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Journal of Clinical Microbiology, January 1999, p. 270-273, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Improvement of Immunoglobulin M Capture Immunoassay Specificity:
Toxoplasma Antibody Detection Method as a Model
Tamara
Tuuminen,*
Helena
Seppänen,
Eeva-Marja
Pitkänen,
Pekka
Palomäki, and
Kirsti
Käpyaho
Labsystems Research Laboratories, Labsystems
OY, 00881 Helsinki, Finland
Received 22 June 1998/Returned for modification 13 August
1998/Accepted 15 October 1998
 |
ABSTRACT |
In the Toxoplasma gondii immunoglobulin M (IgM) capture
fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the
immunoglobulin. This interaction may be mediated through the binding of
human IgM to the HRP moiety of the conjugate. Addition of polymerized
HRP into the reaction mixture reduced nonspecific signals in the
majority of low false-positive serum reactions. Other plausible sites
of interaction are conserved epitopes of mouse immunoglobulins
presenting antigenic similarities with the allotopes of other species.
Fragmentation of mouse antimicrobial IgG to Fab' and selection of
proper conjugation procedure improved assay specificity.
| |
TEXT |
Solid-phase capture
anti-immunoglobulin M (IgM) immunoassays were developed to overcome the
problems with indirect immunoassays caused by rheumatoid factor (RF)
and competition between specific IgG and IgM for antigen binding sites
on the solid phase (3, 4, 7, 8). However, capture type
assays also show immunological interferences. For example, bound to the
anti-IgM capture antibodies, RF can produce false-positive signals by
reacting with antimicrobial-labelled IgGs (3, 7, 8) through
their Fc fragments. The second type of immunological interference
involves specific human IgGs bound to the captured RF via their Fc
fragment which are detected through the microbial antigen bound to the
specific antibody (8, 9). The third type of immunological
interference involves complex reactions of antinuclear antibodies (ANA)
described for immunofluorescence tests (8). The fourth
mechanism of immunological interference which may occur in both
indirect and capture types of immunoassays is mediated through
so-called "naturally occurring antibodies" or "natural
autoantibodies," which are of the IgG and IgM classes and exhibit a
broad range of reactivities (1, 2, 10, 11). Below we
describe two additional interference mechanisms that have not been
previously reported.
The model.
Toxoplasma gondii IgM capture fluorometric
enzyme immunoassay (FEIA) (5) was used as a model. Briefly,
streptavidin-coated microplates (Labsystems, Helsinki, Finland) were
used as a solid phase. Two microliters of each plasma sample was added
to a mixture containing 150 µl of biotinylated polyclonal sheep
anti-human IgM antibody in 0.01 M phosphate-buffered saline (PBS) (pH
7.4)-Tween 20-1% bovine serum albumin. After incubation, the
microplates were washed, and 150 µl of sonicated T. gondii tachyzoites of RH strain which had been premixed with an
anti-T. gondii horseradish peroxidase (HRP)-labelled
mouse monoclonal antibody was added as an antigen (these
tachyzoites were also used for immunoblotting and indirect enzyme
immunoassay [EIA]). The fluorogenic
3-p-hydroxyphenylpropionic acid substrate (150 µl/well) reaction was performed for 30 min. The enzymatic reaction
was stopped, and the signals were measured with a Fluoroskan II
microplate fluorometer (Labsystems). Alternatively, instead of the
anti-T. gondii conjugate with an antigen, the
respective conjugate was used alone or was replaced by a variety of HRP
conjugates (Table 1). All conjugates were
prepared according to Ishikawa et al. (6) by optimized
techniques. The proper preparation of the conjugates was confirmed by
the molar ratio of HRP/IgG based on the spectrophotometric measurements
at optical densities at 403 nm (OD403) and 280 nm
(OD280) from each fraction. Interferences were also studied
by using another model where a monoclonal anti-Toxoplasma gondii antibody was used intact (1 µg/ml) together with specific antigen. The attachment of intact antibody was detected by sequential addition of rabbit anti-mouse HRP-labelled IgG (Dako, Glostrup, Denmark). The reactivities of samples with the rabbit anti-mouse IgG-HRP conjugate alone were also studied.
For all experiments the same controls were used in every run. Negative
control serum was from a staff member, and positive
and low-positive
control sera were from Antibody Systems LTD (Bedford,
Texas). The
latter were proven to be true positives by an indirect
T. gondii IgM EIA (Labsystems). The borderline control was
artificially
prepared by diluting (1:16) the positive control with the
negative
control, resulting in a signal that was approximately
threefold
greater than that of the negative control (
5). To
interpret
the reactivity of samples with each conjugate tested, signals
derived from each individual sample were compared to the signal
of the
borderline
control.
Sixteen
T. gondii IgM false-positive plasma samples
were selected after screening several hundred adult specimens
from the
Arhangelsk Blood Bank (Arhangelsk, Russia). A
T. gondii IgM capture
EIA with
F(ab)
2-
S-acetyl mercaptosuccinic anhydride
(S-AMSA)-
N-succinimidyl-4-(
N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC)-HRP (Table
1) as a conjugate was used for screening. To
test for
the persistency of interfering antibodies a new sample
after 1 year was
requested. A new sample was obtained from only
6 of 16 individuals. All
samples were divided into aliquots and
kept frozen at
20°C.
Repeated freezing and thawing of the aliquots
was difficult to avoid
but the samples did not show signs of bacterial
growth or any other
visible contamination. All samples were tested
with routinely used
methods for the absence of ANA and RF in the
Aurora Hospital (Helsinki,
Finland) and were found to be negative.
Additionally, one
extraordinarily reactive false-positive serum
sample was from a
pregnant woman from France, and due to its small
amount it was used
only in some experiments (with Boehringer blockers
[see Table
3] and
in dilution experiments and reactions with
HRP-coated plates). Also,
one serum sample (Ang) from a staff
member was
T. gondii IgM borderline reactive and presented as
nonspecific. The
conclusion that the tested samples were indeed
false positive was based
on (i) the negative results of immunoblot
analysis where possible, (ii)
the patterns of reactivity with
anti-
T. gondii HRP
conjugates in the presence and absence of specific
antigen in the
T. gondii IgM, IgG, and IgA FEIAs (see Fig.
1),
(iii) nonreactivity in an indirect
T. gondii
IgM EIA (Labsystems),
(iv) discrepant data (samples 11 and 8) from
Platelia Toxo IgM
(Sanofi Diagnostics Pasteur, Marnes la Coquette,
France) and EIAGEN
Toxoplasmosis IgM (CloneSystems, Casaleccio di
Reno, Italy) assays,
and (v) the reactivity of some samples with the
commercial blocker
polyPOD (Boehringer, Mannheim, Germany). To
exclude interference
in our test model by other autoimmune
antibodies, human sera containing
nucleolar, mitochondrial,
histone, ANA-RF, and microsomal antibodies
(Biomedical Resources,
Hatboro, Pa.) were tested too.
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FIG. 1.
Reactivities of samples 1 to 16 from Arhangelsk (Russia)
and sample Ang from a laboratory member by capture T. gondii FEIA. The same reaction conditions were employed throughout
the experiments. (A) Reactivities of samples with anti-human IgG
capture antibody. (B) Reactivities of samples with anti-human IgA
capture antibody. (C to E) Reactivities of samples with anti-human IgM
capture antibody. The conjugates and concentrations are indicated in
Table 1. The samples were tested in the presence (solid bars) and in
the absence (open bars) of T. gondii antigen. Sample 5 was not available for all experiments. The negative, borderline,
low-positive, and positive T. gondii IgM controls are
marked N, B, L, and P, respectively.
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|
To determine the class of interfering antibodies, biotinylated
polyclonal sheep anti-IgM antibody was replaced by anti-IgG
and
anti-IgA for IgG and IgA determinations, respectively. The
concentration of all capture antibodies used was the same (2 µg/ml).
These capture antibodies were used in the assay with specific
F(ab')
2-S-AMSA-SMCC-HRP (Table
1) conjugate with or without
antigen
(see Fig.
1).
To inhibit nonspecific reactions different blockers from Boehringer
(Mannheim, Germany) were tested (see Table
3). Also,
heat-inactivated
(63°C, 30 min) non-
T. gondii mouse
F(ab')
2 fragments
were added to the sample diluent and to
the antigen-conjugate
mixture in concentrations ranging from 0.5 to 100 µg/ml.
To study the reactivity of samples with HRP or anti-
T.
gondii F(ab')
2 fragments, polystyrene microplates
(Labsystems) were
coated overnight at 4°C with HRP or with
anti-
T. gondii F(ab')
2 fragments in
concentrations of 1.25, 2.5, and 5 µg/ml in 0.01
M PBS, pH 7.4. Plasma samples from false-positive Arhangelsk blood
donors,
false-positive serum from France, and
T. gondii
IgM-positive
and -negative samples were added at concentrations of 2 and 10
µl/well. The reaction of heterophilic IgM antibodies to either
HRP or F(ab')
2 was detected by polyclonal antibody against
human
IgM labelled with alkaline
phosphatase.
Mechanisms of interference.
The isotypes of the interfering
antibodies and the effects of conjugation on the intensity of
nonspecific signals are presented in Fig. 1. The immunoreactivity
of each sample is expressed as a double bar on the histogram, and the
signals are compared to the respective signal from the borderline
control. The histograms show that signals exceeding the borderline
level are observed only for the reaction with anti-IgM-coated
microplates (Fig. 1C to E). The Ang sample showed slight reactivity in
the immunoblot assay and high reactivity by the T. gondii IgG indirect method (data not shown), and it was slightly
reactive in the capture IgG assay. Thus, this sample represented a
complex immunoresponse of being IgM true borderline-low positive and
nonspecific (Fig. 1C, lane Ang). Treatment of all tested samples with
sheep anti-human IgG antibody and subsequent centrifugation did not
remove nonspecific responses (data not shown), suggesting that IgG was
not involved in the reaction mechanism. As seen from the comparison of
the reactivities of three anti-T. gondii specific
conjugates (Fig. 1C to E), the conjugation procedure and fragmentation
of antibody to Fab' instead of F(ab')2 had a clear effect
on the reduction of nonspecific signals. Figure 1D shows that the
nonspecific responses of all samples with
F(ab')2-S-AMSA-N-succinimidyl-3(2-pyridyldithio)propionate (SPDP)-HRP were lower than those with
F(ab')2-S-AMSA-SMCC-HRP (Fig. 1C), and at withdrawal of the
antigen the majority, except samples 14 and 16, showed no response.
Fragmentation of F(ab')2 to Fab' further improved the
specificity, without affecting the signals from the true-positive
samples. It is noteworthy that sample 14 also showed some
reactivity with the Fab'-HRP conjugate in the absence of antigen. This
sample was also reactive with other HRP conjugates (Table 1) and
with free HRP (Table 2). Long-term
persistence of interfering antibodies interacting with F(ab')2-S-AMSA-SMCC-HRP was confirmed for six individuals
(samples 2, 4, 6, 9, 10, and 14) from whom paired plasma specimens
taken 1 year apart were available. For those individuals IgM
nonspecific antibodies were detectable, with signals from the first and
the second (1 year later) samples being equal (data not shown). The triple sandwich assay in which mouse monoclonal antibody was used intact with a subsequent detection via rabbit polyclonal antibody revealed lower but clear reactivities of samples 5, 8, 11, 14, and 15 (data not shown). As in previous experiments, sample 14 also
showed reactivity in the absence of antigen. Reactivity with an
HRP-labelled polyclonal rabbit antibody alone was observed only for
sample 14.
Among commercial blockers tested (Table
3) only polyPOD (polymerized HRP) had a
clear effect on the reduction of nonspecific
reactions;
however, complete abolishment of all nonspecific reactions
was not
achieved even at a concentration of 1 mg/ml (the range
of recommended
concentrations is from 0.5 to 100 µg/ml) (data
not shown). The
addition of heat-inactivated non-
T. gondii
F(ab')
2 fragments to the reaction mixture in concentrations
up to 100
µg/ml had no effect on specific positive and false-positive
reactions
(data not shown). Addition to the sample diluent of
anti-
T. gondii F(ab')
2 fragments at a
concentration of 100 µg/ml showed that
no competition of these
fragments with the corresponding HRP conjugate
was observed in
false-positive reactions (data not shown). A comparison
of dilution
curves for the true- and false-positive samples giving
extremely high
signals showed that the shapes of the dilution
curves were similar, and
thus a dilution approach cannot be used
to discriminate the true- from
the false-positive signals. Testing
of commercial sera from patients
with autoimmune diseases did
not reveal nonspecificity (data not
shown), providing further
and stronger evidence that nucleolar,
mitochondrial, histone,
microsomal, and most importantly, ANA-RF
autoantibodies were not
involved in the described interference
mechanisms. The reactivity
patterns of nonspecific samples in a variety
of test systems allowed
us to combine these samples into groups (Table
2).
Our experiments showed that when interference is mediated through the
bridging between captured antibody and a conjugate,
the method of
preparation of the latter has a clear effect on
the reduction of
nonspecific signals (cf. Fig
1C, D, and E). Lower
nonspecific signals
with the F(ab')
2-S-AMSA-SPDP-HRP conjugate
(Fig.
1D)
compared with those with the
F(ab')
2-S-AMSA-SMCC-HRP
conjugate (Fig.
1C) may be
accidental for two reasons. First,
HRP in both conjugate preparations
was linked to amino groups
of immunoglobulin in an unpredictable
fashion. Second, the degree
of conjugation as well as the presentation
of immunoreactive epitopes
on immunoglobulins conjugated by both
chemistries was only a matter
of chance. Conversely, in the conjugation
of Fab' to HRP (via
SPDP), HRP is linked to the sulfhydryl group of the
hinge in a
predictable and reproducible way with a fairly constant
Fab'/HRP
ratio equal to one. Probably, reduction of the epitope density
on the conjugate diminishes binding sites for heterophilic human
IgM
antibodies. The lower content of HRP per conjugate molecule
probably
also contributed to the lower false-positive reactions
with Fab'-HRP
conjugates, although true-positive samples produced
responses that were
as high as those with the F(ab')
2-HRP conjugates
for the
same concentration (per HRP) (compare reactivities of
the borderline,
low-positive, and positive control samples in
Fig.
1). Because
false-positive reactions were observed with a
variety of HRP conjugates
with different idiotopes and of different
origins, it can be speculated
that heterophilic antibodies bind
to allotopes on the constant
domain of Fab' fragments. These allotopes
might represent conserved
amino acid sequences in immunoglobulins
of different species.
Nonreactivity of samples in the indirect
EIA in which the polystyrene
surface was coated with F(ab')
2 fragments
does not exclude
interactions through F(ab')
2 because the attachment
of
F(ab')
2 fragments to the polystyrene surface could have
modified
or covered epitopes. If it is assumed that heterophilic
antibodies
possessing multiple reactivities reacted through
F(ab')
2, conjugation
to HRP might result in the
"stretching" of some amino acid sequences
from the globular
structure of immunoglobulin, thus making them
"approachable."
Because nonspecific reactions were observed with
conjugates employing
different linkage chemistries, we assume
that heterophilic IgM was not
elicited against
linkers.
Another mechanism of interference, clearly observed in this study, is
through the binding to the HRP moiety of the conjugate.
Full
elimination or attenuation of false signals by polymerized
HRP,
indirect reaction with HRP-coated surface, and multiple reactions
with
HRP-containing conjugates suggests that some individuals
have
anti-HRP antibodies. All available samples tested for the
presence of
interfering antibodies showed no decline of the response
after 1 year,
indicating persistence of the antigenic stimulus.
The individuals
might become immunized by HRP, e.g., through the
alimentary
pathway. If the latter interference mechanism predominates,
it becomes
clear why the addition of mouse serum had practically
no effect on the
decrease of nonspecific reactions. The reactivity
merely through HRP
may also explain why in some nonspecific samples
anti-
T.
gondii F(ab')
2 fragments added to the sample diluent
in
concentrations drastically (200-fold) exceeding those of the
conjugate
did not compete with the relevant
conjugate.
| |
ACKNOWLEDGMENTS |
We thank Roger Eaton from the New England Regional Newborn
Screening Program, Massachusetts State Laboratory Institute, Jamaica Plain, Mass., and Eskild Petersen from the Department of Parasitology, Statens Serum Institut, Copenhagen, Denmark, for critical comments and
valuable suggestions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Labsystems
Research Laboratories, Labsystems OY, Sorvaajankatu 15, 00881 Helsinki, Finland. Phone: 358-9-32910484. Fax: 358-9-32910531. E-mail:
tamara.tuuminen{at}thermobio.com.
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Journal of Clinical Microbiology, January 1999, p. 270-273, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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