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.
 |
INTRODUCTION |
Streptococcus pneumoniae
is an important pathogen, and there is currently an effort to develop a
vaccine against S. pneumoniae that would be more effective
than the current 23-valent vaccine (14). Vaccine
development would be facilitated if there were a simple surrogate assay
for pneumococcal vaccine responses. Antibody concentrations estimated
by enzyme-linked immunosorbent assay (ELISA)-type methods are commonly
used as the measure of vaccine responses. However, it was found that
ELISAs for pneumococcal antibodies were sometimes nonspecific, thus
often producing falsely high responses (4, 17). Even if
the concentration of pneumococcal antibody could be accurately
determined by ELISA, ELISA may measure nonfunctional low-avidity
antibodies as well as high-avidity antibodies (3, 6). For
these reasons, assays, such as the opsonophagocytic assay, that measure
the function of pneumococcal antibodies would be useful.
While the opsonophagocytic killing assay is widely accepted as one of
the best surrogate assays for vaccine-induced protection, the assay is
tedious to perform, and several improvements have been made to simplify
the assay. For instance, the classical assay was greatly simplified
with the use of microtiter plates, frozen bacterial aliquots
(1), and differentiated-HL-60 cells (13). We
have recently developed a double-serotype opsonization assay which can
yield the opsonophagocytic titers of two different serotypes in a
single assay with the use of antibiotic-resistant pneumococci (12). While these modifications have reduced the amount of
required effort and serum, the surviving bacteria still need to be
enumerated by counting their colonies. Moreover, the assays need to be
performed for many different serotypes because pneumococcal vaccines
contain up to 23 serotypes. Consequently, the difficulties in counting colonies make the assay impractical for a large-scale use.
To eliminate the need to count bacterial colonies, we investigated the
use of a formazan dye, XTT
(3,3'-[1{(phenylamino)carbonyl}-3,4-tetrazolium]-bis[4-methoxy-6-nitro] benzene sulfonic acid hydrate) (15). XTT is converted to a
colored, water-soluble product by live (but not dead) eukaryotic as
well as prokaryotic cells (15). This property can be
useful for assays measuring the ability of antisera to kill bacteria
with or without neutrophils. However, the conversion of XTT by bacteria
is relatively inefficient, and XTT can be used to measure the ability
of neutrophils to kill various bacteria only when a large number of
bacteria are present in the reaction well (15).
Unfortunately, the standardized pneumococcal opsonization assay uses a
small number (e.g., 1,000 CFU/well) of target bacteria. Therefore, XTT
would not be useful unless the number of pneumococci surviving at the
end of the assay could be increased in proportion to the number of
surviving bacteria. We found that a brief (e.g., 4 to 5-h) culture
prior to adding XTT increases the cell number and that the capacity of
antisera to opsonize S. pneumoniae can be rapidly and easily determined.
| |
MATERIALS AND METHODS |
Serum samples.
Adult volunteers were immunized with a
23-valent polysaccharide (PS) vaccine available from either Merck (West
Point, Pa.) or Wyeth-Lederle Vaccines (Pearl River, N.Y.), and serum
samples were obtained either before and/or 1 month after the
vaccination. A human serum pool (HSP-3) was prepared by mixing equal
volumes of sera from 1,638 blood donors (9). The pool was
stored in aliquots at 
20°C, and the pool was incubated in a 56°C
water bath for 30 min before being added to Todd-Hewitt broth
containing 0.5% yeast extract (THY medium). Another serum pool
(Ppool10) was made by pooling equal volumes of the sera from 10 individuals who received a pneumococcal PS vaccine 1 month before
phlebotomy. Ppool10 was used as a control for opsonophagocytic assays.
Twenty-two postimmune serum samples were mixed with their own preimmune
sera to obtain the samples with low opsonization titer and they were used to produce the data shown below (see Fig. 5).
Bacteria.
Three strains of pneumococci (strains DS221494,
DS2212, and DS2217) were obtained from G. Carlone at the Centers for
Disease Control and Prevention (Atlanta, Ga.). Their serotypes were,
respectively, 14, 6B, and 19F. A streptomycin-resistant variant of
DS2212 was obtained as described before (12) and labeled
R6BSR. An optochin-resistant variant of DS2217 was similarly obtained
and labeled R19FOR. R6BSR and R19FOR were grown in THY broth,
aliquoted, and frozen at 70°C until used.
Double-serotype opsonophagocytic killing assay.
Double-serotype opsonization assays were performed as previously
described (12). Briefly, differentiated HL-60 cells were diluted to 107 cells/ml in Hanks' buffer supplemented with
0.1% gelatin and 10% fetal calf serum (HGF buffer). The serum samples
for the test were serially diluted in HGF buffer. To obtain the maximal
assay sensitivity, undiluted serum was applied to the first well of the
serial dilution. Each strain (R6BSR or R19FOR) of frozen bacteria was
thawed and was diluted to 105 CFU/ml in HGF buffer. The two
bacterial solutions were mixed (1:1 by volume), and 20 µl of the
mixed bacterial solution was added to 10 µl of a diluted serum sample
in a well of 96-well microtiter plate. After a 15-min incubation at
37°C, 40 µl of HL-60 cell suspension and 10 µl of baby rabbit
complement (Pelfreeze, Browndeer, Wis.) were added to each well. The
mixture was incubated for 45 min at 37°C with shaking. A 5 µl
aliquot of reaction mixture was plated in a THY agar plate containing
streptomycin (100 µg/ml), and another 5 µl was plated in a THY agar
plate containing optochin (5 µg/ml). The THY agar plates were
incubated in a candle jar at 37°C for 7 to 9 h, and bacterial
colonies on the plates were counted. The opsonization titer of a serum
is defined as the final dilution of a serum that results in half as
many colonies as are seen with the control well containing all the
reactants except for the serum. For instance, if 10 µl of the neat
serum added to the well killed half of the bacteria, the opsonization
titer is 8, because the serum was diluted with the reactants. Each
sample was analyzed in duplicate.
Chromogenic opsonophagocytic killing assay.
Following the
double-serotype opsonization assay and after replica plating in THY
agar, the remaining reaction mixture was split into two by transferring
about one-half (40 µl) to another microtiter plate. To the wells in
the new plate, 20 µl of THY both containing 0.2% saponin, 10%
HSP-3, and optochin (5 µg/ml) was added. The human serum was added to
minimize the variable nutrient effect of the serum present in the test
wells at various concentrations. Saponin was added to lyse HL-60 cells,
which can degrade XTT. To the wells in the original plate, 20 µl of
THY broth containing 0.2% saponin, 10% HSP-3, and streptomycin (100 µg/ml) was added. The presence of optochin or streptomycin permits selective growth of serotype 19F or 6B, respectively. After incubating the plates for 4.5 h at 37°C, 20 µl of a freshly made XTT
substrate solution was added to each well. The substrate solution is
phosphate-buffered saline containing XTT (0.5 mg/ml; Sigma Chemical,
St. Louis, Mo.) and coenzyme Q (40 µg/ml; Sigma Chemical). The plates
were incubated at room temperature, and the optical density at 450 nm
(OD450) was measured with an ELISA reader (model EL309;
BioTek Instrument, Winooski, Vt.) at 0, 30, and 120 min. "Delta
ODs" were obtained by subtracting the OD obtained at 0 min from the
OD obtained at either 30 or 120 min. The opsonization titer of a serum
is defined as the final dilution of serum that produces delta OD that
was obtained with 500 CFU/well, which is half of the initial number of
bacteria of each serotype. Each sample was analyzed in duplicate.
| |
RESULTS |
Characterization of chromogen reaction step.
To determine the
reaction time and to determine that only the living, but not dead,
pneumococci convert XTT to water-soluble orange formazan, 100 µl of
THY broth containing zero, 2 × 106 living, or 2 × 106 dead pneumococci (strain DS2212) was mixed with 50 µl of the XTT substrate solution in a microtiter well, and its
OD450 was monitored during an incubation at room
temperature. Dead pneumococci were prepared by incubating at 65°C for
1 h. Due to the absorption of the light by the culture medium,
OD450 was readily detectable at the beginning of the
reaction. The OD of the wells containing no bacteria or dead bacteria
slowly increased over time, indicating that XTT spontaneously became
colored product but the dead bacteria did not actively convert XTT to
the colored product. However, the OD450 of the wells
containing the live bacteria increased severalfold faster than those of
the wells with no or dead bacteria for the first 5 h (Fig.
1A). This indicates clearly that the live bacteria produced the chromogenic product. After 5 h of
incubation, the rate of OD change of the wells with the live bacteria
became similar to those containing no bacteria. This may have happened because the bacteria died in 5 h. Based on these results, 1- to 3-h time periods were chosen to be the optimum for the chromogenic reaction.
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FIG. 1.
(A) OD450 (y axis) versus
time of color development (x axis). The wells assayed
contained either 2 × 106 live cells of S. pneumoniae 6B serotype strain DS2212 (solid triangle), 2 × 106 heat-killed cells of S. pneumoniae 6B
serotype strain DS2212 (open triangle), or no bacteria (solid circle).
(B) OD450 (y axis) versus the number of live
pneumococci in the well (x axis). S. pneumoniae serotype 14 (strain DS221494) was added to the
well, and the OD was obtained after a 2-h incubation with XTT and was
expressed in milliabsorbance units.
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To establish the sensitivity of the chromogenic reaction, we determined
the number of pneumococci necessary to produce detectable OD changes by
adding various numbers of bacteria to each well and monitoring the
OD450 after a 2-h incubation. As shown in Fig. 1B, the
increase in OD450 was demonstrable when the wells contained 105 to 107 CFU of pneumococci. Also, within
this range of bacterial density, the rate of OD change was proportional
to the number of live bacteria in the well.
Characterization of culture condition.
Since 1,000 CFU of the
target pneumococci are added to each well in a routine pneumococcal
opsonization assay (13), the number of bacteria must be
amplified by 102- to 103-fold, to
105 to 107 CFU/well, in proportion to the
original number of live bacteria in the well. To achieve this, we
investigated the culture conditions by testing various culture media
and culture periods. Studies of various culture supplements added to
the wells at the end of the opsonization assay verified that THY broth
containing 10% human serum and 0.2% saponin was satisfactory. THY
medium was chosen as the basic supplement because it supported the
growth of S. pneumoniae well and had an OD450
low enough to pose no practical problems. HSP-3 (10%) was added to the
medium because some human serum enhances the growth of bacteria.
Addition of catalase did not change the performance of the medium (data
not shown) and was not added to the medium. HL-60 cells, which can
convert XTT to the colored product, were lysed by adding saponin
(0.2%) to the culture medium. Saponin, up to 0.1%, had little effect
on the growth of pneumococci (data not shown), and the added (0.2%) saponin would be diluted threefold during the assay.
After too long a period of culture, the bacterial density would be
independent of the initial density of bacteria. Thus, we needed to
determine the optimum culture period, i.e., one short enough to reflect
the initial number of bacteria but long enough to produce a sufficient
number of bacteria. To identify the optimum period, we inoculated each
well with 40 µl containing a variable (0 to 1,000 CFU) number of
pneumococci, added 20 µl of the culture supplement, and incubated the
wells at 37°C for 3, 4.5, and 6 h. After the incubation, 20 µl
of XTT substrate solution was added and OD450 was monitored
at 0 and 2 h. To compensate for the light absorption by the
medium, the temporal change in OD (delta OD), obtained by
subtracting the initial OD from the final OD, was used to estimate
the number of bacteria. As shown in Fig.
2 a 3-h culture was too short, and no
signal could be detected even for the wells seeded with 1,000 CFU of
pneumococci. On the other hand, a 6-h incubation was too long and the
OD changes were the same as long as the wells were seeded with more
than 150 CFU (Fig. 2). Incubation for 4.5 h was found to be the
best. The OD450 changes were readily detectable and
remained proportional to the number of bacteria used to seed the wells.
Assuming that bacteria divide every 30 min, the number of bacteria
should have increased by 29 (512)-fold in 4.5 h and
1,000 CFU would have increased to 5 × 105 CFU. Thus,
this incubation period is consistent with our previous conclusion in
Fig. 1B. For all subsequent experiments, the bacteria were cultured for
4.5 h after the culture supplement was added to each well.
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FIG. 2.
Delta OD (y axis) versus the number of
bacteria added to the wells at the beginning of culture (x
axis). After the bacteria were added to the well, the culture
supplement was added and the well was incubated at 37°C for 3 h
(triangle), 4.5 h (circle), or 6 h (square). The culture
supplement contained THY broth, 0.2% saponin, and 10% HSP-3. The
delta OD was expressed in milliabsorbance units and was obtained by
subtracting the OD450 at the beginning of the color
development period from the OD450 obtained at the end of
the color development.
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Comparison of chromogenic opsonization assay with a conventional
assay.
Once the conditions for the bacterial culture and the
chromogenic reaction were determined, we compared the sensitivities of
the chromogenic and the conventional (i.e., colony counting) opsonization assays by performing both assays for 6B and 19F serotypes with four serum samples. The curves describing delta OD versus serum
dilution and the number of CFU of bacteria versus serum dilution had
almost identical shapes for both 6B and 19F serotypes (Fig.
3), and the nearly identical shapes
suggested that the assay sensitivity would be identical. Both assays
produced comparable titers for the four samples. Delta OD is therefore
a good indication of the number of bacteria, and the chromogenic assay
appears to perform as anticipated.
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FIG. 3.
CFU of bacteria versus dilution of serum samples (A and
B) or delta OD versus dilution of serum samples (C and D) for serotype
6B (A and C) and serotype 19F (B and D). One sample is a serum pool,
Ppool10 (solid square), and the others are serum samples from three
persons who received a pneumococcal PS vaccine 1 month prior to
phlebotomy. Delta OD was expressed in milliabsorbance units and was
obtained by subtracting the OD450 obtained at the beginning
of the color development from the OD450 after 2 h of
color development.
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Assay precision was determined by analyzing one serum pool (Ppool 10)
five different times in both ways for 6B and 19F serotypes (Table
1). While the coefficient of variation
for 6B serotype was higher than that for 19F serotype, the two methods
produced comparable coefficients of variation for both serotypes. Thus, the serotype-dependent variability in the precision is not due to the
method, and both methods provide comparable assay precision.
We then examined whether both opsonization assays produce comparable
results by testing 33 human serum samples by both assays for 6B and 19F
serotypes. The samples with undetectable titer were declared to have
titer of 12, or half of the minimum detectable titer. The minimum
detectable titer was 24 because all the serum samples were diluted
threefold and all the samples became diluted eightfold during the
opsonization assay with various reactants. The results of both assays
correlated well for both serotypes (Fig.
4) as shown by the very high r
values (>0.95). One outlier data point was noted for serotype 6B. The
outlier sample had an undetectable opsonization titer by the
conventional assay but had a clearly demonstrable opsonization titer by
the chromogenic assay. When the curve for OD versus serum dilution was
examined, one data point in the middle of the titration had one
abnormally small OD change, and this low OD point yielded a detectable
opsonization titer. Without this low data point, the sample would have
been declared to have an undetectable opsonization titer, and it is likely that there was a technical problem in assaying this
sample.
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FIG. 4.
Comparison of the results obtained with the conventional
method (x axis) and the chromogenic method (y
axis) for serotype 6B (top panel) and serotype 19F (bottom panel). The
correlation coefficients were 0.96 for serotype 6B and 0.99 for
serotype 19F. Seven serum samples for serotype 6B and 10 serum samples
for serotype 19F had undetectable opsonization titers (i.e., <24) by
both methods. One data point near the origin in each figure represents
these samples. Ops, opsonization.
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Most samples tested as described above had relatively high (i.e.,
>100) titers. To evaluate the new method for the samples with low
titers, we diluted 22 postimmune samples with their preimmune sera and
tested them against 6B and 19F serotypes for opsonization titers.
Because the samples have low titers, undiluted samples were used here
as the first sample in the serial dilution and the samples with
undetectable titers were declared to have titer of 4, or half of the
minimum detectable titer. As shown in Fig. 5, the two methods were highly correlated
(r = 0.93 for 6B; r = 0.90 for 19F).
Thus, the new method correlates well with the conventional method for
the samples with low opsonization titers.
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FIG. 5.
Comparison of the results obtained with the conventional
method (x axis) and the chromogenic method (y
axis) for serotype 6B (top panel) and serotype 19F (bottom panel).
Fifteen serum samples for serotype 6B and 12 serum samples for serotype
19F had undetectable opsonization titers (i.e., <8) by both methods.
One data point near the origin in each figure (marked with an arrow)
represents these samples. Excluding these samples with undetectable
titers, the data points have correlation coefficients of 0.93 for
serotype 6B and 0.90 for serotype 19F. Ops, opsonization.
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When the data obtained with 30 min of color development were examined,
the signal-to-noise ratio was slightly less than the ratio with the 2-h
color development period, but the results were similar to those
obtained with 2 h of color development (data not shown). Thus, any
color development time between 0.5 and 2 h should provide the same results.
| |
DISCUSSION |
Chromogenic substrates have often been used to detect the number
of viable cells. They have been used to measure proliferation of
lymphocytes (11), and to enumerate fungal cells
(5), as well as Mycobacterium bovis
(7). It was also used by Stevens and Olsen
(15) to determine the capacity of immune sera to opsonize bacteria. They were able to use XTT without introducing a short culture
step because their opsonization assay used a very large number of
bacteria. Since the current opsonization assay for pneumococcal antibodies uses very few target bacteria (13), we
introduced a short culture step, during which the number of bacteria
was increased about 1,000-fold. We now report that this short culture is practical and the chromogenic opsonization assay yields results that
are comparable to the assay based on the counting of bacterial colonies.
Because the dyes measuring the number of viable cells can be useful for
many different purposes, many different tetrazolium compounds have been
produced. Among them, MTT, which is chemically stable, is commonly
used, but MTT produces nonsoluble products, and its use requires a step
to dissolve the product. This additional step would have increased the
complexity of the assay. We therefore chose to use XTT, which gave us a
simpler assay because it produces water-soluble products that absorb
light at 450 nm (15). The substrate solution did have to
be freshly prepared before each reaction, because XTT is unstable in
solution. An interesting choice of dye would have been 5-cyano-2,3
ditolyl tetrazolium chloride (8). This compound produces a
fluorescent product, which might make the assay more sensitive.
Increased sensitivity may eliminate the need to increase the number of
bacteria by culturing, and therefore the assay could be made even
simpler with this dye. Unfortunately, we could not evaluate this dye,
because the detection of the fluorescence would require a new piece of equipment.
Compared to other assays for measuring opsonic capacity of pneumococcal
antibodies, this chromogenic assay is much easier and simpler to
perform. For instance, the conventional opsonization assay requires a
time-consuming step of colony counting. Another opsonization assay
method uses a radiolabel (16), which is inconvenient and
increasingly more difficult to use because of regulatory burden. Recently, a new method of measuring the opsonophagocytic capacity of
sera was developed in which phagocytosis of fluorescent bacteria by
phagocytes was measured (2, 10). This method, however, requires a flow cytometer, an expensive piece of specialized equipment, so that only certain laboratories can use this method. In addition, this method measures not the opsonophagocytic killing but the opsonophagocytosis. Compared to these alternative opsonization assays,
the present chromogenic assay is simple and requires only commonly
available instruments.
In terms of the ease of the assay, this chromogenic assay compares
favorably even to ELISA. Clearly, this opsonization assay still needs
cell culture facilities to culture the HL-60 cells. However, except for
this, the chromogenic method only uses equipment already used for
ELISA. Furthermore, the chromogenic assay results could be analyzed
with the computer programs used to analyze ELISA data so that only
minimal additional effort would be needed to set up this opsonization
assay in a laboratory which already performs ELISAs. We therefore
conclude that by using the double-serotype opsonization assay, the
chromogenic opsonization assay may require less effort to perform than
the ELISA.
In view of these advantages offered by the chromogenic opsonization
assay, the assay could have a very wide appeal as the primary measure
of pneumococcal vaccine response. Therefore, we believe that our
study described here should be independently confirmed and validated by
other laboratories for additional serotypes included in the
pneumococcal vaccine. Also, both the short culture and the chromogen
can be used to develop a very simple assay for measuring the
bactericidal capacities of sera. If this chromogenic opsonization assay
is found to be useful for determining pneumococcal vaccine responses,
the direct measurement of the opsonophagocytic capacity or the
bactericidal activity may become popular as the primary measure of the
immune responses to various vaccines in the future.
We thank C. Frasch and M. Loeb for their critical reading of the
manuscript and E. Henderson for her secretarial help.
This work was supported with a fund from the National Institutes of
Health (AI-85334). M. H. Nahm is partially supported by National
Institute of Allergy and Infections Diseases contract NO1 AI-45248.
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Yu, X.,
Y. Sun,
C. E. Frasch,
N. Concepcion, and M. H. Nahm.
1999.
Pneumococcal capsular polysaccharide preparations may contain non-C-polysaccharide contaminants that are immunogenic.
Clin. Diagn. Lab. Immunol.
6:519-524[Abstract/Free Full Text].
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Abstract
Introduction
Present address: Faculty of Biological Sciences, College of Natural
Sciences, Chonbuk National University, Chonju 561-756, Korea.
