Vol.1, No.3, 74-79 (2011)
doi:10.4236/oji.2011.13009
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/oji/
Open Journal of Immunology
Comparison of antibody detection with
Blastomycesdermatitidis yeast lysate antigens in serum
specimens from immunized rabbits and infected dogs
Will Christenson, Rachel Horton, Kayla Campbell, Kelly Meacham*, A mber Schroeder,
Gene M. Scalarone
Department of Biological Sciences, Idaho State University, Pocatello, USA; *Corresponding Author: meackel2@isu.edu
Received 9 September 2011; revised 18 October 2011; accepted 8 November 2011.
ABSTRACT
This present study w as designed to evaluate B.
dermatitidis antigens, prepared from two iso-
lates (B5896, 597), when the yeast cells were
allowed to lyse in distilled water for one day or
seven days. The indirect enzyme-linked immu-
nosorbent assay (ELISA) was used to determine
the ability of the lysate reagents to detect anti-
bodies in 30 rabbit and 30 dog serum speci-
mens. Mean absorbance values w ith B5896 lys-
ate antigen ranged from 1.637 (day 1) to 1.461
(day 7) and absorbance values w ith 597 antigen
ranged from 1.579(day 1) to 1.396 (day 7) with
the serum specimens from immunized rabbits.
Serum specimens from infected dogs yielded
absorbance values ranging from 1 .67 2 (day 1) to
1.763 (day 7) w ith the B5896 and values ranging
from 1.909 (day 1) to 1.224 (day 7) with the 597.
Optimal reactivity was obtained with the day 1
lysate using both lysate antigens against the
rabbit sera and with the 597 antigen against the
dog sera. Slightly greater reactivity was evi-
denced with the day 7 B5896 antigen when the
dog sera was tested. Comparative studies are
continuing in order to produce an optimal anti-
genic preparation for antibody detection in
blastomycosis.
Keywords: ELISA; Blastomy cesdermatitidis ;
Antibody Detection; Lysate Antigens
1. INTRODUCTION
Bla stomycesde rmatitidisis a thermally dimorphic fun-
gus that exists in a mycelial mold-like form in nature at
approximately 25˚C, but when incubated at 37˚C, it sur-
vives in the yeast form [1,2]. The organism exists in the
environment as a spore-producing mold and the spores
can be inhaled into the lungs where they develop into
budding yeast cells [3]. In humans, the typical route of
infection is inhalation of aerosolized conidia of B. der-
matitidis. From the respiratory tract, the developing
yeast form may disseminate throughout the body and
affect multiple organ systems, most commonly the lym-
phatic, skeletal, eyes and skin [4]. The disease can be
spread throughout the body in which cutaneous lesions
may occur. Up to 5 to 10 percent of disseminated cases
produce meningitis or epidural/cranial abcesses; if left
untreated, death may result in animals and in humans
[5-7]. Blastomycosis is endemic in North America, Af-
rica, India and parts of Europe. The majority of reported
cases are from North America, primarily in the Great
Lakes area as well as the Mississippi and Ohio River
Valley regions [8,9].
In recent years, there has been an adamant thrust in
understanding the proper diagnosis of this disease be-
cause blastomycosis is often misdiagnosed that can cre-
ate treatment delays [10-14]. As with many other dis-
eases, time is of particular importance in terms of diag-
nosis and treatment [15,16]. Current culture methods
may be effective, but very time consuming. Therefore
researchers have focused on the development of immu-
nodiagnostic assays in attempts to provide improved
methods for the clinical diagnosis of blastomycosis in
humans and animals [2,10,11,14-18]. Our laboratory has
developed various B. dermatitidisyeast phase lysate
preparations and utilized these antigens in the ELISA for
the detection of antibodies in serum specimens from
immunized or infected animals [10,11,13,15]. These
studies have indicated the potential of the yeast lysate
antigens as immunodiagnostic reagents, but additional
studies are required in order to produce an optimal anti-
gen for antibody detection.
The purpose of this present study was to compare the
ability of B. dermatitidisyeast cell lysate antigens, pre-
pared by lysing the cells in water for periods of 1 or 7
W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79
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7575
days, to detect antibodies in serum specimens from im-
munized rabbits or infected dogs. Most of our previous
comparative studies were performed with antigenic
preparations fo l l owi n g 7 day s of ly si s.
2. MATERIALS AND METHODS
2.1. Antigens
Mycelial phase cultures of two B. dermatitidisisolates
(B5896 and 597) from blastomycosis outbreaks in
Mountain Iron, Minnesota and Eagle River, Wisconsin
were converted to yeast cells by culturing at 37˚C on
BHI agar. Yeast phase lysate reagents were prepared by a
method similar to one that was previously used for the
production of antigen from Histoplasmacapsulatum and
modified in our laboratory for B. dermatitidislysate an-
tigen production [10,11,13,19,20]. The yeast phase cells
were grown for 7 days at 37˚C in a chemically defined
medium in an incubator shaker, harvested by centrifuga-
tion (700 ×g; 5 min), followed by washing with distilled
water, resuspend ed in distilled water and then allowed to
lyse for 1 or 7 days at 37˚C in water with shaking. The
preparations were centrifuged, filter sterilized, merthio-
late added (1:10,000) and stored at 4˚C for further use.
Protein determinations were performed on the lysates
using the BCA Protein Assay Kit (Pierce Chemical Com-
pany, Rockford, IL) and dilutions of the antigenic re-
agents to be used in the assays were based on protein
concentration.
2.2. Serum Specimens
Serum specimens (30) from rabbits that were previ-
ously immunized using lysate preparations of B. derma-
titidis and serum specimens (30) from dogs with blasto-
mycosis, provided by Dr. A.M. Legendre (University of
Tennessee College of Veterinary Medicine, Knoxville,
TN) were assayed. In addition 5 serum specimens from
rabbits immunized with Histoplasmacapsulatum killed
whole yeast cells were used in the assays to compare
cross reactivity with the 4 B. dermatitidis lysate antigens.
2.3. ELISA
The ability of each yeast lysate reagent to detect anti-
bodies in the above serum specimens was determined
using the indirect biotin-streptavidin enzyme-linked
immunosorbent assay (ELISA). Each lysate antigen was
diluted (2000 ng of protein) in a carbonate-bicarbonate
coating buffer (pH 9.6) and then added to triplicate wells
(100 ul) of a Costar 96-well microdilution plate
(Thermo-Fisher Scientific) and incubated overnight at
4˚C in a humid chamber followed by washing three
times with phosphate buffered saline containing 0.15%
between 20 (PBS-T). The serum specimens (1:2500 di-
lution; 100 ul) were added to the microplate wells and
incubated for 30 min at 37˚C. Following this incubation
the wells were washed as above and 100 ul of goat
anti-rabbit or goat anti-dog IgG (H & L) biotin labeled
antibody (Kirkegaard and Perry, Gaithersburg, MD) was
added to each well and incubated for 30 min at 37˚C.
The plates were washed as above and 100 ul of peroxi-
dase labeled streptavidin was added to each well and
incubated for 30 min at 37˚C The plates were again
washed as above and 100 ul of 1-step Ultra TMB-ELISA
peroxidase substrate (Thermo-Fisher Scientific) was
added to each well and incubated for approximately 2
min at room temperature. The reaction was stopped by
the addition of sulfuric acid and the absorbance read at
450 nm using a BIO-RAD 2550 EIA reader.
3. RESULTS
3.1. Comparison of Antibody Detection in
Rabbit Sera with the Day 1 and Day 7
L ysate Antigens
Thirty serum specimens from rabbits were immunized
with B. dermatitidis. Two trials were performed using
yeast lysate antigens (B5896 and 597) prepared on day 1
and day 7. Th e results of trial 1 (Figure 1) indicated that
the B5896 day 1 lysate antigen had absorbance values
ranging from 1.122 to 1.759 with a mean value of 1.533,
while the range of the day 7 lysate absorbance values
was 0.914 to 1.672 with a mean value of 1.269. The
second lysate antigen (597) on day 1 had absorbance
values ranging from 1.155 to 1.765 with a mean value of
1.488 and day 7 values ranged from 1.008 to 1.761 with
a mean value of 1.329. In trial 2 the absorbance value
range of day 1 lysate antigen (B5896) was 1.556 to
1.958 with a mean value of 1.740; in contrast the day 7
lysate had a range of 1.498 to 1.859 with a lower mean
value of 1.653. The second trial of the day 1 597 lysate
antigen had absorbance values ranging from 1.487 to
1.863 and a mean value of 1.669. The day 7 absorbance
values of the same antigen ranged from 1.176 to 1.856
with an overall lower mean value of 1.463 (Figure 2).
3.2. Comparison of Antibody Detection in
Dog Sera with the Day 1 and Day 7
L ysate Antigens
Thirty serum specimens from dogs with Blastomyco-
siswere also assayed, as above with the rabbit sera, with
the day 1 and day 7 lysate antigenic preparatio ns. In trial
1 (Figure 3) the B5896 day 1 lysate h ad abso rbance val-
ues ranging from 0.450 to 1.997 with a mean value of
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76
Figure 1. Trial 1 indirect ELISA mean absorbance values using 20 rabbit serum specimens with lysate antigens (B5896 and 597)
prepared on day 1 and day 7
Figure 2. Trial 2 indirect ELISA mean absorbance values using 10 rabbit serum specimens with lysate antigens (B5896 and 597)
prepared on day 1 and day 7.
Figure 3. Trial 1 indirect ELISA mean absorbance values using 20 dog serum specimens with lysate antigens (B5896 and 597) pre-
pared on day 1 and day 7
1.418 and the day 7 antigen values ranged from 0.529 to
2.205 with a greater mean value of 1.714 compared to
the day 1 lysate. Antigen 597 day 1 values ranged from
0.480 to 2.344 with a mean value of 1.765 whereas the
day 7 values ranged from 0.429 to 1.690 with a lower
mean value of 1.124 when compared to 597 (day 1).
Trial 2 data (Figure 4) indicated that the B5896 (day 1)
absorbance values ranged from 1.427 to 2.210 with a
mean value of 1.926 and the day 7 values ranged from
1.639 to 1.994 with a sligh tly lower mean value of 1.812.
Lysate antigen 597 (day 1) had an absorbance range of
1.630 to 2.210 with a mean value of 2.052; in contrast
day 7 lysate values ranged from 0.845 to 1.720 with a
much lower mean val ue o f 1. 32 3.
Openly accessible at
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7777
Figure 4. Trial 2 indirect ELISA mean absorbance values using 10 dog serum specimens with lysate antigens (B5896 and 597) pre-
pared on day 1 and day 7
3.3. Antibody Detectio n Mean Abs orbance
Values Obt ained with the Day 1 and Day
7 Lysate Antigens in Sera from Rabbits
and Dogs
The antibody detection resultswith the day 1 and day
7 lysatesfrom the two trials using sera from immunized
rabbits and infected dogs were combined for compara-
tive purposes (Figure 5). All 30 rabbit serum specimens
tested with B5896 lysate antigen showed a day 1 mean
value of 1.637 compared to the day 7 mean value of
1.461. Thirty dog serum specimens tested using the same
antigen indicated a day 1 value of 1.672 and day 7 value
of 1.763. The same 30 rabbit sera were tested using a
different yeast lysate antigen (597) which yielded a
mean value of 1.579 (day 1) while the day 7 lysate re-
sulted in a mean value of 1.396. Lastly, the same 30 dog
sera assayed with antigen 597 resulted in absorbance
values of 1.909 (day 1) and 1.224 (day 7).
3.4. Specificity Determinations with the B.
dermatitidislysate Antigens
Five serum specimens from rabbits that had been im-
munized with H. capsulatumwhole yeast cells were as-
sayed with both B. dermatitidis and H. capsulatum lysate
reagents in order to obtain initial evidence with regard to
specificity of the day 1 and day 7 lysate antigens. As
shown in Table 1, two of the B. dermatitidis prepara-
tions (Antigen 2: B5896, day 7 and Antigen 3: 597, day
1 exhibited a lower level of cros s reactivity than the An-
tigen 1: B5896 day 1 and Antigen 4: 597 day 7 when
compared to the reactivity evidenced with the homolo-
gous H. capsulatum antigens. As indicated above this
data was obtained with a limited number of H. capsula-
tumsera and further studies are required in order to as-
sess the specificity of the different lysate preparations.
4. DISCUSSION
The production and evaluation of B. d ermatitidis yeast
cell lysate antigenic reagents has been a major thrust of
our research endeavors for several years. Our initial re-
search work on the preparation of B. dermatitidis lysates
was based on previous investigations on the preparation
of lysate antigens for either the detection of antibod ies or
delayed dermal hypersensitivity in coccidioidomycosis
and histoplasmosis. In studies on the development of
skin-testing and serological antigens for coccidioido-
mycosis, Coccidioidesimmitis spherules were lysed in
distilled water for a period of 40 days [19,21,22]. In
contrast a one-day period of lysis of H. capsulatum yeast
cells was sufficient to produce a reagent that detected
both delayed dermal hypersensitivity as well as being
useful for the detection of antibodies in the ELISA
[19,20]. With regard to our work with B. dermatitidis
yeast phase lysate antigens many of our comparative
studies have been done with reagents following growth,
harvesting and allowing the cells to lyse for a p eriod of 7
days [10,11,13,15,23,24].
The primary objective of this present comparative
study was to compare yeast phase lysate reagents that
had been prepared following either 1 day or 7 days of
lysis in water. The lysate antigens were prepared from
human isolates of B. dermatitidis from outbreaks in
Mountain Iron, Minnesota (B5896) and Eagle River,
Wisconsin (597) and assayed using the ELISA to detect
antibodies in serum specimens from immunized rabbits
or infected dogs. The evidence indicated that the day 1
lysates from both isolates reacted optimally with regard
to detecting antibodies in the rabbit sera and the 597 day
1 lysate showed a greater degree of sensitivity than the
day 7 lysate when the dog sera were assayed. In contrast
the B5896 day 7 lysate exhi bited sli ght ly great e r reacti vit y
than the day 1 preparation with th e dog se rum specimens.
W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79
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78
Figure 5. Combined (trial 1 and trial 2) mean absorbance values using rabbit and dog serum specimens with lysate antigens (B5896
and 597) prepared on day 1 and day 7.
Table 1. H.capsulatumserum specimens were teste d with 4 B. dermatitidislysate antigens [B* = (1) B5896 (day 1); (2) B5896 (day 7);
(3) 597 (day 1); (4) 597 (day 7)] and with 4 H. capsulatum lysate antigens [H* = (1) G217B; (2) G217B; (3) G217A; (4) G217B].
Cross-reactivity study Antigen 1 Antigen 2 Antigen 3 Antigen4
B* 1.626 1.597 1.44 1.535
H* 1.713 1.942 1.83 1.785
Therefore when the data from the above sensitivity
comparisons and the specificity determinations against
the limited number of H. capsulatum serum specimens it
appeared that the day 1 597 lysate antigen was an opti-
mal reagent for antibody detection. These results indi-
cate the potential of the day 1 lysates and also provide
evidence for continuing comparative studies on the
preparation of such lysates from additional B. dermatiti-
dis isolates in an effort to develop lysate antigens with a
high degree of sensitivity and specificity for use in the
immunodiagnosis of Blastomycosis in humans and ani-
mals.
5. ACKNOWLEDGEMENTS
This research was supported by the Department of Biological Sci-
ences, Idaho State University.
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