NUTRITION AND CANCER, 43(2), 167–173
Copyright © 2002, Lawrence Erlbaum Associates, Inc.
Detection of Bowman-Birk Inhibitor and
Anti-Bowman-Birk Inhibitor Antibodies in Sera of Humans and
Animals Treated With Bowman-Birk Inhibitor Concentrate
X. Steven Wan, David G. Serota, Jeffrey H. Ware, James A. Crowell, and Ann R. Kennedy
Abstract: The Bowman-Birk inhibitor (BBI) is a soybean-
derived serine protease inhibitor with anticarcinogenic activ-
ities. BBI, in the form of BBI concentrate (BBIC), is currently
being evaluated in clinical trials as a human cancer-
preventive agent. In the present study, an enzyme-linked
immunosorbent assay was used to measure BBI concentra-
tions in serum samples collected from human subjects and an-
imals treated with BBIC. The results demonstrate that the
serum BBI concentration was higher than the baseline level
for the patients after treatment with BBIC at 100-800 chymo-
trypsin-inhibitor units/day for 0.5, 1, 2, 4, and 6 mo. The in-
crease in serum BBI concentration was also observed in dogs
treated with BBIC at 100–1,000 mg/kg/day for 52 wk, and the
increase was dose dependent. The results also indicate that
anti-BBI antibodies were present in animals and the serum
levels of anti-BBI antibodies increased significantly in mice
treated with BBIC at 100–1,000 mg/kg/day for 15 and 26 wk.
The increase in the serum level of anti-BBI antibodies in dogs
treated with BBIC was not statistically significant, and no in-
crease in the serum level of anti-BBI antibodies was observed
in human subjects after BBIC treatment. These results suggest
that orally ingested BBI is absorbed by human subjects and
animals and that some animals develop antibodies to BBI in
response to treatment with BBIC.
Introduction
The Bowman-Birk inhibitor (BBI) is a soybean-de-
rived serine protease inhibitor that inhibits trypsin- and chy-
motrypsin-like proteases, such as trypsin, chymotrypsin,
cathepsin G, elastase, and chymase (1). BBI is also an anti-
carcinogenic agent that inhibits malignant transformation in
vitro and suppresses cancer development in several organ sys-
tems and animal species (1–3). Epidemiological data have
shown that high levels of soybean consumption are correlated
with low incidence rates of colon, breast, and prostate cancers
in human populations (4). These observations suggest that
X. S. Wan, J. H. Ware, and A. R. Kennedy are affiliated with the Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA 19104.
D. G. Serota is affiliated with MPI Research, Mattawan, MI 49071. J. A. Crowell is affiliated with the Chemopreventive Agent Development Research Group,
Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20852.
BBI could be a useful agent for cancer prevention in humans.
BBI, in the form of BBI concentrate (BBIC), is currently be-
ing evaluated as a cancer-preventive agent. Studies in animals
have demonstrated that BBIC is well tolerated by dogs (5),
rats (6), and mice (7) at up to 1,000 mg/kg/day without signif-
icant drug-induced toxicity (5–8). Human studies have also
shown that BBIC is not toxic at ≤800 or 1,066 chymotrypsin-
inhibition (CI) units/day (9–12); 1 CI unit is defined as the
amount of protease inhibitor needed to inhibit 1 mg of bovine
pancreatic α-chymotrypsin (13). In a Phase IIa oral cancer
prevention trial in patients with oral leukoplakia, treatment
with BBIC at 200–1,066 CI units/day for 1 mo resulted in a
dose-dependent decrease in oral lesion size (11). In a Phase I
trial of BBIC in patients with benign prostatic hyperplasia,
treatment with BBIC at 100–800 CI units/day for 6 mo re-
sulted in reductions in serum prostate-specific antigen levels
and prostate volume (12).
In toxicity studies and clinical trials to evaluate BBI as a
cancer-preventive agent, a method for detecting BBI and its
metabolites in body fluids is essential for monitoring the
BBI exposure. We previously demonstrated that BBI can be
detected in the urine of human subjects by an immunoassay
method using a monoclonal antibody (MAb) against re-
duced BBI (10–12,14). In the present study, we measured
BBI concentrations in serum samples collected from human
subjects and animals treated with BBIC. The results demon-
strated an increase of serum BBI concentration in the human
subjects and dogs after BBIC. The results also showed that
anti-BBI antibodies were present in mouse sera and that the
serum titers of the anti-BBI antibodies increased signifi-
cantly in the mice treated with BBIC.
Materials and Methods
Antibodies and Chemicals
The MAb 5G2 was prepared and characterized as previ-
ously described (15). BBI was purchased from Sigma Chemi-
cal (St. Louis, MO) and was found to be >95% pure as
analyzed by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (data not shown). The reduced BBI (rBBI) anti-
gen was prepared by a radiochemical procedure described
previously (15–17). BBIC was purchased from Central Soya
(Ft. Wayne, IN); methods for the production of BBIC have
been described elsewhere (13). Horseradish peroxidase-con-
jugated goat anti-mouse IgG was purchased from Southern
Biotechnology (Birmingham, AL). Horseradish peroxidase-
conjugated rabbit anti-dog IgG and rabbit anti-human IgG
were purchased from Sigma Chemical.
Serum Samples From Patients Treated With BBIC
Human serum samples used in the present study were ob-
tained from 19 male patients enrolled in a 6-mo Phase I
BBIC clinical trial, which has been described in detail else-
where (9,12). In that trial, placebo or BBIC tablets were ad-
ministered orally at 100, 200, 400, or 800 CI units/day for 6
mo. Nonfasting blood samples were collected from patients
before and during the 6-mo treatment period. Serum was
separated from the whole blood and stored at −20°C before
analyses.
Serum Samples From Animals With BBIC
Mouse serum samples used in the present study were ob-
tained from animals in a 6-mo BBIC toxicity study, which
was performed by D. G. Serota (Laboratory Study Identifi-
cation No. 560-057) and reported to the National Cancer In-
stitute, Division of Cancer Prevention, Chemoprevention
Branch on 3 January 2000 (7). In this study, 100 B6C3F1
mice were divided into 4 groups of 25 animals per group,
and BBIC was orally administered at 0, 100, 500, or 1,000
mg/kg/day for 6 mo. During the 6-mo treatment period, the
animals were fed a diet that was modified from Certified Ro-
dent Diet 5002 (PMI Feeds, St. Louis, MO) by replacement
of the soybean component with an equivalent meat by-prod-
uct component. Blood samples were collected from the first
10 animals of each gender and treatment group at Week 15
and at the termination of the study.
Dog serum samples used in the present study were ob-
tained from animals in a 1-yr BBIC toxicity study performed
by D. G. Serota (Laboratory Study Identification No. 560-
058) and reported to the National Cancer Institute, Division
of Cancer Prevention, Chemoprevention Branch on 27 Janu-
ary 2000 (8). In this study, BBIC was orally administered to
16 male and 16 female beagle dogs at 0, 100, 500, or 1,000
mg/kg/day for 1 yr. During the 1-yr treatment period, the an-
imals were fed modified Certified Canine Diet 5LC7 (PMI
Feeds), in which the soybean component was replaced with
an equivalent meat by-product component. Blood samples
were collected from animals 1–2 days before BBIC treat-
ment and in Weeks 13 and 52 of the BBIC treatment period.
All animal serum samples were stored frozen at −20°C
before analysis.
168
Determination of Serum BBI Concentration
In the present study, the serum BBI concentrations were
determined with an enzyme-linked immunosorbent assay
(ELISA), essentially as described previously (14) with minor
modifications. To prepare antigen-coated plates for the exper-
iments, rBBI antigen solution [10 µg/ml in 10 mM phosphate
buffer (PB), pH 7.0] was applied to 96-well polystyrene plates
at 50 µl/well and incubated at 4°C overnight to allow the anti-
gen to attach. The plates were fixed with 0.1% glutaraldehyde
for 10 min, neutralized with 0.1 M glycine for 10 min, incu-
bated with 1% bovine serum albumin (BSA) in 20 mM PB,
pH 7.0, for ≥1 h to block nonspecific binding sites on the
plates, and then frozen and stored at −20°C before use. To
perform the experiments, 50 µl of MAb 5G2 (diluted 1:2,000
in BSA-PB) were mixed with an equal volume of the serum to
be tested in a well on the 96-well plates precoated with rBBI
and incubated for 90 min. The antibody solution was also
mixed with BSA-PB containing 0 (negative control), 15.6,
31.3, 62.5, 125, 250, 500, or 1,000 ng/ml of rBBI and in-
cluded in the experiments to generate standard curves. After
the incubation, the plates were washed three times with water
and further incubated with horseradish peroxidase-conjugated
goat anti-mouse IgG (50 µl/well) for 1 h. The plates were
again washed three times with water and incubated with tetra-
methylbenzidine substrate for 30 min. The color development
was stopped by addition of 100 µl of 1.0 N HCl to each well.
All incubations were carried out at room temperature, and the
plates were read at a wavelength of 450 nm with a Power-
Wave 340 microplate scanning spectrophotometer (Bio-Tek
Instruments, Winooski, VT). In each experiment, the serum
BBI concentrations were estimated using a standard curve
generated in the same experiment.
Detection of Anti-BBI Antibodies in Sera
The anti-BBI antibodies in the serum samples were also
detected by the ELISA method. To carry out the experiments,
10 µl of each serum sample were diluted with 40 µl of BSA-
PB in a well on the 96-well plates precoated with rBBI and in-
cubated for 90 min. For negative controls, BSA-PB was
added in place of serum sample. The plates were then washed
three times with water and further incubated for 1 h with
horseradish peroxidase-conjugated antibodies against mouse,
dog, or human IgG. The plates were again washed three times
with water and incubated with tetramethylbenzidine substrate
for 30 min. The color development was stopped with 1.0 N
HCl, and the plates were read at 450 nm as described above.
The serum levels of the anti-BBI antibodies were expressed as
absorbance readings after subtraction of the mean of the
absorbance readings for the negative controls.
Data and Statistical Analysis
The standard curve utilized for estimating the serum BBI
concentrations was established by semilogarithmic regres-
sion analysis using the logarithm of BBI concentration as the
Nutrition and Cancer 2002
independent variable and the absorbance at 450 nm as the
dependent variable. The BBI concentrations (ng/ml) in the
serum samples were determined using the standard curves
generated as part of each experiment. The group means of
the serum BBI concentrations and the anti-BBI antibody lev-
els were analyzed by the regression analysis or one-way
analysis of variance (ANOVA) followed by the Student’s t-
test and/or Tukey’s test using Prism version 2.0 statistical
software (GraphPad Software, San Diego, CA).
Results
We previously demonstrated that MAb 5G2, which was
developed against rBBI (15), can be utilized to detect BBI in
the urine of human subjects after oral administration of
BBIC (10–12) or ingestion of BBI-containing soy milk (14).
The present study involves measurements of BBI concentra-
tions in sera of human subjects and animals treated with
BBIC. BBI concentrations in human serum were determined
for 10 patients who were treated with placebo (n = 4) or
BBIC at 100 (n = 3), 200 (n = 1), 400 (n = 2), or 800 (n = 4)
CI units/day for 6 mo. There are small but varying numbers
of missing data points because of inadequate or unavailable
samples. Given the small number of patients, those in the
four BBIC dose groups were combined into a single BBIC
treatment group for the purpose of statistical analysis. Be-
fore BBIC treatment, the baseline values for the serum BBI
concentration measured in the 10 patients assigned to the
BBIC treatment group and the 4 patients assigned to the pla-
Figure 1. Serum Bowman-Birk inhibitor (BBI) concentrations in patients treated with BBI concentrate (BBIC) or placebo tablets for 6 mo. Placebo or BBIC
tablets were orally administered to patients with benign prostatic hyperplasia twice a day at 100–800 chymotrypsin-inhibitor units/day for 6 mo. Blood was
drawn from patients before and during treatment. Serum BBI concentrations were determined by enzyme-linked immunosorbent assay (ELISA).
Vol. 43, No. 2
cebo control group were equivalent to 140 ± 155 and 97 ± 99
(SD) ng/ml, respectively. The baseline values of serum BBI
concentrations varied substantially among the individuals,
as indicated by the relatively large standard deviations. The
high baseline value for the BBIC treatment group was en-
tirely due to one patient whose serum BBI concentration
(536–685 ng/ml before and after BBIC treatment) was ex-
ceptionally and persistently higher than that of all other pa-
tients enrolled in the study. Without this patient, the baseline
value for the BBIC treatment group was 96 ± 72 ng/ml of
BBI, which was very close to that of the placebo control
group. At 0.5, 1, 2, 4, and 6 mo of BBIC treatment, the aver-
age serum BBI concentration for the BBIC treatment group
was 66.37%, 56.05%, 51.24%, 25.70%, and 13.98% higher
than the baseline value (Fig. 1). The serum BBI concentra-
tions at 0.5 or 1 mo of BBIC treatment were significantly
higher than the baseline value (P ≤ 0.025, paired Students’ t-
test). In contrast, the serum BBI concentrations measured for
the control group during the 6-mo placebo treatment period
were mostly below the baseline value and did not differ sig-
nificantly from the baseline value (P > 0.44, paired Stu-
dent’s t-test) at any of the five time points (Fig. 1).
The BBI concentrations in dog serum were determined in
16 male and 16 female beagle dogs that had been treated
orally with BBIC at 0 (carrier control), 100, 500, or 1,000
mg/kg/day for 13 or 52 wk. Before BBIC administration, the
baseline values of serum BBI concentration were not signifi-
cantly different among the dogs assigned to the four treat-
ment groups (P > 0.80, 1-way ANOVA, data not shown).
169
Table 1. BBI Concentrations in Sera From Dogs Treated
With BBIC for 52 Weeksa
BBIC Dose, BBI Concn,b Increase,
mg/kg/day n ng/ml % P (t-test)

0 8 202.53 ± 61.95
100 7 253.34 ± 96.94 25.08 0.107
500 8 248.64 ± 65.14 22.77 <0.05
1,000 8 301.48 ± 85.71 48.86 <0.001

a: Serum samples from dogs treated with Bowman-Birk inhibitor (BBI)

concentrate (BBIC) at 0 (control), 100, 500, or 1,000 mg/kg/day for 52
wk were mixed with monoclonal antibody 5G2 solution and incubated
in microwell plates precoated with reduced BBI (rBBI) antigen. Anti-
bodies bound to rBBI antigen were detected with a horseradish
peroxidase-conjugated goat antibody against mouse IgG. n, Number of
animals.
b: Values are means ± SD.

Figure 3. Standard curve of inhibitory ELISA used to measure serum BBI

The serum BBI concentrations were not significantly differ-
ent for the dogs of the four treatment groups after 13 wk of
BBIC treatment (P > 0.10, 1-way ANOVA, data not shown).
After 52 wk of BBIC treatment, the serum BBI concentra-
tions were ~25% (P = 0.107), 23% (P < 0.05), and 49% (P <
0.001) higher for the animals treated with BBIC at 100, 500,
and 1,000 mg/kg/day, respectively, than for the control
group (Table 1). The increase in the serum BBI concentra-
tions was dose dependent (Fig. 2).
We also attempted to measure the BBI concentrations in
the sera of mice treated with BBIC at 0 (carrier control), 100,
500, and 1,000 mg/kg/day for 15 or 26 wk. In the inhibitory
ELISA used for this study, the absorbance reading was in-
versely correlated with the logarithm of BBI concentration
(Fig. 3), and the highest absorbance reading was expected
for the negative control, which contained no free BBI anti-
gen to inhibit the binding of MAb 5G2 to the immobilized
concentration. Reduced BBI was diluted in bovine serum albumin-
phosphate buffer at different concentrations and included in each run of
ELISA to generate standard curves. Relationship between BBI concentra-
tion (in logarithm) and absorbance reading was established by a regression
analysis.
BBI antigen. In the present study, however, the absorbance
readings of most mouse serum samples were higher than the
reading of the negative control, and the absorbance readings
of the animals treated with BBIC were significantly higher
than those of the control animals (Table 2). These results
suggested that the serum samples from mice treated with
BBIC might have contained anti-BBI antibodies that bound
to the immobilized BBI antigen. Because of the interference
by the anti-BBI antibodies in the mouse serum samples, it
was not possible to meaningfully measure BBI concentra-
tions in the mouse sera by the inhibitory ELISA used in the
present study.
The levels of anti-BBI antibodies in the mouse serum
samples were determined by an ELISA method using the

Table 2. Absorbance Data From an ELISA Experiment

Intended to Measure Mouse Serum BBI Concentrationsa
Time of Blood BBIC Dose, Absorbance at
Collection mg/kg/day n 450 nmb P (t-test)

Week 15 0 10 0.903 ± 0.288

100 10 1.324 ± 0.564 <0.01
500 9 1.440 ± 0.546 <0.001
1,000 10 1.303 ± 0.530 <0.01
Week 26 0 10 0.947 ± 0.476
100 10 1.663 ± 0.491 <0.001
500 10 1.300 ± 0.579 <0.05
1,000 10 1.388 ± 0.474 <0.001
Negative control 0.709 ± 0.054

Figure 2. Serum BBI concentrations in dogs treated with BBIC for 52 wk.
BBIC was orally administered to a total of 16 male and 16 female beagle
dogs at 0 (carrier control), 100, 500, and 1,000 mg/kg/day for 52 wk (4 male
and 4 female dogs for each dose group). Blood was drawn from dogs at end
of the treatment, and serum BBI concentrations were determined by ELISA.
a: Serum samples from mice treated with BBIC at 0 (control), 100, 500,
or 1,000 mg/kg/day for 15 or 26 wk were mixed with monoclonal anti-
body 5G2 solution and incubated in microwell plates precoated with
rBBI antigen. Antibodies bound to rBBI antigen were detected with a
horseradish peroxidase-conjugated goat antibody against mouse IgG.
ELISA, enzyme-linked immunosorbent assay; n, number of animals.
b: Values are means ± SD.

170 Nutrition and Cancer 2002


Figure 4. Detection of anti-BBI antibodies in serum from mice treated
with BBIC. BBIC was orally administered to a total of 100 B6C3F1 mice at
0 (carrier control), 100, 500, or 1,000 mg/kg/day for 26 wk (25 mice for
each dose group). Blood was drawn from first 10 animals in each dose
group after 15 and 26 wk of BBIC treatment, and serum levels of anti-BBI
antibodies were determined by ELISA. P values represent comparison be-
tween control group and each treatment group (1-way analysis of variance
followed by Tukey’s test). A450, absorbance at 450 nm.
mouse sera as the primary antibodies. The serum levels of
anti-BBI antibodies were 52–125% higher in the mice
treated with BBIC for 15 wk than in the control mice (Fig.
4). The differences in the serum levels of anti-BBI antibod-
ies between the control group and each of the three BBIC
dose groups were statistically significant (P < 0.05). The se-
rum levels of anti-BBI antibodies were 87–161% higher in
the mice treated with BBIC for 26 wk than in the control
mice (Fig. 4), and the increases were also statistically signif-
icant (P < 0.0005).
To determine whether anti-BBI antibodies were also
present in dogs and human subjects treated with BBIC, the
ELISA experiments were performed using sera from BBIC-
treated dogs and human subjects as the sources of primary
antibodies. In dogs treated with BBIC at 100, 500, or 1,000
mg/kg/day for 13 or 52 wk, the serum levels of anti-BBI an-
tibodies increased by <15%, and the increase was not statis-
tically significant compared with the control group (data not
shown). In human subjects treated with BBIC at 100, 200,
400, or 800 CI units/day (equivalent to ~12.5, 25, 50, or 100
mg/kg/day) for up to 6 mo, the serum levels of anti-BBI anti-
bodies did not show any increase compared with the base-
line values measured in the serum samples collected before
the BBIC treatment or the values measured in the placebo
control group (data not shown).
Discussion
MAb 5G2 was produced against the reduced form(s) of
the BBI molecules (15) and was able to detect BBI in human
urine samples after BBIC administration (10–12) or inges-
Vol. 43, No. 2
tion of BBI-containing soy milk (14). The present study has
demonstrated that this antibody can also be used to measure
BBI in human and dog serum samples. In human subjects,
the serum BBI concentration varied greatly, even before
BBIC was administered. This is reflected in the large coeffi-
cients of variation (SD ÷ mean) for the serum BBI concen-
tration, which are 1.021 (99 ÷ 97) and 1.107 (155 ÷ 140),
respectively, for the patients in the placebo control group
and the BBIC treatment group. The large individual varia-
tions in the serum BBI concentration could have been
caused by the difference in diets consumed by the patients,
because BBI is present at various levels in soybean-con-
taining foods. The large individual variations could also
have been caused by the difference in the time between
BBIC administration and blood sample collection, because
the length of time between BBIC administration and blood
sample collection was not controlled in the clinical trial.
Because of the large individual variation among the pa-
tients, the serum BBI concentrations measured in individual
patients after BBIC treatment were compared with the pre-
treatment serum BBI concentrations in the same patients by
the paired Student’s t-test to avoid masking of treatment-
associated changes by the individual variations. The results
demonstrated that the serum BBI concentration was in-
creased significantly in patients after 2 and 4 wk of BBIC
treatment. The average serum BBI concentration for patients
after 2, 4, or 6 mo of BBIC treatment was also higher than
the baseline value, although the magnitudes of increase in
the serum BBI concentration were not as large as that ob-
served after 2 or 4 wk of BBIC treatment. These results are
consistent with the previous observation that BBI levels in
urine were increased in these patients after BBIC treatment
(12). These findings suggest that BBI was absorbed after
oral ingestion.
In contrast to the large individual variation observed in
the human subjects, the individual variation for the serum
BBI concentration in dogs before BBIC treatment was
small, as reflected by the smaller coefficient of variation (62
÷ 203 = 0.305). This is not surprising, because the dogs were
fed the same food before and during the study, and the blood
samples were collected at the same time. The serum BBIC
concentration displayed a dose-dependent increase in dogs
after 52 wk of treatment with BBIC. Such an increase was
not observed in the animals after 13 wk of BBIC treatment.
This is different from the time course observed in the human
BBIC clinical trial, in which the increase in the serum BBI
concentration was most significant after 2–4 wk of BBIC
treatment and then gradually diminished during the remain-
ing 5 mo of BBIC treatment. The cause for such a difference
is not known.
In the present study, the serum BBI concentration in mice
could not be measured by the inhibitory ELISA method be-
cause of the existence of anti-BBI antibodies in the mouse
serum. The binding of the anti-BBI antibodies to the immo-
bilized rBBI antigen made it impossible to measure the se-
rum BBI concentration in mice by the inhibitory ELISA
method. It is unlikely that this problem can be overcome by
171
changing the design of the ELISA method, because the anti-
BBI antibodies in the mouse serum will most likely mask the
BBI antigen and prevent its detection by other immunoassay
methods. The levels of anti-BBI antibodies were signifi-
cantly higher in the mice treated with BBIC than in the mice
treated with empty capsules, suggesting that the mice pro-
duced anti-BBI antibodies in response to the BBIC treat-
ment. The increase in serum levels of anti-BBI antibodies in
dogs after BBIC treatment was not significant, and no
change in the serum levels of anti-BBI antibodies was ob-
served in human patients after BBIC treatment.
The different antibody responses to orally ingested BBI
in dogs and mice could reflect a species difference, because
the dogs and mice were treated with BBIC at the same doses
in relation to their body weight. It is also possible that the
difference in the anti-BBI antibody responses between these
two species is related to their prior exposure to BBI in the
diet. In this study, the dogs and mice were fed diets devoid
of soybean products during the BBIC treatment periods;
however, the diets used before the treatment periods were
unmodified Certified Canine Diet and Certified Rodent Diet,
which contain soybean as a major ingredient. Because the
soybean content in the unmodified Certified Rodent Diet is
~40% higher than in the unmodified Certified Canine Diet
(personal communication, Dr. Dorrance Haught, Purina
Mills), the antibody response to orally ingested BBI ob-
served in mice could be a result of the relatively higher prior
exposure to BBI in the diet. The lack of antibody response to
orally ingested BBI in human patients may or may not be
due to a species difference, because the highest BBIC dose
received by the patients was only equivalent to ~10% of that
received by the dogs and mice. The average level of soybean
consumption in the US population has been estimated to be
2.5 g/capita/day (18) or 0.16% assuming a normal daily di-
etary intake of 1,600 g, which is about two orders of magni-
tudes below the levels of soybean constituents in the canine
and rodent diets and is unlikely to be sufficient to elicit an
anti-BBI antibody response.
The development of antibodies to orally ingested proteins
has been reported previously for proteins other than BBI.
For instance, orally administered bovine milk lactoferrin
was shown to induce the production of anti-lactoferrin anti-
bodies in female BALB/c mice (19). In germ-free rats fed
ovalbumin-containing food, the levels of antiovalbumin an-
tibodies increased (20). In young piglets abruptly weaned
onto soy food at 3 wk of age, antibodies against soybean
protein were produced, and the serum levels of the anti-
soybean protein antibodies were almost comparable to those
induced by immunization with soybean protein in adjuvant
at 7 wk (21).
The levels of the anti-BBI antibodies in the mice treated
with BBIC were relatively low, and a relatively high concen-
tration (20% in this study) of the mouse serum was needed to
demonstrate the existence of the anti-BBI antibodies. This is
consistent with the observations on the antibody responses
to other orally ingested proteins. For example, the antibody
response to orally ingested ovalbumin was very weak and
172
was mostly IgM (20). In the piglets primed by feeding with
soybean food, no further increase in the levels of antibodies
occurred after subsequent immunization with soybean pro-
teins, suggesting the development of specific unresponsive-
ness to soy proteins in neonates fed soybean food (21).
Given that the levels of the anti-BBI antibodies were rela-
tively low in the mice treated with BBIC and were below the
detection limit in human patients after BBIC treatment, it is
not expected that oral administration of BBIC will result in
clinically meaningful complications related to an immune
response to BBI antigen.
In previous studies, BBI was shown to be effective in
suppressing cancer development in the lung (22), liver (23),
and colon (24) of mice exposed to 3-methylcholanthrene
(22) or dimethylhydrazine (23,24). The results of the present
study have demonstrated that mice developed antibodies
against BBI after 15 wk of treatment with BBIC. It is not
clear why BBI is effective as an anticarcinogenic agent in a
species of animals that develop anti-BBI antibodies after
BBI exposure. One possible explanation is that the levels of
anti-BBI antibodies developed in the mice might be too low
to completely neutralize the anticarcinogenic effect of BBI.
Another possible explanation is that BBI might have already
exerted its anticarcinogenic effect by the time the anti-BBI
antibodies were developed. If this is the case, a diminished
effect of BBI could be expected in mice with prior BBI ex-
posure. Because the anti-BBI antibodies were not detected
in human patients after 6 mo of treatment with BBIC, the
anticarcinogenic effect of BBI is not expected to be dimin-
ished in humans by prior BBI exposure.
Acknowledgments and Notes
This work was supported by a contract from National Cancer Institute
to MPI Research and a grant from ProtoMed (Lake Forest, IL). Address
correspondence to X. S. Wan, University of Pennsylvania, 195 John Mor-
gan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6072. Phone:
(215) 898-0270. FAX: (215) 898-0090. E-mail: xswan@mail.med.upenn.
edu.
Submitted 7 February 2002; accepted in final form 8 April 2002.
References
1. Kennedy AR: Chemopreventive agents: protease inhibitors. Pharma-
col Ther 78, 167–209, 1998.
2. Kennedy AR: In vitro studies of anticarcinogenic protease inhibitors.
In Protease Inhibitors as Cancer Chemopreventive Agents, Troll W
and Kennedy AR (eds). New York: Plenum, 1993, pp 65–91.
3. Kennedy AR: Anticarcinogenic activity of protease inhibitors: over-
view. In Protease Inhibitors as Cancer Chemopreventive Agents, Troll
W and Kennedy AR (eds). New York: Plenum, 1993, pp 9–64.
4. Fontham E and Correa P: The epidemiologic approach to the study of
protease inhibitors. In Protease Inhibitors as Cancer Chemopreventive
Agents, Troll W and Kennedy AR (eds). New York: Plenum, 1993, pp
1–8.
5. Page JG, Rodman LE, Giles HD, Farnell DR, and Wood RD: Thirteen-
Week Toxicity Study of Bowman-Birk Inhibitor Concentrate (BBIC) in
Nutrition and Cancer 2002
 Dogs. Birmingham, AL: Southern Research Institute, 1994. (SRI-CBE
Rep. No. 94-135-7482)
6. Page JG, Heath JE, May RD, and Martin JF: Thirteen-Week Toxicity
Study of Bowman-Birk Inhibitor Concentrate (BBIC) in Rats. Birming-
ham, AL: Southern Research Institute, 1994. (SRI-CBE Rep. No. 94-
060-7482)
7. Serota DG: Six-Month Oral Toxicity Study of Bowman-Birk Inhibitor
Concentrate in Mice. Mattawan, MI: MPI Research, 2000. (Rep. No.
560-057)
8. Serota DG: One-Year Oral Toxicity Study of Bowman-Birk Inhibitor
Concentrate in Dogs. Mattawan, MI: MPI Research, 2000. (Rep. No.
560-058)
9. Malkowicz SB, Broderick GA, Zoltick B, Marks SHF, Treat A, et al.:
Phase I study of oral Bowman-Birk inhibitor concentrate (BBIC), a soy
protein, on patients with lower urinary tract symptoms (abstr). J Urol
161, 228, 1999.
10. Armstrong WB, Kennedy AR, Wan XS, Atiba J, McLaren CE, et al.:
Single dose administration of Bowman-Birk inhibitor concentrate
(BBIC) in patients with oral leukoplakia. Cancer Epidemiol
Biomarkers Prev 9, 43–47, 2000.
11. Armstrong WB, Kennedy AR, Wan XS, Taylor TH, Nguyen QA, et
al.: Clinical modulation or oral leukoplakia and protease activity by
Bowman-Birk inhibitor concentrate in a Phase IIa chemoprevention
trial. Clin Cancer Res 6, 4684–4691, 2000.
12. Malkowicz SB, McKenna WG, Vaughn DJ, Wan XS, Propert KJ, et
al.: Effects of Bowman-Birk inhibitor concentrate (BBIC) in patients
with benign prostatic hyperplasia. Prostate 48, 16–28, 2001.
13. Kennedy AR, Szuhaj BF, Newberne PM, and Billings PC: Preparation
and production of a cancer chemopreventive agent, Bowman-Birk in-
hibitor concentrate. Nutr Cancer 19, 281–302, 1993.
14. Wan XS, Lu L-J, Anderson KE, Ware JH, and Kennedy AR: Urinary
excretion of Bowman-Birk inhibitor in humans after soy consumption
as determined by a monoclonal antibody-based immunoassay. Cancer
Epidemiol Biomarkers Prev 9, 741–747, 2000.
Vol. 43, No. 2
15. Wan XS, Koch CJ, Lord EM, Manzone H, Billings PC, et al.: Mono-
clonal antibodies differentially reactive with native and reductively
modified Bowman-Birk protease inhibitor. J Immunol Methods 180,
117–130, 1995.
16. Kock CJ and Raleigh JA: Radiolytic reduction of protein and non-
protein disulfides in the presence of formate: a chain reaction. Arch
Biochem Biophys 287, 75–84, 1991.
17. Lord EM, Harwell L, and Koch CJ: Detection of hypoxic cells by
monoclonal antibody recognizing 2-nitroimidazole adducts. Cancer
Res 53, 5721–5726, 1993.
18. Sipos E: Dietary trends for vegetable proteins in foods. Inform 1,
753–757, 1990.
19. Debbabi H, Dubarry M, Rautureau M, and Tome D: Bovine lactoferrin
induces both mucosal and systemic immune response in mice. J Dairy
Res 65, 283–293, 1998.
20. Dahlgren UI, Wold AE, Hanson LA, and Midtvedt T: Secretory anti-
body response against bacterial antigens and food proteins. Immunol
Res 10, 437–440, 1991.
21. Bailey M, Miller BG, Telemo E, Stokes CR, and Bourne FJ: Specific
immunological unresponsiveness following active primary responses
to proteins in the weaning diet of piglets. Int Arch Allergy Immunol
101, 266–271, 1993.
22. Witschi H and Kennedy AR: Modulation of lung tumor development
in mice with the soybean-derived Bowman-Birk protease inhibitor.
Carcinogenesis 10, 2275–2277, 1989.
23. St. Clair WH, Billings PC, Carew J, Keller-McGandy C, Newberne P,
et al.: Suppression of dimethylhydrazine-induced carcinogenesis in
mice by dietary addition of the Bowman-Birk protease inhibitor. Can-
cer Res 50, 580–586, 1990.
24. Billings PC, Newberne P, and Kennedy AR: Protease inhibitor sup-
pression of colon and anal gland carcinogenesis induced by dimethyl-
hydrazine. Carcinogenesis 11, 1083–1086, 1990.
173