidal antibodies, but all of them have three main elements: bacteria, antibody, and complement. Available SBAs differ in the number of CFU per well (1, 9, 18), assay buffer (9, 18, 26), growth of the target strain (9, 12, 18), assay incubation time (18, 19, 26), complement source (5, 12, 15, 18, 12, 28, 29), complement concentration (11, 15, 26), and starting serum dilution (5, 25, 26).
The minimum level of protection by antibodies was estab-lished by Golschneider et al. (12) for serogroup C using human complement at a titer ofⱖ4. Recently, Borrow et al. (2)
rees-tablished these correlates with baby rabbit complement. The potential effectiveness of polysaccharide vaccines is evaluated through detection of the induction of bactericidal antibodies. SBA is a well-established correlate for protection from sero-group A and C meningococcal disease (13). This criterion has been extended to nonpolysaccharide vaccines like those devel-oped against serogroup B. Several studies support a relation-ship between SBA and clinical protection from serogroup B meningococcal disease (3, 6, 15, 20, 29). However, data from one recent study suggest that SBA may underestimate the clinical efficacy of serogroup B vaccine (22).
The traditional SBA is considered labor intensive and not workable for large numbers of samples. The major problem with traditional SBAs lies with the techniques, which involve plating and counting of target bacteria. New protocols have been developed to replace the traditional SBAs; for example, Kriz et al. described a modification of the bactericidal microas-say using triphenyltetrazolium chloride solution (TTCmSBA) as a germination indicator for visualizing the results (17). Re-cently, Mountzouros and Howell described a fluorescence-based SBA (fSBA) for serogroup BN. meningitidis(21). More investigation is needed to standardize a universally accepted SBA for the detection of serogroup BN. meningitidis.
The purpose of this study was to select assay parameters (target strain preparation, number of CFU per well, and com-plement concentration) that led us to standardize a colorimet-ric SBA (cSBA) based on the ability ofN. meningitidis sero-group B to consume glucose, leading to acid production. We added glucose and a pH indicator to the medium in order to
* Corresponding author. Mailing address: Finlay Institute, 27 ave-nue, % 198 and 202, La Lisa, P.O. Box 16017, Havana City, Cuba. Phone 53 (7) 217597 or 218321. Fax: 53 (7) 286075. E-mail: trodriguez @finlay.edu.cu.
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estimate growth of SBA target cell survivors through color change. Thereafter, we optimized the assay to obtain intral-aboratory reproducible titers with a variety of sera from im-munized adult volunteers and compared the results generated by the cSBA with those generated by a traditional colony-counting microassay (mSBA).
MATERIALS AND METHODS
Bacteria.The Cuban vaccineN. meningitidisserogroup B strain Cu385-83 (B:4:P1.19.15;L3,7,9) was used as the target strain in the standardization of the bactericidal assay. Three other strains of serogroup B were used as target strains in the standardized cSBA. They were 511/91 (B:2b:P1.10), isolated in Argentina; 490/91 (B:14:P1,7), isolated in Uruguay; and 44/76 (B:15:P1.7.16), isolated in Norway. These strains were stored in skim milk (Oxoid Ltd., Basingstoke, Hamp-shire, England) at⫺70°C in multiple aliquots. A fresh aliquot was obtained for each experiment to eliminate differences in the passage history of the strain.
Serum samples.Sera obtained from 13 young adults (18 to 23 years old) immunized 7 years before with two doses of VA-MENGOC-BC, the Cuban antimeningococcal vaccine against serogroups B and C ofN. meningitidis, were evaluated by a standardized meningococcal enzyme-linked immunosorbent assay (ELISA) and by Western blot. The serum with the higher titer of immunoglob-ulin G (IgG) antibodies by ELISA that also reacted against main proteins in the Western blot (proteins present in the outer membrane vesicles [OMV] of VA-MENGOC-BC) was selected as the positive control. This serum was used for the evaluation of assay parameters and as the positive control in all of the assays performed to standardize the test in which the other 12 sera were also used. Those sera were heat inactivated (56°C, 30 min) and stored at⫺70°C.
Normal human serum (NHS) from two adults who had never been immunized with any meningococcal vaccine and had never had meningococcal disease were evaluated by ELISA and Western blot to ensure that they did not respond against OMV and were also validated by use in the standard SBA at 25 and 50% concentrations. Those sera were processed (quick-frozen with dry ice and etha-nol) to preserve hemolytic complement activity and stored at⫺70°C.
The positive control serum and the two NHS (collected as possible sources of complement) were obtained by plasmapheresis. All sera were collected after informed consent was obtained from the participants.
Standardized cSBA. All the steps of the cSBA were performed in safety cabinets. The test strain was streaked for isolated colonies and incubated over-night at 37°C with 5% CO2on Mueller-Hinton (Oxoid)–10% fetal bovine serum
(Sigma) (MH-FBS) agar plates. The strain was subcultured by spreading cells over the entire surface of another MH-FBS agar plate and incubated for 4 h at 37°C with 5% CO2. Cells were suspended to an optical density (OD) of 0.5 to
0.55 at 600 nm in physiological saline solution pH 7.2 to 7.4. The cell suspension was diluted to yield 100 CFU/12.5l. Heat-inactivated serum samples (25l) were serially diluted twofold in Hanks’ balanced salt solution (HBSS; Flow Laboratories, Irvine, Scotland) containing 0.1% bovine serum albumin (BSA; Sigma), pH 7.2 (HBSS-BSA), in 96-well, flat-bottom tissue culture-treated plates (Costar, Inc., Cambridge, Mass.). Then 12.5l of cell suspension and 12.5l of complement were added. The final concentration of complement in each well was 25%.
Control wells included on each microtiter plate contained (i) bacteria and buffer (suspension control), (ii) buffer, bacteria, and complement (complement-dependent control), and (iii) heat-inactivated test serum, bacteria, and buffer (complement-independent control). The positive control serum of known titer was included on each plate as a criterion to accept the plate. The final volume in each well was 50l. The first reciprocal serum dilution as well as the final dilution of the complement-independent serum control was 2. The number of CFU added to each well before incubation with complement (time zero [T0]) was
obtained by plating 12.5l of bacterial stock solution on MH-FBS agar plates. The microtiter plate was incubated at 37°C without CO2for 30 min. After
incubation, 150l of Mueller-Hinton broth (Oxoid) (MHB) containing 2% glucose (BDH Laboratory Supplies, Poole, England), 2% bromocresol purple (Fluka Chemika, Switzerland), and vancomycin-colimycin-nystatin (VCN) inhib-itor (bioMerieux SA, Marcy I’Etoile, France) were added to each well. The VCN inhibitor was used to ensure the absence of contaminant microorganisms that could affect the results of the assay. The plate was further incubated for 20 h at 37°C with 5% CO2. The color change was detected visually as well as
spectro-photometrically in a microplate reader (Titertek Multiscan Plus) at 405 nm. This equipment was placed into the safety cabinet because in the final step there are still viable broth cultures.
The bactericidal titer for each serum sample was expressed as the reciprocal
serum dilution yieldingⱖ90% killing, that is, total inhibition of bacterial growth marked by the color invariability of the pH indicator, and corresponds to an OD in a range of 0.1 to 0.4 at 405 nm of.
Standardization of assay components.The assay components (target strain preparation, number of CFU per well, and complement) described above were evaluated on the basis of reported methods and the particular characteristics of the cSBA. The different variants were compared and optimized with sera from immunized volunteers. The results were obtained from at least three indepen-dent assays run on separate days.
(i) Target strain preparation.The influence on the cSBA titers of growth (broth or agar plate growth) of the working cell culture and the number of CFU per well used were evaluated. Target strain Cu385-83 was grown to log phase (2 to 3 h) in MHB at 37°C in a shaker at 150 rpm until the OD was 0.25 to 0.3 at 620 nm. The titers of five sera obtained with these log-phase cultures were compared with the titers obtained with the same strain grown for 4 h on MH-FBS agar plates. Each culture was diluted to give 100 CFU per well atT0. The effect
of different CFU numbers on the cSBA titers in relation to the incubation time for visualizing the color change was evaluated. The strain was diluted to give 50, 1⫻102, 5⫻102, 1⫻103, and 1⫻106CFU per well atT
0. Four sera were run
against each target cell number.
(ii) Complement.Sera from two adults that lacked bactericidal activity against the serogroup B target strain were tested to determine its suitability (the CFU per well in the complement control well wasⱖ80% of the CFU per well atT0and
color change of the pH indicator in this well) as a complement source. The strain was grown and diluted in HBSS-BSA as described above. Eight replicates of complement-dependent control wells were prepared, and the microtiter plate was incubated for 30 min as described above. The minimal concentration of the NHS selected as the complement source needed for efficient bactericidal killing was estimated by using target strain Cu385-83. The bactericidal titer of the positive-control serum was measured with various concentrations of the comple-ment source (25, 20, 16.8, 13.8, 12.5, 11.3, 8.8, and 6.3%).
Intralaboratory evaluation.The standardized assay described above was used to test 12 sera and one positive control serum from vaccinated volunteers for bactericidal activities to serogroup B strain Cu385-83. Each serum sample was tested in duplicate on five different days. After visual determination of the titers, the absorbance was measured at 405 nm to confirm the titers by quantitative determination. For each evaluated serum, titration curves were drawn using the OD obtained at each serum dilution.
Specificity of cSBA. The numbers of CFU in blue and green wells were counted and compared in order to demonstrate that the color change of the pH indicator was specifically related to the growth of cSBA target cell survivors. Samples of 10l from some wells before and after the color change from different dilutions of seven positive sera as well as from six negative sera, the suspension control wells, and the complement-dependent control wells were removed at the end point of the cSBA, after 20 h of incubation at 37°C with 5% CO2. They were conveniently diluted, added as droplets to MH-FBS agar, and
incubated as described above.
ELISA.A standardized ELISA was performed in triplicate in microtiter plates (Maxisorp; Nunc, Rothskild, Denmark) with a peroxidase-conjugated anti-hu-man IgG detection system (Sigma, St. Louis, Mo.). OMV (20g/ml) from group B strain Cu385-83 was used as the antigen. As an internal antibody standard, a twofold dilution series of a positive postvaccination serum sample was used in all experiments. The mean value of the observed OD was transformed to arbitrary units per milliliter by a sigmoidal standard curve. Initially, all serum samples were analyzed at a 1:200 dilution. Samples with OD values ofⱖ90% of the maximum OD of the standard were further diluted and analyzed again (7).
mSBA.A traditional serum bactericidal microassay was performed in a labo-ratory with broad experience in performing mSBA as previously described (4, 19). Briefly, 25l of Hanks’ solution from the 2nd to 11th columns of wells in a 96-well microtiter plate and 25l of serum samples in the first well of each line were added, making a twofold dilution. Then, 12.5l of bacterial suspension (containing around 50 CFU) and 12.5l of human serum as complement source were added. The plate was incubated at 37°C without CO2for 30 min. After
incubation, Mueller-Hinton agar (150l/well) was added to the whole plate. The plate was incubated overnight at 37°C with 5% CO2, and the colonies were
counted in all wells with a stereoscopic microscope. The results were expressed as the reciprocal serum dilution yielding less than or equal to 50% survival compared to the bacterial counts from control wells that contained only com-plement and bacteria. The human serum used as a source of comcom-plement was from a Cuban blood bank and shown to be negative when tested for anti-N. meningitidisgroup B antibodies.
Statistical analysis.The Pearson product-moment correlation coefficient (r) was calculated by using the log2-transformed intralaboratory median cSBA titer
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and log2-transformed ELISA antibody concentration for each serum sample. It
was also calculated by using the log2-transformed cSBA titer and the log2
-transformed mSBA titer for each serum sample (27). Student’sttest was used to examine the significance level of differences between the number of CFU per well corresponding to each color of the pH indicator, blue (bactericidal activity zone) and green (growth zone).
RESULTS
Standardization of assay components. (i) Target strain preparation.There were no differences between broth- and agar plate-grown serogroup B target strain Cu385-83. The ef-fect of the CFU concentration per well on cSBA titer and its relation to incubation time for visualizing the color change was evaluated. The highest evaluated concentration of CFU per well (106) resulted in the lowest cSBA titer after 8 h of
incu-bation. Little or no difference in titers of evaluated sera with 100 and 50 CFU per well were found; the color change was visualized after 20 h (Fig. 1). The target of CFU per well selected for the cSBA was 100 CFU/12.5l.
(ii) Complement source. Serogroup B target strain Cu 385-83 was tested for sensitivity to two NHS lacking bacteri-cidal activity against this strain. The strain was sensitive to one of the two evaluated sera as a possible source of complement. This serum reduced the cell count by at least 90% from the CFU per well at T0. The strain had acceptable cell growth (ⱖ80% of CFU per well atT0) with the other evaluated serum, and it was selected as the complement source.
Various complement concentrations were tested with the target strain to ensure that complement was not limited in the cSBA. Complement concentrations between 25 and 16.3% were optimal, with no difference in cSBA titers. Reductions of 2 dilutions in titer were observed with complement concentra-tions of 13.8 and 11.3% and of 3 diluconcentra-tions with 8.8 to 6.3% complement concentrations.
Intralaboratory evaluation. The standardized assay de-scribed above was used to test 12 sera from immunized adults for bactericidal activity to serogroup B (strain Cu385-83). In the cSBA, blue and green meant serum bactericidal activity and bacterial growth, respectively. The OD in all dilutions of
the negative sera as well as in complement-dependent and suspension control wells were in the range of 0.5 to 0.9. How-ever, lower OD values in the area of bactericidal activity of the positive sera were observed, in the range of 0.1 to 0.4, followed by an abrupt increase in OD corresponding to serum dilutions where there was growth of target cell survivors (Fig. 2A). The same titer to each serum was found in 78% of 10 replicates of five positive sera and ⫾1 dilution in the remaining 22% of replicates. The negative sera always had the same titer (⬍2).
Specificity of cSBA.The count of CFU of each color showed that the change from blue to green in all evaluated positive sera corresponded to an abrupt increase in CFU. The average CFU in blue wells of evaluated positive sera was 2.87⫻107,
while the average CFU in green wells was 4.93⫻108. In all
negative sera, the color of the pH indicator changed from blue to green at all dilutions, and the average CFU count was 6.4⫻ 108. In complement-dependent and suspension control wells, FIG. 1. Geometric mean (GM) bactericidal antibody titers for
pos-itive-control serum titrated with different numbers of CFU. Different concentrations of the serogroup B target strain were incubated with the serum and the complement at 37°C for 30 min. The cSBA titers were defined as the reciprocal of the serum dilution that totally inhib-ited bacterial growth, marked by the color invariability of the pH indicator. The standard errors of the mean, calculated with three replicates of titration of the positive-control serum for each CFU per well, were computed using the log2data.
FIG. 2. (A) Spectrophotometric determination of cSBA titers. Ti-tration curves of the positive and negative control sera and of three other sera titrated in triplicate are shown. Sera A and B are from vaccinated adults, and serum C is from a nonvaccinated adult. All negative sera had the same OD corresponding to the green color in all dilutions at which bacterial growth was observed. All positive sera changed OD, and this was related to the bactericidal activity. The titers determined for the three positive sera are shown to point out where the endpoint is in relation to the color change. (B) Relationship be-tween OD values and number of CFU per well in positive and negative sera. Solid symbols, CFU; open symbols, OD. Triangles and squares represent positive and negative control sera, respectively. Triplicate assays were performed.
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the number of CFU was approximately the same as that in green wells for all positive and negative sera (Fig. 2B). The average CFU in blue and green wells was calculated, and significant differences (P⬍0.01) between them were found.
Use of different target strains on standardized cSBA.After the assay parameters were optimized, growth of target cells (MH-FBS agar plates), target cell number (100 CFU/per well), human complement source used at a final concentration of 25%, and assay incubation time (20 h), three disease strain isolates were used as the target for the cSBA. Three positive sera and the positive control serum were titrated. Titers varied by 6 dilutions (Fig. 3). Titer differences were serum dependent. Strain 511-91 produced titers consistently lower than those of the other strains evaluated, but for the evaluated sera, the highest titers were produced against the vaccine strain Cu385-83. Each strain gave consistent (⫾1 dilution) intrastrain titers with the evaluated sera.
cSBA titers correlate to titers obtained in an mSBA and to ELISA IgG concentrations. The 13 human sera used in the standardization of the cSBA and the serum used as the source of complement were evaluated by ELISA and by mSBA. The results of those assays were compared with the cSBA titers in order to determinate the relationship between them. There was high correlation between ELISA and cSBA titers (r ⫽ 0.910,P⬍0.01). The bactericidal titers generated by the two assays were nearly identical; 50% of evaluated sera generated the same titer in both assays, and in the other 50% the cSBA titers were in a range of⫾1 dilution of the titers obtained for the mSBA. A direct correlation (r⫽0.974,P⬍0.01) between the cSBA and mSBA titers was demonstrated.
DISCUSSION
The bactericidal assay is a good laboratory surrogate for predicting protective titers for meningococcal vaccines (3, 6, 20). The main drawback with traditional SBAs is that the colony counts cannot be totally automated (1, 9, 15, 18), and that is why they have been described as difficult to standardize,
labor-intensive, and cumbersome (21). In this study, we opti-mized the parameters for intralaboratory reproducibility of a serogroup BN. meningitidiscSBA and compared it to a tradi-tional mSBA.
A number of bactericidal buffers have been used by different workers, but HBSS-BSA was selected for this cSBA because this has been the most widely used for serogroup B SBAs (9, 15). In order to avoid a pH increase in HBSS-BSA with stor-age, we used it for only 5 days after preparation. Maslanka et al. (19) reported that different lots of BSA gave different re-sults; in our study, we used the same source of BSA in all assays. Further studies will show the influence of BSA in the cSBA buffer and the possibility of using HBSS without BSA, as was suggested by Maslanka et al. (19).
An incubation time that gives maximum titers with a mini-mum reduction in the number of CFU in control wells must be selected for each strain. Many researchers have shown that the optimal incubation time of target strains with complement in SBA is strain dependent (1, 18, 19, 26). We used a 30-min incubation time, as reported by Milagres et al. (20), who used the same target strain (Cu385-83).
Although various researchers reported different methods for growing target cells prior to use, most agree that cells should be in log phase. Frasch and Robbins (9) have stated that serogroup B strains grown on agar plates for 4 h are in log phase. Our data showed that growth of the serogroup B target strain Cu385-83 to log phase in broth gave titers that were more or less equivalent to those for the same strain grown for 3 to 5 h on agar plates, suggesting that the organisms were in the same metabolic state. Goldschneider et al. (12) reported that assay results are dependent on the lot of Mueller-Hinton agar used to grow target strains. We did not observe any noticeable difference in assay titers when we used different lots of MH-FBS agar plates.
The number of CFU per well is a very important parameter on cSBA because it is closely related to assay sensitivity and time to visualization of the results. This assay uses 100 CFU per well, which allowed color change to be observed after 20 h of incubation of the plate. When 106CFU per well was
eval-uated, the incubation time was reduced to 8 h, but lower titers were detected. The cell number used in previously reported SBAs varies between the different growth methods of target cell survivors of each technique. In bactericidal assays, which are carried out in microtiter plates on solid medium, 40 or 50 to 130 CFU per well is regarded as acceptable to facilitate the counting of target cells in microtiter plates (4, 9, 15). In con-trast, in bactericidal assays in which colony counting is done in large agar plates, the cell number is higher (1, 18).
SBA is a functional measure of the ability of antibodies in conjunction with complement to kill bacteria. The age-related inverse relationship between meningococcal bactericidal activ-ity and meningococcal disease was first demonstrated with a human complement source (12). We used as the complement source a serum from an individual who had never been immu-nized with any meningococcal vaccine and never had menin-gococcal disease. The optimal final complement concentration in the assay was 25%. Ideally, this assay would combine anti-bodies and complement derived from the same individual to give a complete assessment of an individual’s ability to fight infection. However, it would be difficult to ensure that all sera
FIG. 3. Effects of using different target strains on the cSBA titer (reciprocal serum dilution) of positive sera. The four serogroup B strains were incubated with serum and complement at 37°C for 30 min. The cSBA titers were defined as the reciprocal of the serum dilution that totally inhibited bacterial growth, marked by the color invariability of the pH indicator. Triplicate assays were performed.
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were treated to preserve complement, particularly in remote areas. That is why the majority of studies of serogroup B meningococcal vaccines have used donor (nonvaccinee) hu-man complement for the measurement of bactericidal titers. Such sera may, however, be difficult to find for some strains (18).
Some researchers have evaluated the use of other comple-ment sources, mainly pooled baby rabbit serum (BRS). Those reports (18, 29) have noted differences in the level of bacteri-cidal activity when rabbit serum was replaced with nonimmune human serum. Mandrell et al. (18) suggested that the differ-ence in the level of serogroup B polysaccharide bactericidal activity between human complement and rabbit complement may be due to IgM antibody, since it appeared that there was no difference in activity between complement sources with IgG antibody. One recent study in which SBA on the same speci-mens using both donor human complement and the vaccinee’s own complement was measured found no difference between the bactericidal titers in tested sera using donor or the vaccin-ee’s own complement source. These findings support the va-lidity of using properly screened human donor complement in SBAs against serogroup B meningoccoci (8).
When all assay parameters were selected, the intralabora-tory evaluation was done. This study has confirmed the estab-lished intralaboratory reproducibility of⫾1 dilution (12). Fur-ther study with pre- and postimmunization sera is necessary to confirm that the cSBA is a useful method to detect functional antibody produced in response to VA-MENGOC-BC or other existing and new serogroup B meningococcal vaccines.
We demonstrated that the color change of the pH indicator was directly related to the growth of target cell survivors when significant differences between the number of CFU on each color were found. That is why we can affirm that the color change allows us to differentiate unequivocally the points where there was growth inhibition due to the serum bacteri-cidal activity from those where bacterial growth was not af-fected. The surviving bacteria in blue wells was minor, 8% of the number of CFU in green wells. It means that more than the 90% of target bacteria were killed in each blue well in the cSBA. That is why we expressed the titer as the highest serum dilution which caused total inhibition of bacterial growth marked by the color invariability of the pH indicator. While the Cuban vaccine strain Cu385-83 was used for assay standard-ization, three other serogroup B strains were used as targets for the cSBA, showing that this assays works with group B strains other than Cu 385-83.
The cSBA was compared with the mSBA. The main differ-ence between these assays lies in how the surviving bacteria are detected. The comparison of the results generated by these methods demonstrated a high correlation despite the interven-tion of two different operators, the use of two different human sera as source of complement, and two different laboratories. Various methods (agar overlay, tilt, and spot) have been used for growing the surviving cells after incubation with serum and complement. All of them, as well as the microassay com-pared in this study to the cSBA, employ colony-counting tech-niques to determine the results. Those techtech-niques of titration are labor-intensive, and the evaluation of a large number of samples in one assay is a hard task. A recently described fSBA for group BN. meningitidisuses the reduction-oxidation
(re-dox) indicator in Alamar Blue to detect surviving bacteria after SBA components are allowed to react in a microtiter plate (21). The cSBA, like the fSBA and the TTCmSBA, eliminates the labor-intensive steps of plating reaction mixtures and counting numbers of individual CFU, which are critical for interpretation of the results of traditional SBAs (17, 21).
The 96-well format makes both fSBA and cSBA potentially adaptable to robotic systems similar to those used for ELISA but taking appropriate safety stops because both assays use live bacteria until the end point. Although the fSBA has the ad-vantage of quantitatively detecting viable microorganisms more rapidly than a traditional SBA, the cSBA gives the pos-sibility of titrating by just looking at the plate, without the use of any equipment. Furthermore, the titration can be confirmed spectrophotometrically. Those new protocols allow investiga-tors to evaluate serum bactericidal activity against serogroup B
N. meningitidiswithout the limitations of traditional SBAs. In conclusion, the standardized cSBA can potentially be used to screen several serum samples, allowing an extremely easy and quick determination of results. This assay promise to be a useful tool in further studies to elucidate the correlates to protection in animal models, vaccine efficacy trials, postmar-keting surveillance studies, and epidemiological studies.
ACKNOWLEDGMENTS
We gratefully acknowledge Sandra Steiner (Respiratory Diseases Branch, Centers for Disease Control and Prevention) and Pierre Voet (R&D Bacteriology DAP, GlaxoSmithKline) for the acute revision of and important suggestions about the manuscript. We also thank Maria A. Camaraza (Finlay Institute) for performing the colony counting microassay.
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