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Clinical and Diagnostic Laboratory Immunology, May 2000, p. 422-426, Vol. 7, No. 3
Institut für Hygiene und
Laboratoriumsmedizin, Klinikum Krefeld,
Krefeld,1 and Abteilung für
Pädiatrie, Klinikum Wuppertal,
Wuppertal,2 Germany
Received 15 November 1999/Accepted 24 January 2000
Measuring antibodies to Bordetella pertussis antigens
is mostly done by enzyme-linked immunosorbent assays (ELISAs). We
compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for
evaluation of the kits. The specimens contained 20 paired serum samples
from patients with clinical pertussis, 15 samples were from children
vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven
specimens were taken from an interlaboratory comparison of ELISAs, and
there were three reference preparations from the Food and Drug
Administration's (FDA's) Laboratory of Pertussis and from our
laboratory. Reference values were obtained from the FDA or from results
obtained with an in-house ELISA. Commercial ELISAs were compared with
respect to their reproducibility and variability, their ability to
detect significant titer rises in paired serum samples, their ability
to detect an immune response after vaccination, and the comparability
of semiquantitative and quantitative results. Reproducibility was
generally good (>89%), intra-assay variation ranged from 2.4 to
28.7%, and indeterminate results were recorded in up to 18.5% of all
specimens. Most kits correctly identified the antibody response to an
acellular pertussis vaccine. None of the commercial kits identified all
cases of pertussis correctly, and the sensitivity ranged between 60 and
95%. All five commercial ELISAs showed great discrepancies when
comparing semiquantitative results and contained obviously different
antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.
According to the World Health
Organization case definition, the diagnosis of pertussis is based on
clinical symptoms ( In addition to vaccine trials, serology plays a key role in the
diagnosis of pertussis in adolescents and adults (3, 26), as
well as for epidemiologic surveys (1, 6, 17, 19). Furthermore, the diagnosis of pertussis based on a single serum sample
using age-specific reference values for different populations is
increasingly being used (25).
In 1995 a total of 33 research laboratories and vaccine producers
participated in an international collaborative study for the evaluation
of ELISAs to measure antibodies to B. pertussis antigens
which showed differences between different noncommercial assays of
similar format (15). However, the results of this study also
indicated that results from different laboratories can be compared when
a common reference serum is used, when the antigen preparations are
similar, and when comparable techniques are employed. Given the broad
use of commercially available ELISAs for detecting antibodies to
B. pertussis antigens in Germany, we decided to compare five
commercially available ELISAs with an in-house ELISA, which has been
extensively evaluated. ELISA kits were selected according to their
market share in private laboratories, which was evaluated in a
telephone poll by one of us (C.H.W.V.K.).
We compared the reproducibility and variability of the tests, as well
as their ability to detect significant titer rises in paired serum
samples and to detect an immune response after vaccination with a
diphtheria-tetanus-acellular pertussis (DTaP) vaccine and the
comparability of semiquantitative and quantitative results.
Serum specimens.
Specimens included 20 paired serum samples
from a recent pertussis vaccine trial (20, 27), 15 samples
from an immunogenicity study (Hib 032, kindly provided by SB
Biologicals, Rixensart, Belgium), 7 samples from an international
collaborative study for the detection of antibodies to B. pertussis antigens (15) (kindly provided by the
Laboratory of Pertussis, Center for Biologics Evaluation and Research,
U.S. Food and Drug Administration [FDA], Bethesda, Md.), the FDA
reference serum lots 3 and 4, and a lyophilized in-house reference
preparation (lot 2).
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparison of Five Commercial Enzyme-Linked Immunosorbent Assays
for Detection of Antibodies to Bordetella
pertussis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
21 days of paroxysmal cough) in combination with
the isolation of Bordetella pertussis and/or a positive
serology and/or contact with a culture-confirmed case of pertussis
(28). Enzyme-linked immunosorbent assays (ELISAs) currently
are the method of choice for detection of antibodies to B. pertussis antigens (16). Various ELISA formats with
different antigens have been developed (5, 8, 12, 14, 23, 24, 29,
30) and were evaluated intensively in vaccine trials (7, 11,
18, 27).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C. All samples were
retested after thawing.
20°C, to be used in our study.
Specimens (MPI-1 to MPI-7) from an international study to evaluate the
comparability of immunoassays for the detection of antibodies to
B. pertussis antigens were kindly donated by Bruce Meade
(FDA). Five of the seven recalcified plasma samples (MPI-1, -2, -4, -5, and -6) had been characterized in the past by the Laboratory of
Pertussis, by our in-house ELISA, and by various other laboratories
(15). For this study, IgG anti-PT and IgG anti-FHA levels
were determined as the mean of the results obtained by the Laboratory
of Pertussis and our laboratory. IgA anti-PT and IgA anti-FHA levels
for these sera, as well as IgG and IgA anti-PT and anti-FHA for MPI-3
and MPI-7, were determined by 15-fold testing with the in-house assay only.
U.S. Reference Pertussis Antiserum lots 3 and 4 were obtained from the
Laboratory of Pertussis with 200, 15, 200, and 100, U of IgG anti-PT,
IgA anti-PT, IgG anti-FHA, and IgA anti-FHA, respectively, per ml for
lot 3 and 393 U of IgG anti-PT and 270 U of IgG anti-FHA per ml for lot
4. No FDA reference values existed for lot 4 IgA antibodies. These
levels were determined with the in-house ELISA (IgA anti-PT, 10 U/ml;
IgA anti-FHA, 62 U/ml).
The in-house reference preparation lot 2 consisted of pooled
recalcified plasma from healthy blood donors, adjusted to the FDA
reference sera. It contained 32 U of IgG anti-PT, 5.5 U of IgA anti-PT,
and 123 U of each IgG and IgA anti-FHA per ml as estimated by our ELISA.
All samples were coded so that the technicians performing the tests
were blinded. The assays were evaluated independently. Before analysis
of the samples they were divided into seven replicate aliquots for
single day's use and stored at 20°C.
Each specimen was tested with each commercial kit on two consecutive
days (one determination per day), except for the in-house reference 2
which was tested 10 times in order to assess intra-assay variation.
Specimens from the vaccine trial and from the immunogenicity study, FDA
reference lots 3 and 4 and the in-house reference preparation were
tested only once for each antigen and isotype with the in-house ELISA,
since results from earlier analyses of the same sera were available for
comparison. Sufficient data existed for specimens from the
international collaborative study which were not repeated with the
in-house ELISA for the present study.
All tests were able to give quantitative (ELISAs 0 and 1) or
semiquantitative (ELISAs 2 to 5) results.
Indeterminate results, i.e., neither positive nor negative, were
regarded as indeterminate for determination of the assay reproducibility and as negative for the specimens of the immunogenicity study. In all other cases, results were not affected by the number of
indeterminate results because semiquantitative or quantitative results
were used for evaluation.
In-house ELISA (ELISA 0). For this study the in-house ELISA was considered the reference assay due to the amount of data accumulated with this test (27) and its validation in an international collaborative study (15). The procedure has been described elsewhere in detail (25, 27). In brief, the assay consists of absorption of PT and FHA (SB Biologicals) to separate microtiter plates, incubation of patient samples in eight dilutions with both antigens, addition of alkaline phosphatase-conjugated anti-IgG or anti-IgA (Kirkegaard and Perry), incubation with substrate, and cessation of the reaction with NaOH. The absorbance values were transformed to ELISA units per milliliter by using an internal reference (lot 2, adjusted to the FDA reference sera) for constructing the reference line. Reference preparations 3 and 4 of the FDA served as controls. The lower level of detection was arbitrarily set at 2 U/ml.
Interpretation of the assay was performed as for the vaccine trial, i.e., recent contact with B. pertussis antigens was assumed when there was an IgG or IgA conversion to8 U of PT, FHA, or both
per ml or when IgG or IgA antibodies to PT or FHA increased 100% to
at least 8 U/ml (27).
Commercial assays. (i) ELISA 1. For the Serion ELISA classic (Institut Virion Serion GmbH, Würzburg, Germany), microtiter plates are coated with purified acellular B. pertussis components (FHA and PT). Two ELISAs to determine IgG and IgA antibodies were performed according to the manufacturer's instructions. Interpretation of the results was performed with the help of the Serion Easy Base 4PL-Software, and results were expressed in FDA units/milliliter, although no detailed information about the source of the reference sera was given (IgG, >30 U/ml [positive], <20 U/ml [negative], 20 to 30 U/ml [indeterminate]; IgA, >22 U/ml [positive], <15 U/ml [negative], 15 to 22 U/ml [indeterminate]).
(ii) ELISA 2. For the B. pertussis IgG-, IgM-, IgA-Enzym-Immunassay (Innova Labordiagnostik GmbH, Darmstadt, Germany), the antigens used to detect IgG and IgA antibodies to B. pertussis are not stated on the test insert. The results were determined by a mathematic conversion supplied by the manufacturer and were expressed as inU/milliliter (IgG, >18 inU/ml [positive], <14 inU/ml [negative], 14 to 18 inU/ml [indeterminate]; IgA, >26 inU/ml [positive], <19 inU/ml [negative], 19 to 26 inU/ml [indeterminate]). No information about the source of reference sera was given for this test.
(iii) ELISA 3. For the B. pertussis ELISA (Genzyme Virotech GmbH, Ruesselsheim, Germany), the antigens absorbed to the microtiter plates are not specified. The assay was performed according to the manufacturer's instructions, and the results were converted to Virotech units (VE) (IgG and IgA, >11 VE [positive], <9 VE [negative], 9 to 11 VE [indeterminate]). No information about the source of reference sera was given for this test.
(iv) ELISA 4. For the B. pertussis/toxin IgA/IgG ELISA (Novum Diagnostica GmbH, Dietzenbach, Germany), the antigens used are PT and FHA. The results were transformed to Novum units (NE) as described in the test protocol. (IgG and IgA, >11 NE [positive], <9 NE [negative], 9 to 11 NE [indeterminate]). No information about the source of reference sera was given for this test.
(v) ELISA 5. For the B. pertussis IgG/IgA ELISA Test (HRP) (Hycor Biomedical GmbH, Kassel, Germany), the antigens employed are not specified. For the determination of IgG antibodies to B. pertussis, the optical densities were converted to arbitrary units (AU) by an ELISA reader software and a standard curve according to the manufacturer's instructions (IgG, >40 AU [positive], <27 AU [negative], 27 to 40 AU [indeterminate]). IgA antibodies were expressed as ratios. Samples that were weakly positive according to the manufacturer were regarded as positive: >45%, positive; <35%, negative; and 35 to 45%, indeterminate. No information about the source of the reference sera was given for this test.
Statistical analyses. Data were tabulated and ranked. Data were analyzed by the linear regression method (Sigma-Plot; Jandel Scientific, Erkrath, Germany) and by ranking specimens according to antibody levels.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Intra-assay variation.
The intra-assay variation of tests was
estimated twice for each commercial assay, except for ELISA 2, IgG, by
10-fold determination of standard serum 2. For the in-house assay a
variation from 3.8% (anti-FHA IgA) to 14.0% (anti-PT IgA) was found.
Intra-assay variation for the commercial tests ranged from 2.4% (ELISA
3, IgA) to 28.7% (ELISA 4, IgA) (Table
1).
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Reproducibility.
Reproducibility of the test results on two
consecutive days with respect to their interpretation, i.e., positive,
negative, and indeterminate, is shown in Table
2. The number of discrepant results
ranged from 1 (1.5%) (ELISA 1, IgG) to 7 (10.8%) (ELISA 2, IgG) for
65 specimens.
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Indeterminate results.
The number of indeterminate results
ranged from 5 (7.8%) (ELISA 1) to 12 (18.5%) (ELISA 5) for 65 specimens (Table 3).
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Paired serum samples from pertussis children. According to the case definition (27), all 20 paired sera showed evidence of recent contact with B. pertussis when analyzed with the in-house ELISA in 1998 as well as in 1995. A total of 11 samples showed a significant titer increase or seroconversion of IgG and IgA antibodies; 8 were identified due to the rise of isotype IgG, and 1 was identified due to the increase of isotype IgA only.
None of the five commercial assays gave any rules as to how to interpret the results obtained. Thus, we applied the following criteria: recent contact with B. pertussis antigens was assumed when levels of at least one antibody isotype (IgG and/or IgA) increased100% to a level above the cutoff value or when there was
an IgG and/or IgA seroconversion from negative to positive. An increase
of 100% but not to a level above the cutoff value or seroconversion
from indeterminate to positive without a 100% increase in antibody
levels was regarded as no evidence of recent contact with B. pertussis antigens. The results are presented in Table
4. Overall sensitivity for the five
commercial assays ranged from 60 to 95%.
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21 days duration was
ELISA 1. Of 20 serum pairs, 19 were correctly identified as showing
evidence of recent contact to B. pertussis on both days: 11 serum pairs were regarded as positive because of a significant rise in
the titer of IgG antibodies only, 7 were regarded as positive on day 1 (8 on day 2) due to a combined rise of IgG and IgA, and 1 (0 on day 2)
serum pair was regarded as positive due to a rise in isotype IgA only
(Table 4). ELISA 1 was unable to detect a significant rise in titer of
the same paired specimen on both days. This sample pair showed
seroconversion for IgA anti-PT and anti-FHA only with the in-house ELISA.
ELISA 2 was able to detect 19 of 20 significant rises in titer on day 1 and 18 on day 2. Most serum pairs showed a significant rise in IgG
isotype only, whereas five pairs on day 1 and four on day 2 were
diagnosed by a titer rise for IgA and IgG.
ELISA 3 judged only 16 pairs
the same pairs on duplicate testingas
positive for contact with B. pertussis antigens.
Only 12 pairs were identified as having had recent contact with
B. pertussis antigens using ELISA 4. On the second day two other serum pairs were classified as positive for contact with pertussis than on the first day.
Seventeen serum pairs on the first day were shown to be positive for
recent contact with B. pertussis antigens when assayed with
ELISA 5. On the second day two sera which showed a significant increase
of IgG antibodies only on the first run gave no evidence of recent
contact with B. pertussis.
Table 4 also indicates that ELISAs 4 and 5 had more problems in
diagnosing titer increases than seroconversions.
Samples from an immunogenicity study.
Sensitivity for
detecting IgG antibodies to B. pertussis after three
injections of an acellular three-component pertussis vaccine ranged
from 79% (ELISA 5) to 100% (ELISAs 1, 2, 3, and 4) (Table
5). With both ELISAs 4 and 5, one serum
was excluded from evaluation because it showed discrepant results on
the two occasions it was tested. One equivocal sample (ELISA 5, IgG)
was regarded as negative. None of the assays detected IgA antibodies in
these samples.
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Serum samples from an interlaboratory study, FDA references 3 and 4. For each ELISA the antibody levels (IgG and IgA) were plotted against IgG anti-PT and anti-FHA or IgA anti-PT and anti-FHA, respectively, as determined by the FDA and the in-house ELISA. Regression coefficients ranged between 0.87 (ELISA 1, IgG versus IgG-PT) and 0.02 (ELISA 5, IgA versus IgA-anti-PT) with a median of 0.21 (mean, 0.33), indicating that no correlation was found between the different commercial ELISAs measuring IgA or IgG to B. pertussis antigens and the reference values obtained by ELISA 0, which distinguished between antibodies to PT and FHA.
In a second step, sera were ranked according to their antibody level. Results for IgG assays were compared to the IgG anti-PT and IgG anti-FHA levels established as above. Table 6 shows that ELISAs 1, 2, and 4 rank the sera according to their IgG anti-B. pertussis antigen content relatively homogeneously, and the results were comparable to the ranking obtained by measuring IgG anti-PT; ELISAs 3 and 5, however, ranked the sera in a different order. Table 7 displays the same ranking now compared with the IgG anti-FHA content. Here, all commercial ELISAs display a different ranking compared to ELISA 0 and the Laboratory of Pertussis. Similar results were obtained when IgA anti-PT and IgA anti-FHA were ranked according to the results of ELISA 0.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In recent years, several vaccine efficacy trials and one immunogenicity study with acellular pertussis vaccines were performed (4, 9, 10, 13, 18, 20). As a by-result from these studies, serological tests measuring antibodies to Bordetella antigens have been well standardized under the conditions of controlled clinical trials, and a comparison among various laboratories that did these tests has helped to set performance criteria for ELISAs measuring antibodies to pertussis antigens (15).
In this study we have tried to apply these criteria to commercially available test kits, and we have compared five ELISAs for the detection of antibodies to Bordetella antigens with the help of several serum panels characterized by the Laboratory of Pertussis and/or our in-house ELISA.
Day to day reproducibility in respect to interpretation was acceptable with discrepant results ranging between 1.5 and 10.7%. Intra-assay variation was acceptable, i.e., <10% for ELISAs 3 and 5 only. Since all of the assays were performed by the same technician, thereby reducing technologist's influence on precision, intra-assay coefficients of variation are a good reflection of assay precision. Because the protocols for the commercial assays are very similar, variability may be antigen dependent as suspected by Lynn et al. (15) for ELISAs being compared in an international collaborative study.
The number of indeterminate results is important when comparing the suitability of different ELISA kits for laboratory routine. All kits suggest repeat analysis of an equivocal serum together with a second serum sample after 1 to 2 weeks. This makes assays with a significant number of indeterminate result less efficient and impedes final results.
The sensitivity and specificity for detecting IgG and IgA antibodies, respectively, after immunization was acceptable. No IgA antibodies were detected with any of the five commercial ELISAs after immunization, thus confirming results with the reference ELISA and supporting the findings that IgA antibodies to B. pertussis antigens are evidence of natural infection and not immunization (2). All commercial ELISAs except ELISA 5 were able to detect an IgG antibody response after immunization with DTaP in all 15 serum samples tested. Pending confirmation with larger sample numbers, commercial ELISAs may be a useful tool for immunogenicity studies.
Regarding the sensitivity for detection of IgA, it was noted that ELISA 4 found only one specimen of all 65 samples (1.5%) to contain IgA antibodies, whereas all other commercial assays showed that 23 to 34% of the specimens were IgA positive.
None of the kits was able to identify all cases of pertussis in paired serum samples correctly. This result was based on the assumption that a significant increase in specific IgG or IgA correlated with infection. Sensitivity ranged between 60 and 95%. The lower figure is unacceptable, assuming that the main application of the ELISAs is to play an important role in the diagnosis of pertussis.
In recent years the diagnosis of pertussis based on a single serum sample has gained increasing importance (25). A prerequisite for this kind of testing is a reliable semiquantitative or quantitative method for detecting antibody levels and comparison with an age-matched reference group. So far, the assays we tested showed discrepant results when serum samples were ranked according to antibody level, making diagnosis from just one serum sample dependent on the ELISA used and not on the amount of antibodies to purified antigens.
None of the commercial assay ranked the nine specimens in the same order as the reference ELISAs, although some kits distinguished correctly between specimens with high and low levels of antibodies. It has to be considered though that the results of the commercial assays were compared to a ranking obtained by measuring antibody levels to FHA and PT independently. These results, however, suggest that some kits (ELISAs 1, 2, and 4) preferentially measured anti-PT and none preferentially measured anti-FHA, while ELISAs 3 and 5 seemed to employ a different antigen composition.
None of the commercial assays distinguished between antibodies to different antigens of bordetellae. However, all clinical trials and many other published studies have used ELISAs which could distinguish between antibodies to PT and FHA (7, 22, 25, 31). Thus, it is difficult if not impossible to extrapolate from their results and conclusions on the interpretation of the assays investigated in this study.
Overall, our results show that the commercial ELISAs tested here still need further improvement and efforts should be made to standardize performance, antigen contents, and reference material in order to guarantee accuracy of diagnosis and interlaboratory comparability for serodiagnosis of Bordetella infections.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Hygiene und Laboratoriumsmedizin, Klinikum Krefeld, Lutherplatz 40, 47805 Krefeld, Germany. Phone: 49-2151-322431. Fax: 49-2151-322079. E-mail: koesters_hyg{at}klinikum-krefeld.de.
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Materials and Methods
Results
Discussion
References
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Clinical and Diagnostic Laboratory Immunology, May 2000, p. 422-426, Vol. 7, No. 3
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