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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 446-446, Vol. 6, No. 3
1071-412X/99/$04.00+0
LETTERS TO THE EDITOR
Interlaboratory Reproducibility of an Enzyme-Linked
Immunosorbent Assay for Quantitation of Antibodies for
Haemophilus influenzae Type b Polysaccharide
 |
LETTER |
In a recent article, Mariani and collaborators (2)
described a competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of serum antibodies to Haemophilus
influenzae type b (Hib) which correlated very well with the
traditional radioantigen binding assay (RABA) and a previously
described direct ELISA (3).
While their analyses were very comprehensive, the basis for their
comparison of methods was data generated in the same laboratory. Mariani et al. did not reference an interlaboratory study evaluating the direct ELISA which indicated that the reproducibility of the HbO-HA
assay is laboratory dependent (1). In the HbO-HA direct ELISA interlaboratory study, 7 of 11 laboratories reported higher antibody concentrations for low-titered sera than expected based on the
RABA. Based on the data generated by Mariani et al., it appears that
their laboratory encountered this previously described difficulty. The
fact that four laboratories in the previous interlaboratory study
generated data by the HbO-HA direct ELISA that were consistent with the
RABA indicates that the variation is a laboratory-specific phenomenon.
The reasons why some laboratories encounter difficulty with
low-titered sera in the HbO-HA ELISA are unclear. We speculate that direct ELISAs are sensitive to low levels of endotoxin
contamination, which are introduced via buffers and glassware used
during the antigen-coating steps; human sera contain antibodies which
can bind to these contaminants.
Another possibility is that the Mariani et al. study used sera which
were more concentrated (1:20 dilution) than the 1:50 dilution
recommended by Phipps et al., which may enhance the nonspecific binding of human immunoglobulins (2). In our
experience, a starting dilution of 1:50 provides sufficient
sensitivity in the HbO-HA ELISA to quantitate to 0.1 µg/ml.
Thirdly, we have noted that plates vary by lot and by manufacturer in
their performance characteristics, requiring prescreening for
optimal and specific antigen-binding capacity.
For those laboratories that are unable to correct this background
binding which affects low-titered specimens, the Mariani et al.
competitive ELISA appears to provide a sensitive alternative method for
quantitation of human antibodies to Hib polysaccharide.
| |
REFERENCES |
| 1.
|
Madore, D. V.,
P. Anderson,
B. D. Baxter,
G. M. Carlone,
K. M. Edwards,
R. G. Hamilton,
P. Holder,
H. Kayhty,
D. C. Phipps,
C. C. A. Peeters,
R. Schneerson,
G. R. Siber,
J. I. Ward, and C. E. Frasch.
1996.
Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay.
Clin. Diagn. Lab. Immunol.
3:84-88[Abstract].
|
| 2.
|
Mariani, M.,
E. Luzzi,
D. Proietti,
S. Mancianti,
D. Casini,
P. Costantino,
P. van Gageldonk, and G. Berbers.
1998.
A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b.
Clin. Diagn. Lab. Immunol.
5:667-674[Abstract/Free Full Text].
|
| 3.
|
Phipps, D. C.,
J. West,
R. Eby,
M. Koster,
D. V. Madore, and S. A. Quataert.
1990.
An ELISA employing a Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate correlates with the radioantigen binding assay.
J. Immunol. Methods
135:121-128[Medline].
|
| | | | |
Dace V. Madore
Sally A. Quataert
Wyeth-Lederle Vaccines and Pediatrics
West Henrietta, New York 14586-9728
|
| |
AUTHOR'S REPLY |
Dr. Madore and Dr. Quataert in their interlaboratory study conducted
with an indirect ELISA evidenced and confirmed the laboratory dependence of the assay and therefore the variation of the assay as we
also found and attempted to solve. The fact that 7 of 11 participating
laboratories (63.6%) reported higher antibody concentrations for
low-titered sera than was expected on the basis of the RABA provides
clear evidence that the problem does exist. We cannot agree with Dr.
Madore's and Quataert's conclusion that such a high percentage of
well-referenced laboratories is not able to correct for background
binding. Of course the assumption that indirect ELISAs may be sensitive
to low levels of endotoxin contamination can be a valuable hypothesis
that should be demonstrated. In our case, we have performed the assays
in a sterile and pyrogen-free environment. Perhaps in some laboratories
it would be more cumbersome to work in a sterile and pyrogen-free
environment than to perform a competitive assay. The background
problem, which varied from serum to serum, was encountered in both our
laboratories (Chiron and RIVM), thus increasing the percentage of
laboratories with the problem. We encountered the background problem
both with 1:20 and 1:50 starting dilutions as well as with high-titered
samples, where the impact on the result was less profound. As the
background binding was not observed with all serum samples, the problem
also seems to be related to the quality of the serum.
The background problem might not really be so important indeed, but we
wanted to provide a solution to this phenomenon since the results we
obtained were used to evaluate the effectiveness of anti-Hib vaccines.
Therefore we preferred to exclude doubtful or borderline results from
the percentages of vaccinees. The more restrictive the assay the more
reliable the effectiveness of the vaccine will be.
In conclusion, we think that a more stringent assay may be useful and,
perhaps, can avoid the laboratory dependence of the assay
evidenced in the interlaboratory study as well as the dependence of the
assay on the quality of the serum samples used.
| | | | |
M. Mariani
Immunology Department
Chiron Research Centre
Siena, Italy
|
| | | | |
G. A. M. Berbers
Laboratory for Clinical Vaccine Research
RIVM
Bilthoven, The Netherlands
|
Clinical and Diagnostic Laboratory Immunology, May 1999, p. 446-446, Vol. 6, No. 3
1071-412X/99/$04.00+0
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