Clinical and Diagnostic Laboratory Immunology, September 1998, p. 667-674, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Competitive Enzyme-Linked Immunosorbent Assay for
Measuring the Levels of Serum Antibody to Haemophilus
influenzae Type b
Massimo
Mariani,1,*
Enrico
Luzzi,1
Daniela
Proietti,1
Silvia
Mancianti,1
Daniele
Casini,1
Paolo
Costantino,2
Pieter
van
Gageldonk,3 and
Guy
Berbers3
Laboratorio di Immunochimica e Sierologia
Sperimentale, Dipartimento Immunologia, Centro
Ricerche,1
Laboratorio di
Biochimica, Centro Sviluppo Biotecnologie,2
CHIRON S.p.A., Siena, Italy, and
Laboratory for Clinical
Vaccine Research, RIVM, Bilthoven, The Netherlands3
Received 12 February 1998/Returned for modification 4 May
1998/Accepted 9 June 1998
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ABSTRACT |
A competitive ELISA method is described for the measurement of
total antibodies to the capsular polysaccharide of
Haemophilus influenzae type b (HibCPS) in human sera. The
competitive method showed an excellent correlation to the radioantigen
binding assay (RABA, or Farr assay) and improved correlation of sera
with low titers with respect to the more conventional noncompetitive
method. Overestimation of samples in the low concentration range was no longer observed with the competitive ELISA method. The free HibCPS competition allowed us to eliminate the day-to-day background variation
typical of some sera; thus, only values representing the true
anti-HibCPS response were determined. The use of precoated microplates,
which could be stored up to 8 months, greatly improved the speed of the
procedure. An overall correlation coefficient of 0.9660 was found when
407 serum samples with a wide variety of anti-HibCPS antibody levels
were tested with the competitive ELISA and RABA. The regression line
was very close to the ideal line, with a slope of 1.0045 and an
intercept of 
0.1996. A subset of 96 serum samples representative of
all pre- and postimmunization samples was used to compare the
competitive ELISA with a previously described ELISA method. The
competitive method performed in two laboratories in different countries
showed a better correlation with the RABA. The correlation factors were
0.9770 and 0.9816, respectively, while a factor of 0.9547 was found
with the previously described noncompetitive procedure, which was
better for this method than previously reported (r = 0.917). Therefore, the competitive ELISA is proposed for the assay of
anti-HibCPS titers in sera from vaccinated subjects.
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INTRODUCTION |
Haemophilus influenzae
type b (Hib) has been a leading cause of bacterial meningitis among
infants and young children worldwide. The organism also causes other
invasive infections, including epiglottitis, cellulitis, pneumonia,
pericarditis, arthritis, bacteremia, empyema, and osteomyelitis
(5, 19). Antibodies to the capsular polysaccharide of Hib
(HibCPS) protect against invasive disease from this organism (15,
18).
Serum antibodies to HibCPS have been quantitatively determined by using
the radioantigen binding assay (RABA) technique described by Farr in
1958 (6), modified for specificity and labelling (2, 5,
12). The concentration of serum anti-HibCPS antibody sufficient
to confer protection is not known (10). Estimates have
varied from concentrations of 0.1 µg/ml to concentrations of 1.0 µg/ml (1, 11, 14, 17). Because of qualitative differences
in antibody functions attributable to a combination of differences in
isotype and avidity (1, 11), precise estimates are probably
not possible. However, vaccinated subjects are considered protected
when a level of 1.0 µg of anti-HibCPS antibodies per ml is found
(1, 11), although the use of conjugate Hib vaccines able to
elicit a T-cell-dependent immune response may lower this limit in the
future because of their ability to prime for memory serum antibody
responses, as recently suggested by Kayhty (10).
In 1990, Phipps et al. proposed an enzyme-linked immunosorbent assay
(ELISA) measurement of total immunoglobulin (Ig) to HibCPS that
correlated well with RABA results (16). This ELISA
procedure, although unable to resolve the dependence of the assay on
antibody avidity (3), was an improvement in terms of the
feasibility of assaying large numbers of serum samples, while avoiding
the use of radioisotopes. However, in our hands, this assay showed a
high variability in serum antibody background levels. Therefore, we
developed and standardized an improved ELISA measuring total specific
Ig levels with a competitive assay, in which the specific binding to
HibCPS was measured in each sample by subtraction of the uninhibited
fraction after addition of a saturating amount of soluble HibCPS. Here,
we describe the competitive ELISA method for quantitative measurement
of serum antibodies to HibCPS.
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MATERIALS AND METHODS |
Preparation of conjugated HibCPS.
A procedure to prepare
human serum albumin-HibCPS conjugate (HSA-Hib) was developed and
standardized to provide antigen to coat microtiter plates. Twenty
percent (wt/vol) HSA (Sclavo S.p.A., Siena, Italy) was first
characterized for protein content according to the method of Lowry et
al. (13) and then was characterized for amino group content
(8). Fifty milliliters of an aqueous solution of 500 mg of
HibCPS (40% [wt/wt] ribose content; lot 12/89; CHIRON S.p.A., Siena,
Italy), corresponding to 1,335 µmol in ribose was added to 0.4 M
NaIO4 at a ribose/NaIO4 molar ratio of 8. The
mixture was maintained for 20 min in the dark at room temperature and
then stored at 4°C. The content of ribose and the aldehyde groups was
determined according to conventional colorimetric assays (4,
9). A volume containing 100 µmol of the aldehyde groups was
added, while being stirred, to a volume of the HSA solution equivalent
to 50 µmol of amino groups (about 50 to 60 mg of protein) and a
volume of a 1 M pyridineborane (PyBH3) solution in methanol
corresponding to 1,310 µmol (molar ratios: CHO-NH2 = 2, PyBH3-CHO = 13, and PyBH3-NH2 = 26). The mixture was continuously stirred overnight at room
temperature. (NH4)2SO4 was added to a final concentration of 22.5% (wt/vol), and chromatography was performed with a phenyl-Sepharose (26 by 10 mm) column previously equilibrated with 10 mM phosphate buffer (pH 7.2) containing 22.5% (wt/vol) (NH4)2SO4. The column was
washed with about 10 volumes of equilibration buffer, and then the
conjugate was eluted with 10 mM phosphate buffer (pH 7.2). The elution
profile was monitored at 280 nm, and 2-ml fractions were collected. The
respective fractions of the first peak (washing) and of the second peak
(elution) were pooled and assayed for ribose and protein content. The
elution peak solution also was evaluated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (7.5% polyacrylamide) under
reducing conditions and silver staining in order to exclude the
presence of free albumin. The solution containing the elution peak was
then divided into aliquots and stored at 20°C.
Sera.
The correlation between the ELISA and RABA was done by
assaying 407 serum samples collected pre- or postvaccination from
children who had participated in clinical trials with three conjugated Hib vaccines (VAXEM-Hib [Chiron], HibTITER [Lederle-Praxis], or ACT-Hib [Pasteur-Merieux]). Assays also were done with pre- and postvaccination serum samples from 28 adults (13.2%) who received a
plain HibCPS investigational vaccine. The sera were collected prior to
immunization (93 samples [22.9%]) and at different times after
immunization
post-1 (8 samples [2.0%]), pre-3 (21 samples [5.2%]), post-3 (111 samples [27.3%]), pre-4 (56 samples
[13.2%]), and post-4 (62 samples [15.2%])according to different
clinical protocols. This variety of sera ensured a broad range of
antibody concentrations elicited by different vaccines. All sera were
stored at 80°C in small aliquots to avoid repeated freeze-thawing.
A subset of 96 serum samples of the panel consisting of 26 preimmunization samples (27.1%), 26 post-3 samples (27.1%), 19 pre-4
samples (19.8%), and 25 post-4 samples (26.0%) was used for further
comparison with the Phipps' ELISA as described by Phipps et al. in
1990 (16). Hib serum lot 1983, with an assigned value of 70 µg/ml, was obtained from Carl Frash (Center for Biological Evaluation
and Research [CBER], Food and Drug Administration) and used as a
reference serum. Serum pools A110 (90 µg/ml), A143 (1.7 µg/ml),
A144 (49.1 µg/ml), A146 (13.1 µg/ml), and A148 (6.8 µg/ml) were
kindly provided by Dan Granoff (Chiron Vaccines, Emeryville, Calif.,
and Children's Hospital Oakland Research Center, Oakland, Calif.) and
used as internal standard (A110) and quality control sera.
RABA.
All sera were analyzed by RABA to determine the total
anti-HibCPS antibody concentration. The general procedure of Robbins et
al. was followed (17). 125I-labelled HibCPS was
obtained according to the chloramine-T method (7) by
labelling a HibCPS-tyramine conjugate to conform to the specifications
established in 1987 by Carl Frash. Purified HibCPS from strain Eagan
was fractionated on a Sepharose CL-2B (Pharmacia, Uppsala, Sweden)
column, and fractions in the 0.3- to 0.7-kDa range were pooled to
exclude the very-high-molecular-weight outer membrane
protein-containing material. The purity of the radiolabelled antigen
was assayed for binding to rabbit serum prepared against a
capsular-deficient variant (S-2) of strain Eagan (average of 3.8%
binding). The antigen concentration was estimated in RABA by
competition with cold HibCPS at concentrations ranging from 7.8 to
2,000 ng/ml in the presence of an antibody concentration of 0.875 µg/ml. The antigen concentration used in the RABA was about 50 to 60 ng/ml. Antibody concentrations of test sera were determined by
comparison of binding with that of the standard CBER reference serum
(70 µg/ml). For calculation, we used the average of values determined
by serum dilutions within the 15 to 80% binding range.
Competitive ELISA.
Phosphate-buffered saline (PBS) was
prepared with sterile, nonpyrogenic water. HSA-Hib conjugate was
diluted in PBS (pH 7.4) to a concentration of 1.0 µg/ml (based on
saccharide concentration). One hundred-microliter aliquots of this
solution were dispensed into the wells of polystyrene microtiter plates
(Maxisorp, Nunc, Roskilde, Denmark). After overnight incubation at
4°C, plates were washed with PBS (pH 7.4) containing 0.01% (vol/vol)
Tween 20. Wells were overcoated with 250 µl of a 1% (wt/vol) gelatin solution in PBS (pH 7.4) for 3 h at 37°C. After washing, 250 µl of a fixative solution (saline containing 4% [wt/vol]
polyvinylpyrrolidone and 10% [wt/vol] saccharose) was added to each
well. After 2 h of incubation at room temperature, the fixative
solution was removed from the wells, and the plates were dried
overnight at room temperature. Dried plates, hermetically sealed in
plastic bags, could be stored for up to 8 months.
All sera were plated in duplicate. Each plate contained reference
serum, quality control sera, and sample sera diluted in a mixture of 10 mM phosphate buffer and 150 mM NaCl (pH 7.4) containing 0.05%
(vol/vol) Tween 20, and 1% (wt/vol) bovine serum albumin (PBS-BSA).
Each plate contained eight replicates of PBS-BSA (50 µl) to determine
general nonspecific binding. The reference serum was diluted in
duplicate from 0.250 to 0.0019 µg/ml (final dilutions; 50 µl);
there were 4 internal quality control serum samples (50 µl) and 15 serum samples at four fourfold serial dilutions (50 µl). Fifty
microliters of PBS-BSA was then added to the first wells (final
dilutions of quality control and sample sera were 1:50, 1:200, 1:800,
1:3,200). Fifty microliters of a 100-µg/ml HibCPS solution (final
dilution 50 µg/ml) in PBS-BSA was added to the duplicate wells.
Plates were then incubated for 3 h at 37°C (or overnight at
4°C). After incubation, the wells were washed three times with a
mixture of 10 mM phosphate, 150 mM NaCl, (pH 7.4), and 0.05% Tween 20 (PBS-Tween).
Next, 100 µl of alkaline phosphatase-conjugated goat IgG-anti-human
Ig (IgG, IgM, and IgA) (Sigma Chimica, Gallarate, Milan, Italy) diluted
1:10,000 in PBS-BSA was added to each well. Plates were incubated for
3 h at 37°C and then washed as described above. To prepare the
chromogen-substrate solution, p-nitrophenylphosphate tablets
(Sigma Chimica) were dissolved in 1 M diethanolamine-0.5 mM
MgCl2 (pH 9.8) according to the manufacturer's
instructions. One-hundred-microliter aliquots of the solution were
distributed into the wells. After 35 min, the reaction was stopped with
100 µl of 4 N NaOH, and the plates were read at
A405 with a reference filter at 595 nm.
The analysis of the positive values (
0.10 µg/ml) for sera from
children (86.8% of the samples) presented a median for specific binding after inhibition of 91.2% with a minimum value of 28.6%, while for sera from adults (13.2% of the samples), the analysis presented a median of 83.55% with a minimum value of 23.26%. The percentage of inhibition was calculated, assuming the optical density
(OD) reading in the noninhibited well as 100% of binding, according to
the formula % inhibition = 100 [(ODinhibited
well × 100)/ODnoninhibited well]. The higher the
percentage was, the more specific the calculated result was. Therefore,
as a precaution, low-inhibition samples were considered negative,
regardless of their absorbance, when the percentage of specific binding
after inhibition obtained by comparison of inhibited and noninhibited wells was lower than 20%, since noninhibitable binding cannot be
considered specific.
ELISA specificity.
Competition with purified capsular
polysaccharide of Neisseria meningitidis serotype C
(MenCCPS) was carried out to confirm the specificity of HibCPS
inhibition. Six serum samples representative of a wide range of serum
titers were serially diluted from 0.250 to 0.0019 µg/ml. The binding
was then inhibited by addition of a constant excess of purified HibCPS
or MenCCPS (100 µg/ml, final dilution). The first dilutions of the
sera were as follows: A110 (90 µg/ml) was diluted 1:360, 14/2 (55.42 µg/ml) was diluted 1:222, 26/2 (9.94 µg/ml) was diluted 1:40, 25/2
(2.81 µg/ml) was diluted 1:11, 18/2 (2.45 µg/ml) was diluted 1:10,
and 15/2 (1.26 µg/ml) was diluted 1:5. It should be underlined that
the sera showing low titers were diluted at dilutions lower than the
first serum dilution in the assay (1:50) to obtain the inhibition
curve.
Data selection and statistical analysis.
A titration curve
was obtained for each serum sample by plotting the absorbance values as
a function of the logarithm of the reciprocal of the serum dilution.
Sample concentrations were determined by using only absorbance values
in the linear portion of the standard curve (OD of 0.050 to the reading
plateau of an OD of about 2.600). To calculate serum anti-HibCPS
antibody concentrations, absorbance values in wells containing serum
dilutions incubated with soluble HibCPS were subtracted as background
from the corresponding values of the wells in which sera were diluted
with buffer alone. Only sera with absorbance values which were
inhibited at least 20% by soluble HibCPS and giving a difference
between noninhibited and inhibited A405 values
of 0.050 were considered positive. Antibody concentrations in sera
were calculated from the standard curve and were expressed in
micrograms per milliliter.
Noncompetitive ELISA results were obtained from the same data generated
with competitive ELISA, but with the average absorbance of the buffer
subtracted as background instead of the absorbance of the inhibited
serum sample. At a 1:50 dilution of serum, the ELISA sensitivity limit
in undiluted sera corresponded to 0.10 µg/ml. For data analysis,
samples with an antibody concentration of <0.10 µg/ml were assigned
50% of the minimum (0.05 µg/ml). Logarithmically transformed values
of antibody concentrations were used for determination of the
correlation coefficients between the different assay methods.
Comparison of competitive ELISA and Phipps' ELISA.
The
Phipps' ELISA was performed as described previously (16).
Two minor differences were the starting dilution of the samples, which
was 1:20, and the fact that the alkaline phosphatase-conjugated polyspecific goat anti-human Ig was purchased from Caltag (San Francisco, Calif.). The main differences between the two procedures are
summarized in Table 1 and seem to be the
incubation time, buffer, and temperature, in addition to the materials
used. For the comparison, the panel of 96 serum samples described above was used.
| |
RESULTS AND DISCUSSION |
HSA-Hib conjugate and plate coating.
Three lots of HSA-Hib
conjugate were prepared. As summarized in Table
2, the results were consistent.
Therefore, the lots were pooled. The activity of the pooled lot was
tested against that of a previous lot by comparing standard reference
serum curves and medium-titer (6.5 µg/ml) serum dilution curves in an
ELISA. A t test showed that the difference between the mean
values of the two groups was not great enough to exclude the
possibility that the difference was just due to random sampling
variability. No statistically significant difference between the groups
analyzed was found. In fact, statistical analysis gives
t = 0.133, with an associated P = 0.896 for the standard reference serum curves, and t = 0.019, with an associated P = 0.985 for the
medium-titer serum curves. The same results were also obtained with a
nonparametric analysis according to the Mann-Whitney rank-sum test.
The final lot gave a total amount of 133 mg (in protein) of HSA-Hib
conjugate that was stored in aliquots at 20°C. The HSA-Hib conjugate was stable for at least 8 months at 4°C and could be frozen
at 20°C for prolonged storage, since at least four freeze-thawing cycles did not affect antigen stability.
HSA-Hib conjugate-coated plates prepared as described in Materials and
Methods were stored for up to 8 months at 4°C in sealed plastic bags.
Plates were tested simultaneously after different storage times: when
freshly prepared (0 months) and at 1.5, 4, 6, and 8 months. Coated
plates were proved stable by the consistency of a standard curve with
A110 serum in the different plates, as depicted in Fig.
1. The HSA-Hib conjugate was also
successfully tested for equivalence against Phipp's HbHOA conjugate
(data not shown).
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FIG. 1.
Stability of microtiter plates coated with HSA-Hib
conjugate. Results represent the consistency of the anti-HibCPS total
Ig standard curve in plates stored at 4°C for different times. Ab,
antibody.
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ELISA specificity.
The use of competitive ELISA ensured the
specificity of antibody measured for each serum sample. Competition
with purified MenCCPS was carried out in parallel to that with HibCPS
to confirm the specificity of HibCPS inhibition; the results are shown
in Fig. 2. The curves obtained with the
six diluted serum samples representative of a wide range of serum
titers showed that the excess concentrations of the specific
Haemophilus polysaccharide were able to completely inhibit
the specific binding in all cases, while the noncorrelated
Neisseria polysaccharide did not interfere with the specific
antibody binding to the plate wells.
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FIG. 2.
Specificity of competitive ELISA for total anti-Hib Igs.
Results are shown for sample serum dilution curves in the presence of
buffer (solid squares), HibCPS (open squares), and MenCCPS (open
circles). Abs, antibodies.
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ELISA results.
With sera containing low (<2.0 µg/ml)
antibody concentrations, day-to-day variability and background
variability were observed with the conventional indirect ELISA.
Therefore, a specific binding inhibition with purified HibCPS was
evaluated. Figure 3 shows the correlation
between competitive and noncompetitive ELISA results. The overall
correlation was very good (r = 0.9714;
n = 407). This correlation was even better
(r = 0.9973; n = 232) when calculated for samples at 2.0 µg/ml, but decreased when calculated for samples at <2.0 µg/ml (r = 0.7618; n = 175).
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FIG. 3.
Correlation of competitive versus noncompetitive ELISA
results. Low-titer values resulted in overestimation by the
noncompetitive assay, thus causing possible false-positive results in
vaccinated individuals. Abs, antibodies.
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Antibody concentrations in low-titer samples were often overestimated
when the noncompetitive assay was used, which is illustrated in Fig.
4. This is also illustrated by comparison
of the geometric means. The geometric means for samples with
concentrations 2.0 µg/ml were 13.36 µg/ml for the competitive
assay versus 14.05 µg/ml for the noncompetitive one, while for
samples with concentrations <2.0 µg/ml, they amounted to 0.16 and
0.28 µg/ml, respectively. Discrepancies in the results from low-titer
sera with antibody concentrations of <1.0 µg/ml, as determined by
RABA, are greater with the noncompetitive ELISA (Fig. 4A) than with the
competitive ELISA (Fig. 4B). As a result, the percentages of samples
with antibody concentrations >1.0 µg/ml were overestimated after
analysis with the noncompetitive ELISA, as shown in Table
3 for preimmune sera, especially for
adults. Thus, the noncompetitive ELISA might overestimate the
percentage of subjects with an antibody concentration >1.0 µg/ml.
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FIG. 4.
Scatter plots of noncompetitive (A) and competitive (B)
ELISA/RABA value ratios versus RABA values. The wider dispersion in the
low data range of 0.1 to 2 µg/ml found with noncompetitive ELISA (A)
was largely reduced with the competitive assay (B). Abs, antibodies.
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Reproducibility.
The standard curve, quality control sera,
three negative samples, and three low-concentration samples were used
to determine the consistency of the assay results from day to day and
with different operators. These sera were measured by competitive ELISA on three different days by two different operators testing four replicates of each point of the standard curve, six replicates of each
quality control point, and four replicates of each sample serum
dilution. The results, expressed as percentages of coefficients of
variation (%CVs), were calculated by the absorbances read for each
point and are summarized in Tables 4 to
7.
For the standard curve (Table 4), the %CVs were calculated as the mean
of the averages of the absorbances of all of the individual points
before and after background (buffer) subtraction. For the quality
control (Table 5), negative (Table
6), and low-titer (Table
7) sera, the %CVs were calculated as the
averages of the absorbancies of the individual point both in the
noninhibited and in the inhibited wells. Good reproducibility of data
was observed, and the calculated %CVs were always in an acceptable
ELISA range.
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TABLE 5.
Quality control serum reproducibility of the ELISA used
in this study with sera A143, A144, A146, and A148
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The reference values of the quality control sera (Table 5) were
calculated by using the values obtained from 15 different experiments
performed by different operators on different days. The averages of the
two replicates of each quality control serum were subtracted from the
averages of the corresponding inhibited wells, the resulting absorbancy
values were interpolated on the corresponding standard curves, and the
values obtained were multiplied by the respective dilution factors and
then expressed as micrograms of specific Igs per milliliter. The
calculated values were very close to those determined by RABA reported
in parentheses as follows). A143 serum gave a mean (±standard
deviation) value of 1.05 ± 0.41 µg/ml (1.09 ± 0.32 µg/ml), that for A144 was 55.8 ± 16.8 µg/ml (51.90 ± 10.16 µg/ml), that for A146 was 10.1 ± 3.2 µg/ml (14.40 ± 2.59 µg/ml), and that for A148 was 3.57 ± 1.17 µg/ml
(5.06 ± 1.36 µg/ml).
Comparison of competitive ELISA and RABA.
Competitive and
noncompetitive ELISA results were compared to the RABA results by
analyzing a panel of 407 serum samples which belong to different
groups, as shown in Table 3. In Fig. 4, the ratios of noncompetitive
(Fig. 4A) and competitive (Fig. 4B) ELISA values versus RABA values are
illustrated as a function of calculated RABA antibody concentrations,
confirming that the specific binding inhibition allowed a decrease in
low-concentration sample overestimation. The overall competitive ELISA
versus RABA correlation was good, with a correlation coefficient of
0.9660 (n = 407), indicating that no significant
difference between competitive ELISA and RABA was found. The regression
line of competitive ELISA versus RABA is y = 1.0045x 0.1996, which is very close to the ideal
line, because it is shown by a slope very close to 1.0 and an intercept close to origin. This correlation was well conserved (r = 0.9251) when calculated for samples with RABA values 2.0 µg/ml
(n = 232) and lightly decreased (r = 0.7910) when calculated for samples with RABA values <2.0 µg/ml
(n = 175).
The reproducibility of the competitive ELISA was also demonstrated with
the subset of 96 serum samples being assayed in two laboratories in
different countries. This measurement revealed correlation factors of
0.9770 and 0.9816, respectively, compared to those of the RABA. The
correlation between the two ELISAs yielded a comparable factor of
0.9734.
Comparison of competitive ELISA and Phipps' ELISA.
With the
same subset of 96 serum samples, the competitive ELISA was also
compared with the Phipps' ELISA (16). This yielded correlation factors of 0.9376 and 0.9516 as found in comparison with
the competitive ELISA which was performed in the two laboratories. The
correlation factor with the RABA was 0.9547, which is even better than
the factor (r = 0.917) reported by Phipps
(16). Although the correlation found with the Phipps' ELISA
and RABA is quite good, it is lower than the factors observed with the competitive ELISA and RABA. When the Phipps' ELISA was performed with
the incubation times, buffer, and temperature used in the competitive
ELISA, the correlation with the RABA and competitive ELISA improved
only slightly. The overestimation of sera in the lower concentration
range was still present in the results of the Phipps' ELISA performed
with the change in incubation conditions, as illustrated in Fig.
5.
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FIG. 5.
Scatter plots of Phipps' ELISA (A), Phipps' ELISA with
competitive ELISA incubations (B), and competitive ELISA performed in
two laboratories (C and D). The wider dispersion in the low data range
(0.1 to 2.0 µg/ml) was confirmed with the noncompetitive ELISAs (A
and B). The data dispersion was largely reduced with the competitive
assay performed in two different laboratories (C and D). Abs,
antibodies.
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Conclusions.
The effectiveness of vaccination of individuals
against Hib is conventionally determined with a RABA. This assay is
time-consuming and involves the problems connected with handling
radioactivity and waste. In 1990, an ELISA was proposed by Phipps et
al. (16) that was clearly an improvement in term of assay
feasibility and also paved the way to the replacement of RABA with
ELISA for the estimation of anti-HibCPS antibody titers in human sera.
In our hands, human sera were found to give different background
values, with day-to-day variability. The noncompetitive ELISA might
overestimate the percentage of subjects with antibody titers of >1.0
µg/ml. This fact caused discrepancies in the evaluation of low-titer
sera, and although the problem is essentially restricted to preimmune
sera, it may not exclude a low response after vaccine administration.
In these cases, a marked overestimation was introduced when the
conventional indirect (noncompetitive) ELISA was used, because it was
clearly shown in the low range of titers with the noncompetitive-competitive ELISA data correlation. To solve this problem, we decided to test the serum samples in duplicate: one well
with dilution buffer and, in parallel, one well with dilution buffer
containing an excess of purified HibCPS. To determine serum anti-HibCPS
antibody concentrations, absorbance values in wells containing serum
dilutions incubated with soluble HibCPS were subtracted as specific
background from the corresponding values of the wells in which sera
were diluted with buffer only. Antibody concentrations of sera were
calculated from the standard curve and were expressed in micrograms per
milliliter. This approach has been shown to be useful in obtaining a
very good correlation between ELISA and RABA values on a panel of sera
from different clinical trials representative of a wide range of
antibody levels with various isotype and subclass compositions. The
correlation of the competitive ELISA with RABA was better than that
found for the ELISA from Phipps. Moreover, the overestimation of sera in the lower concentration range was still present when the Phipps' ELISA was performed under the incubation conditions of the competitive ELISA, indicating that competition with free HibCPS is necessary to
avoid this false-positive binding.
The reliability, ruggedness, and reproducibility of the competitive
ELISA and the absence of background, plus the good correlation and
regression line with RABA, demonstrate that the proposed competitive ELISA can replace RABA for measuring levels of anti-HibCPS total Ig in
vaccinated populations.
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ACKNOWLEDGMENTS |
We are indebted to Dan Granoff (Chiron Vaccines, Emeryville,
Calif., and Children's Hospital Oakland Research Institute, Oakland, Calif.) and Giuseppe Del Giudice (Chiron S.p.A., Siena, Italy) for
helpful discussion and suggestions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratorio di
Immunochimica e Sierologia Sperimentale, Dipartimento Immunologia,
Centro Ricerche CHIRON S.p.A., Via Fiorentina 1, I-53100 Siena, Italy. Phone: 39-577-243227. Fax: 39-577-243317. E-mail:
mariani{at}iris02.biocine.it.
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Clinical and Diagnostic Laboratory Immunology, September 1998, p. 667-674, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
