Journal of Clinical Laboratory Analysis 4:126-129 (1990)

An Enzyme-Linked lmmunosorbent Assay for the

Detection of Antitetanus Toxoid Antibody Using
Aluminum-Absorbed Coating Antigen
G. M. Thiele,' J. Rogers,2 M. Collins,* N. Y a ~ u d aD.
, ~ Smith: and T. L. McDonald2
'Experimental Immunology Laboratoty Department of Internal Medicine, and *Department of Pathology
and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska;
3Kyoto Teshin Hospital, Rokkan-Douri, Nakagyo-Ku, Kyoto, Japan
~

An enzyme-linked immunosorbent assay
(ELISA) for the detection of antitetanus
antibodies was developed using aluminum-
absorbed tetanus toxoid as the coating anti-
gen. The assay was tested by measuring
antitetanus antibody levels in serum obtained
from subjects before and after immunization
with the aluminum-absorbedtetanus suspen-
sion. The specificity of the antibodies for tet-
anus antigen was tested both before and after
antitetanusactivity was removedwith tetanus
antigen-coated beads. Also, the activities of
the sera were compared with those of a corn-
mercially available test. Our results indicated
that the aluminum-absorbedtetanus suspen-
sion can be coated onto standard polystyrene
ELISA plates and used to measure antibody
titers to tetanus toxoid.

Key words: Tetanus toxoid, ELISA, antibodies, aluminum-absorbedcoating

INTRODUCTION MATERIALS AND METHODS

The response to tetanus toxoid challenge, as measured by Serum

levels of antitetanus antibodies in serum, is a way of deter-
mining immunological competence or status of a patient (1,2).
Immunization is performed by vaccination with aluminum-
absorbed tetanus toxoid, and antitetanus antibody is most often
quantified by use of an enzyme-linked immunosorbent assay
(ELISA) (3-5). However, in the ELISA, the coating antigen
is a purified preparation of tetanus toxoid or toxin rather than
the aluminum-absorbed preparation used in immunization.
However, the purified tetanus preparations are not always
readily available and are usually quite costly. Therefore, an
ELISA that utilizes the same aluminum-absorbed preparation
as is used in the immunization would have two distinct ad-
vantages over purified preparations of tetanus toxoid. First,
the aluminum-absorbed tetanus antigen is inexpensive and
readily available. Second, the ELISA may more accurately
measure the immune response if the immunizing antigen and
the coating antigen are the same preparation. The purpose of
this study was to demonstrate whether the aluminum-absorbed
tetanus that is used for in vivo immunization could also be
used as the coating antigen in an ELISA. Additionally, we
assessed whether the results obtained with this procedure cor-
related with those obtained using a commercially available
tetanus ELISA that utilizes purified tetanus toxoid as the coat-
ing antigen.
Five subjects were chosen who had not received a tetanus
vaccination in the previous 5 yr. Blood was drawn (30 cc) in
a clot tube from each subject, and the serum was harvested,
aliquoted, and frozen at - 135°C. Twenty-four hours later, each
subject was given a tetanus booster (Wyeth Labs, Marietta,
PA). On days 7, 14, and 21, blood was drawn, and the serum
was harvested and frozen at - 135°C until used. All subjects
signed informed consent forms.
Preparation of Dynabeads
Iron core polystyrene beads (5 X 10'; Dynabeads 14002,
Dynal, Norway) were washed twice in saline and resuspended
in 5.0 ml of ELISA coating buffer (0.05 M Na2C03, 0.035
M NaHC03, pH 8.5) containing aluminum-absorbed tetanus
suspension (Wyeth Labs, Marietta, PA) diluted 1:20. The mix-
ture was left undisturbed at room temperature qvernight. The
beads were then washed twice in saline, resuspended in 5.0
ml of phosphate-buffered saline (PBS) containing Tween 20
and 10% FCS (PBS-FCS), and incubated for 2 hr. Beads pre-
Received July 31, 1989; accepted August4, 1989
Address reprint requests to Geoffrey M. Thiele, Ph.D.. University of Nebraska
Medical Center, Department of Internal Medicine, Hospital Rm 6162,42nd
St and Dewey Ave, Omaha, NE68105-1065.

0 1990 Wiley-Liss, Inc.


pared in the same manner without the aluminum-absorbed
tetanus were used as a negative control.
Bead ELISA
Both the antigen-coated and control beads were incubated
for 30 min with serum from day 21 postboost. The beads were
washed twice in PBS containing Tween 20 (PBS-Tween 20)
and resuspended in peroxidase-labeled goat antihuman IgG
(Fc-specific) diluted 15,000 (Jackson ImmunoResearch Lab-
oratories, Avondale, PA) in PBS-Tween 20 with 2% FCS and
incubated for 30 min. The beads were then washed five times
in PBS-Tween 20 and resuspended in 1.0 ml of ELISA
peroxidase substrate (Kirkegaard and Perry Laboratories,
Gaithersburg, MD). Following a 2-min incubation, the beads
settled, and 50 p1 of the substrate was removed and placed in
two wells of a 96-well plate (NUNC, Midlands Scientific,
Omaha, NE). The plate was then read on a Dynatech Micro-
ELISA Reader (Dynatech, Alexandria, VA).
ELSA
A 96-well polystyrene plate (NUNC, Midlands Scientific,
Omaha, NE) was coated with 50 p1 of aluminum-absorbed
tetanus toxoid (Wyeth Labs, Marietta, PA) diluted 150 in
sodium bicarbonate buffer, pH 8.0, overnight at room tem-
perature. The next day, the coating solution was removed and
300 p1 of PBS-FCS was added to the plate and absorbed for
15 min at room temperature. The PBS-FCS was removed,
and the plate was washed once with PBS-Tween 20. To
remove any possibility of nonspecific binding of' antibody to
the plate or FCS, the serum used in the assay was diluted 1:2
in PBS-FCS. The serum was titrated onto the plate using PBS
containing 2% FCS as the diluent and incubated for 30 rnin
at room temperature. The plate was then washed twice with
PBS-Tween 20, and 50 pl of a 1:5,000dilution of peroxidase-
labeled goat antihuman IgG (Fc-specific) or IgM (Fc5p-
specific) antibody (Jackson ImmunoResearch Laboratories,
Avondale, PA) in PBS containing 2% FCS was added and
incubated for 30 min at room temperature. The plate was
washed five times in PBS-Tween 20, and 50 ~1 of ELISA
peroxidase substrate solution (Kirkegaard and Perry Labora-
tories, Gaithersburg, MD) was added. After 15 min, the plate
was read on a Dynatech MicroELISA reader (Dynatech, Alex-
andria, VA). An optical density (OD) reading of 0.060 rep-
resented the mean plus 2 SD above negative controls, and
the last dilution to give a reading above 0.060 was consid-
ered positive and reported as the final antibody titer.
Adsorption of Antibody
As it is difficult to obtain serum from individuals who are
seronegative for tetanus, we made our own preparation for fell, although not significantly, after boosting (to 1:4,000)
use as a negative control. This was performed by taking 10 for the next 3 wk. While the IgG antibody titers decreased,
pI of a low-positive serum (as determined by our ELISA), the IgM titers rose from 150 preboosting to 1:200 3 wk fol-
Detection of Antibodies to Tetanus Toxoid 127
adding it to 290 pl of PBS-FCS, and incubating at 56°C for
10 rnin to inactivate complement. To this, 100 pl of the
aluminum-absorbed tetanus suspension was added and incu-
bated for 4 hr at 37°C. The suspension was pelleted by cen-
trifugation, and the supernatant was transferred to a tube
containing 100 p1of the aluminum-absorbed tetanus suspen-
sion and incubated at 37°C for 4 hr. The suspension was again
pelleted and the supernatant removed (final dilution, 1 :50).
The supernatant was shown to be negative for antibodies to
tetanus toxoid by the aluminum-absorbed tetanus ELISA and
was aliquoted and frozen for future use.
Antibody to Tetanus Toxoid by Commercial Testing
The aluminum-absorbed tetanus ELISA was compared with
a commercially available enzyme immunosorbent assay (EIA)
(Lab Systems, Chicago, IL) that detects antibody to purified
tetanus toxoid. The test was performed as described in the
package insert. The 3.5-hr assay was used and gave a sensi-
tivity range of 5- 160 mIU/ml. The patient serum was diluted
until the levels fell into assay range, and the final data were
expressed in international units per milliliter.
RESULTS
Serum IgM Antibody Response to Tetanus
All sera from subjects utilized in this study demonstrated
IgM antibody titers in the aluminum-absorbed tetanus ELISA.
Although these subjects had not received a booster shot within
5 yr, it is conceivable that they had recently been exposed to
Clostridium tetani and produced an immune response. Alter-
natively, it is possible that either IgM antibody is produced
continuously in response to daily contact to the bacteria or,
more likely, that the assay system is too sensitive and the cut-
off point should be raised. The antibody titers to IgM appear
to be real as there is a definite titration effect observed in
each ciise.
Interestingly, following the administration of the booster
shot, there was no change in the IgM antibody titers in four
of five subjects. In the fifth subject (subject 3), a 10-fold
increase 14 days postinoculation that reverted to normal within
1 wk (week 21) was demonstrated.
Serum IgG Antibody Response to Tetanus
In three subjects, we observed only a 2-4-fold increase in
IgG antibody titers (subjects 1, 2, and 3 ) . One subject (sub-
ject 5 ) demonstrated a 32-fold increase in antibody titer, from
1: 1,000prior to boosting to 1:32,000 at 14 days postboosting.
However, as can be seen in Table 1, the IgM antibody response
did no1 change.
In contrast, in subject 4, the IgG antibody titers (1:8,000)
128 Thiele et al.

TABLE 1. IgM Antibody Titers by ELISA in the Serum of TABLE 3. LgG Antibody to Tetanus Toxoid by a Commercial
Subjects Prior to and Following Tetanus Booster Immunization Enzyme Immunoassay
IgM antibody titers IgC (IUlml)
Days postboost$ Davs Dostboosti
Subject* Preboostt 7f 14 21 Subject* Preboostt 7$ 14 21

1 1:2008 1:lOO 1:IOO 1:200 I 1.6 10.5 14.0 10.0

2 1:200 1:200 1:200 1:200 2 0.3 1.7 3.3 3.9
3 1:loo 1:200 1:1,000 1:400 3 2.0 10.0 14.0 13.0
4 150 1:loo 1:IOO 1 :200 4 5.5 6.8 5.3 5.8
5 I :200 I :200 1 :200 1 :400 5 0.7 34.0 58.0 54.0

*Five healthy volunteers with no history of receiving a tetanus booster in
the previous 5 yr.
+Blood was drawn and serum collected prior to booster.
$Blood was drawn and serum collected 7, 14, and 21 days after booster.
IIgM antibody titers a5 determined by aluminum-absorbed tetanus ELISA.
Serum dilution was considered positive if the OD reading was > 0.060.
lowing the booster injection. These data suggest that this sub-
ject had a predominantly IgM response to the challenge. Why
IgG antibodies were not produced 1 wk later is unclear. The
increase in IgM antibody titer demonstrates that the assay is
capable of detecting this isotype of immunoglobulin.
Comparison of Tetanus Toxoid EIA to Aluminum-
Absorbed Tetanus Toxoid ELISA
It was found that the two tests mimicked each other in rel-
ative increases following boosting. For example, subject 5
had a prebooster antibody titer of 1 : 1,000 (Table 2) that cor-
responded to 0.7 IU/ml (Table 3). On the seventh day post-
aluminum-absorbed tetanus challenge, the antibody titer rose
to 1 :16,000which corresponded to 34 IU/ml. Subject 2 showed
a similar profile but with a lesser degree of reactivity in both
assays. Subjects 1 and 3 demonstrated similar antibody and
international unit totals. Subject 4 did not show a decrease in
the international units as shown by antibody titers in the
aluminum-absorbed tetanus. However the aluminum-absorbed
tetanus ELISA demonstrated only a slight decrease and the
EIA showed a very slight increase at day 7 (6.8 IUiml) that
dropped back to prebooster (5.5) levels (day 14, 5.3; day 21,
5.8). These data suggest that the assays are comparable.
TABLE 2. Summary of the ELISA Test Results for IgG in the
Serum of Subjects Before and After Tetanus Immunization
IgM antibody titers
Days postboostf
Subject* Preboostt 7$ 14
1 1:8,000 1 : 16,000 1 :16,000
2 1 : 1,000 1:2,000 1:4,000
3 1:4,000 1 :8,000 1 :8,000
4 1 :8,000 1:4,000 1:4,000
5 I :I ,000 1:16,000 1:32,000
See footnotes for Table I
See footnotes for Table 1 .
Absorption of Seropositive Serum
Serum from two subjects demonstrated that there is no
nonspecific binding to the noncoated beads preabsorption
and after (Table 4). However, the serum antibody titer was
decreased when incubated with antigen-coated beads. Also,
the beads demonstrated active binding of IgG antibodies.
These data suggest that a highly specific immune response
had occurred.
DISCUSSION
Results from this study indicate that the ELISA specifi-
cally tests for antitetanus antibodies and is comparable with a
commercial assay that measures international units. The assay
detects changes in serum antibody titers following the admin-
istration of a tetanus booster immunization. The IgG titers,
with one exception, all rose after immunization while the IgM
titers were low and showed no clear trend with some subjects
showing a rise and others not. This result is expected since
all subjects had been immunized at some time in the past
and should now have, to one degree or another, more IgC-
secreting cells than IgM-producing cells.
Additionally, the assay detects changes in serum antitetanus
antibody levels after the serum has been absorbed with beads
that have been coated with tetanus antigen. Moreover, after
the activity has been absorbed out of the serum, it can then
be shown to be on the beads. In the two subjects that were
tested in this manner, both showed that when the serum was
absorbed on beads coated with antigen, the serum titer was
markedly reduced as compared with the same serum absorbed
onto control beads. Correspondingly, the antigen-coated bead
ELISA was positive when compared with the control beads.
The greatest difficulty in developing a new assay for the
measurement of antibody levels is determining the specific-
21 ity of the assay. Although specificity can be demonstrated by
1 : 16,000
showing that the assay can detect differences between posi-
1:4,000 tive and negative controls, in the case of tetanus, negative
1 : 16,000 controls are virtually nonexistent since the majority of peo-
1:4,000 ple in this country receive a tetanus toxoid immunization at
1:32.000 approximately 6 mo of age (6). Additionally, even before this
first immunization, infants are often positive for antitetanus
 Detection of Antibodies to Tetanus Toxoid 129

TABLE 4. Summary of the Bead ELISA and Solid-Phase ELISA Results Before and After Adsorption of Day 21 Serum
Preadsorption Serum plus beadst Serum plus antigen-coated beads$
Subject* Serum tited Beads * * Serum titertt Beads Serum titer Beads$$
1 16,000 0.040 16,000 0.036 4000 0.824
5 32.000 0.032 32.000 0.044 8000 > 1.999
*Two healthy volunteers with no history of receiving a tetanus booster shot in the previous 5 yr.
tSerum from day 21 was added to beads that had active sites blocked with FCS only. This served as a negative control for nonspecific binding of serum
proteins to the beads.
$Serum from day 21 was added to beads coated with the aluminum-absorbed tetanus and incubated as described in text.
$The serum was checked for IgG antibody to aluminum-absorbed tetanus toxoid prior to use in the assay.
**Fetal calf serum-blocked beads were incubated with alkaline-phosphatase-labeledgoat antihuman IgG (Fc-specific), washed, and substrate added. This
was used as the second antibody control.
??The serum was checked for IgG antibod to aluminum-absorbed tetanus following incubation with FCS-blocked beads or antigen-coated beads.
$$The beads were processed for binding of IgG antibody as described. Supernatants were harvested and read on the MicroELISA reader.

antibody due to the presence of maternal antibodies (7). This
is particularly true if the mother had been recently immunized
or had received immunoglobulin therapy (8).
In general, although there was a good deal of comparability
between our assay and the commercial assay, there were
some differences, which may have been due to the antigen
preparations. In general, the availability and low cost of the
aluminum-absorbed tetanus suspension makes it a good can-
didate for use as an ELISA coating antigen. In this article,
we have provided evidence that the aluminum-absorbed teta-
nus antigen can be coated onto standard polystyrene ELISA
plates and that antitetanus antibody will bind to this antigen
in a specific and consistent manner.
ACKNOWLEDGMENTS
Supported in part by PHS CA30196 and CA36727, awarded
by the National Cancer Institute, DHHS, and the Lympho-
proliferative Research Fund.
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