APPLIED MICROmOLOGY, Mar., 1967, p. 350-356 Vol. 15, No.

2
Copyright @ 1967 American Society for Microbiology Printed In U.S.A.

Soluble Antigens for Immunofluorescence Detection

of Histoplasma capsulatum Antibodies
WILLIAM A. HOOK AmN EARL H. FIFE, JR.
Department of Serology, Walter Reed Army Institute of Research, Walter Reed Army Medical
Center, Washington, D.C.
Received for publication 20 October 1966

Soluble antigens from Histoplasma capsulatum in the mycelial and yeast phase
were purified by gel filtration, fixed onto paper discs, and employed in an indirect
immunofluorescence procedure to detect antibody in sera from individuals infected
with H. capsulatum. The elution patterns of crude histoplasmin passed through
Sephadex G-200 revealed two minor peaks of protein showing immunofluorescence,
complement fixing, and precipitating-antigen activity. A large peak containing the
pigment and other low molecular weight materials showed no serological activity.
A polysaccharide antigen obtained from fragmented, deproteinized yeast-phase cells
was reactive in the fluorescent-antibody test but showed no antigen activity in com-
plement fixation or precipitin tests. Although certain sera from culturally proven
cases of blastomycosis, coccidioidomycosis, and cryptococcosis reacted with the
purified Histoplasma antigens, preliminary evaluation indicated that the immuno-
fluorescence technique may be of value as a screening procedure for the serodiagno-
sis of histoplasmosis.

Histoplasmin, a soluble culture filtrate of myce-
lial-phase Histoplasma capsulatum, has been
widely used in complement fixation (CF), pre-
cipitation, and latex agglutination tests for the
serodiagnosis of histoplasmosis (9). However,
histoplasmin has not been commonly employed in
fluorescent-antibody techniques which generally
require the use of whole-cell antigen. When histo-
plasmin was adsorbed to a cellulose acetate filter
paper matrix and dried according to the soluble
antigen fluorescent antibody (SAFA) technique
of Toussaint (hla), it showed no serological ac-
tivity (Toussaint, unpublished data). This problem
was resolved in the present study which revealed
that removal of inhibitory substances by gel filtra-
tion yielded a sensitive antigen for immuno-
fluorescence detection of Histoplasma antibodies.
Intact yeast-phase cells of H. capsulatum have
been used as antigen in fluorescent-antibody tests
to detect Histoplasma antibodies in anticomple-
mentary sera (4). However, the results necessarily
are dependent on antigens which reside on the
insoluble outer surface of the cells (8). This
restriction precludes immunofluorescence studies
with purified, soluble preparations of yeast-phase
cells which may be necessary for definitive studies
on the problems of sensitivity and specificity of
serological tests for histoplasmosis. In the present
work, a polysaccharide antigen extracted from
fragmented yeast-phase cells reacted well in the
350
SAFA procedure with sera from patients with
histoplasmosis. Preliminary evaluation of both
mycelial- and yeast-phase antigens in the SAFA
procedure indicated that this method was worthy
of consideration by those laboratories requiring a
sensitive procedure for the detection of Histo-
plasma antibodies but not equipped to perform
the technically more complex CF test.
MATERIALS AND MErHODS
Sera. Blood was obtained from humans infected
with H. capsulatum, Blastomyces dermatitidis, Cocci-
dioides immitis, Cryptococcus neoformans, or Myco-
bacterium tuberculosis, and the sera were collected by
centrifugation. These sera were used to evaluate the
sensitivity and specificity of the standard and experi-
mental Histoplasma antigens. Equal volumes of serum
from six individuals with chronic pulmonary histo-
plasmosis were combined, and the pool was used to
determine antigen activity in the various fractions ex-
tracted from H. capsulatum. In addition, 12 specimens
of sera collected from healthy adult males and shown
to be nonreactive in SAFA tests with Histoplasma
antigens were combined in equal amounts to provide
a negative serum pool control for the SAFA pro-
cedure. All sera were stored at -60 C until tested.
Mycelial-phase antigens. The histoplasmin was a
pooled filtrate of H. capsulatum strains G-8, G-92,
and G-95 grown for 6 months at 20 C in the synthetic
medium described by Smith et al. (11). A 25-ml
amount of the crude histoplasmin was passed through
a column (35 by Z.5 cm) of Sephadex G-200 dextran
VOL. 15, 1967 DETECTION OF H. CAPSULATUM ANTIBODIES
gel (Pharmacia Fine Chemicals, Inc., Piscataway,
N.J.), in 0.15 M NaCl (saline) to separate serologically
reactive, high molecular weight antigen fractions from
lower molecular weight medium constituents and
metabolic products. Relative protein contents of the
eluates were estimated by optical-density measure-
ments at 280 m,u. Fractions of 5 ml were collected and
used to sensitize 0.25-inch (0.6 cm) circular discs
punched from grid-lined cellulose acetate filter sheets
(HAWG 00010; porosity, 0.45 ,; Millipore Filter
Corp., Bedford, Mass.). After drying, antigen-coated
discs were fixed for 10 min at 4 C in 1% acetic acid
in 95% ethyl alcohol. Fixative was removed by wash-
ing discs in three changes of 0.05 M tris(hydroxy-
methyl)aminomethane (Tris) buffer in 0.85% NaCl
adjusted to pH 8 (12). Fractions showing maximal
SAFA activity (tubes 8 to 19, Fig. 1) were pooled and
used to sensitize a large number of discs to evaluate
the mycelial-phase SAFA antigen.
Unfractionated histoplasmin, diluted 1:10, was
used as antigen in CF tests. For use in immunodiffu-
sion studies, the histoplasmin was concentrated five-
fold by passing the crude -antigen through an ultra-
filtration membrane (UM-1, Amicon Corp., Cam-
bridge, Mass.) which retained molecules above a
molecular weight of 10,000.
Yeast-phase antigens. Soluble antigens were ob-
tained from yeast-phase strains G-8, G-66, G-92, and
G-95 grown for 48 hr on Brain Heart Infusion Agar
(Difco) at 37 C. Harvests were suspended in 0.5%
Formalin, and the cells were washed three times in
sterile distilled water, and resuspended to a concen-
tration of S X 108 cells per milliliter in sterile distilled
water containing 0.01% Merthiolate. Fragmentation
was achieved by twice passing a 40-ml amount of the
suspension through a French pressure cell (American
Instrument Co., Inc., Silver Spring, Md.) at a pressure
of 18,000 psi. After rupture of the cell walls, protein
was precipitated at 4 C by adding an equal volume
of 0.5 M trichloroacetic acid and allowing the mixture
to stand for 3 hr. After centrifugation at 2,000 X g
for 15 min, the clear supernatant fluid was decanted
and treated with 2 volumes of absolute ethyl alcohol.
The precipitate that formed upon standing overnight
at 4 C was centrifuged at 2,000 X g for 15 min, re-
suspended to 5 ml in saline, and adjusted to pH 7;
a 2.5-mil quantity was fractionated by passage through
a column (1.5 by 22 cm) of Sephadex G-200 in saline.
The effluent was collected in 2-ml fractions. Discs
then were dipped in each fraction, and the antigen
was fixed according to the procedure used for the
histoplasmin fractions. Those showing maximal im-
munofluorescence activity with homologous antiserum
(fractions 5 and 6, Fig. 2) were combined and used
to evaluate the yeast-phase SAFA test. For CF tests,
whole yeast-phase cells of the same strains were sus-
pended in saline containing 0.01% Merthiolate and
were diluted to concentration optimal for the CF test.
Antigens and discs sensitized with antigen were
stored at 4 C.
Serological tests. The SAFA test (l la, 12) was per-
formed in the wells of plastic Disposotrays (Limbro
Chemical Co., New Haven, Conn.). Antigen-coated
discs, prepared as described above, were immersed in
351
0.2 ml of test serum diluted 1: 8 in Tris-saline contain-
ing 1% Tween 80, and the plates were agitated by
rotation at 120 rev/min for 45 min. The serum then
was removed by aspiration, and the discs were washed
three times in 1-ml volumes of Tris-saline by rotation
for 10 min at 180 rev/min. Fluorescein-conjugated
goat anti-human globulin (50-780, Microbiological
Associates, Inc., Bethesda, Md.) diluted 1:20 in Tris-
saline containing 2% Tween 80 and clarified by filtra-
tion through a cellulose acetate filter of 0.45-;& porosity
was used for the secondary reaction. A 0.2-ml volume
of conjugated antiserum was added to each disc. The
tests then were agitated for 30 min at 120 rev/min and
again washed three times. After drying, fluorercence
of discs was measured in a photoelectric fluorometer
(G. K. Turner Associates, Palo Alto, Calif.) equipped
with a 7-54 primary filter, a 2A-12 secondary filter,
and a 10% neutral density filter.
CF tests were performed by use of precisely stand-
ardized reagents prepared according to the method of
Kent and Fife (5). Titer was recorded as the reciprocal
of the highest serum dilution giving not greater than
50% hemolysis in the presence of an optimal amount
of antigen, five 50% hemolytic units of complement,
and 1.5 X 108 optimally sensitized sheep erythrocytes
in a total volume of 1.5 ml. Sera showing CF titers
less than 1:8 were considered negative.
Precipitin tests were performed in agar gel on
microscope slides containing a 2.0-ml layer of 0.6%
purified agar in 0.85% NaCl and 0.01% Merthiolate.
Wells 3 mm in diameter were separated by a distance
of 6 mm. Slides were examined for the presence of
precipitin bands after 2 and 4 days at 22 C.
A commercially prepared tube agglutination test,
in which histoplasmin was adsorbed onto latex parti-
cles (11-58J, Colab Laboratories, Inc., Chicago
Heights, Ill.), was used to titrate agglutinating anti-
body. Titers of 1:8 or greater were considered
positive.
Chemical analysis. Chemical tests of the yeast- and
mycelial-phase SAFA antigens purified by gel filtra-
tion included determination of protein by the Folin-
Ciocalteau reagent (6), carbohydrate by the Molish
reaction (10), deoxyribonucleic acid (DNA) by the
method of Dische (3), and lipid by reduction of
potassiumi dichromate after extraction with alcohol
and petroleum ether (1). Heat stability of the SAFA
antigens was determined by placing 0.5 ml of the ap-
propriate purified antigen in a stoppered tube, incu-
bating for 30 min at 70 C or 100 C in a constant-
temperature water bath, cooling, and using as antigen
to coat discs in the usual manner.
RESULTS
Purification of histoplasmin. In initial experi-
ments, it was observed that crude histoplasmin
lost serological activity when dried on cellulose
acetate discs for use in the SAFA procedure
(Toussaint, unpublished data). This problem was
overcome by passing histoplasmin through
Sephadex G-200 dextran gel to separate serologi-
cally active, high molecular weight components
from inhibitory culture constiuents or metabolic
352 HOOK AND FIFE APPL. MICROBIOL.
products (Fig. 1). Tubes 8 to 25 contained the
antigens which reacted in the immunofluorescence
test, with the earlier fractions showing the greatest
activity. The characteristic green-brown color of
histoplasmin was found to be associated with a
serologically inactive major peak of protein ma-
terial eluted in tubes 33 to 40. Figure 1 also shows
two discrete areas of maximal complement-fixing 150 _
activity (between tubes 10 to 16 and tubes 20 to -I

22). Precipitating antigen was also found in the

same general region.
Polysaccharide from yeast-phase cells. A sero- 50
100 l X i 1
logically active, soluble polysaccharide was ob-
tained from fragmented yeast-phase H. capsula-
twn. These cells, however, were found to be un-
usually resistant to fragmentation and required at 2 6 10 14 18 22 26 30 34
least two passages through the pressure cell to Tube Number
assure rupture. Phase-contrast microscopy of the
final effluent from the press revealed broken cells FIG. 2. Carbohydrate elution pattern of yeast-phase
soluble antigen from Histoplasma capsulatum passed
that had lost intracellular constituents, but many through a column of Sephadex G-200 in 0.85% NaCl.
of the empty cell walls retained their oval shape. Fractions showing strongest immunofluorescence ac-
Treatment of the suspension with trichloroacetic tivity against histoplasmosis serum pool are designated
acid gave a heavy precipitate. Addition of ethyl by a line of double thickness.
alcohol to the trichloroacetic acid supernatant
solution produced a precipitate which was highly TABLE 1. Chemical characteristics of soluble,
active as antigen in the SAFA procedure but mycelial-, and yeast-phase antigens from
showed no CF or precipitating activity. Gel filtra- Histoplasma capsulatum used in im-
tion of this ethyl alcohol-insoluble material munofluorescence (SAFA) tests
yielded several fractions containing carbohydrate,
but immunofluorescence activity was found Antigen Carbo-
Protein hydrate DNA Lipid
almost exclusively in the high molecular weight
fractions obtained in tubes 5 and 6 (Fig. 2). None pg/mi pg/mi pg/mi
of these fractions, however, was active in CF or Mycelial phase .... 270 250 17 +
Yeast phase . ........<2
138 <5 +
2.00

precipitin tests. All of the above fractionations

were performed three times, and the results ob-
1.50
tained were nearly identical.
Chemical composition of SAFA antigens. The
data presented in Table 1 suggest that the purified
E
0o
immunofluorescent antigen obtained from histo-
plasmin consisted primarily of protein and carbo-
-6
@.210
hydrate with some DNA and lipid. On the other
c:
cL
SA FA
hand, the principal component of antigen purified
CF from yeast-phase cells was carbohydrate. Both
PPTN. mycelial- and yeast-phase antigens retained sero-
logical activity after heating to 70 C for 30 min,
and both contained lipid (Table 1). Although the
colorimetric method used for lipid assay has been
used by others to quantitate total blood lipids by
calculation of the oxidation of a cholesterol stand-
Tube Number
ard, use of this standard curve was not considered
to be directly applicable to the quantitative meas-
FIG. 1. Elution pattern ofhistoplasmin passed through urement
a column of Sephadex G-200 in 0.85% NaCI. Fractions
of lipids from Histoplasma. Therefore,
showing strongest serological activity against histo- in the present studies, the methods were used to
plasmosis serum pool are designated by lines of double provide qualitative evidence of the presence or
thickness. absence of lipid.
VOL. 15, 1967 DETECTION OF H. CAPSULATUM ANTIBODIES
Preliminary evaluation of mycelial- and yeast-
phase antigens. Sera from culturaUy proven cases
of histoplasmosis were tested with both mycelial-
and yeast-phase SAFA antigens, and the results
were compared with those obtained in CF, pre-
cipitation, and latex agglutination tests (Table 2).
In the SAFA procedure, 11 of 12 serum specimens
from individuals infected with H. capsulatum gave
fluorometer dial readings (FDR) of 10 or greater
with both mycelial- and yeast-phase SAFA anti-
gens, which were considered positive reactions.
On the other hand, only six of the specimens had
CF titers toward histoplasmin. These latter sera
also were positive in precipitin tests with histo-
plasmin. Positive yeast-phase CF tests were ob-
served with 8 of the 12 sera, and titers seemed
generally to parallel the magnitude of the yeast-
phase SAFA reactions. Results with sera from 12
healthy individuals showed no positive reactions
in CF, precipitin, and latex agglutination tests.
On the other hand, SAFA tests of 12 normal sera
showed 3 positive reactions with mycelial phase
antigen and 5 observable FDR with yeast-phase
353
antigen. Of these five, however, only one serum
specimen was considered to give a positive reac-
tion, i.e., FDR greater than 10.
Table 3 gives results obtained with the same
antigens tested against sera from human cases of
blastomycosis, coccidioidomycosis, cryptococco-
sis, and tuberculosis. Four of the six individuals
infected with B. dernatitidis showed significant
FDR with both mycelial- and yeast-phase anti-
gens from H. capsulatum. Three of these sera also
reacted in CF tests with yeast-phase antigen; one
was anticomplementary, and one had a histo-
plasmin latex agglutination titer of 1: 8.
The findings presented in Table 3 also suggest
that sera from humans infected with C. immitis
may give positive reactions with SAFA antigens
from H. capsulatum. One of the six coccidioido-
mycosis sera had a CF titer of 1:8 against the
yeast-phase Histoplasma.
Of the six cryptococcosis sera tested, four gave
positive SAFA values with mycelial-phase H.
capsulatum antigen, and one showed a titer of
1:32 in the histoplasmin latex test (Table 3). In

TABLE 2. Comparison of immunofluorescence (SAFA) tests with complement fixation, agar gel
precipitation, and latex agglutination tests for the detection of antibodies toward
Histoplasma capsulatum
Mycelial-phase antigens' Yeast-phase antigens
Diagnostic status Serm no.
SAFA CF PPTN LA SAFA CF

Histoplasmosis Hi 28 1:32 + _ 52
H2 99 - 1:8 77
H3 97 1:32 + - 13 -
H4 16 - - 48 1:64
H5 58 - - 33 1:16
H6 87 - 52 1:64
H7 20 - 28 1:16
H8 20 - 1:8 45 1:32
H9 53 1:32 + 59 1:16
H10 25 1:16 + 19 1:8
Hit 43 1:256 + 1:256
H12 39 1:32 + - 33 _
Healthy Ni 13------
N2------
N3------
N4------
N5 ----7-
N6 - - - - - -

N7 ----5-
N8 55 - - - 26 -
N9 11 - - 9 -
N10 - - - - 7 -

N12 - - - - -

Numbers in SAFA column are fluorometer dial readings. Complement fixation (CF) and latex
agglutination (LA) values are serum titers. Precipitin tests (PPTN) were recorded as positive when one
or more bands of precipitation were observed.
354 HOOK AND FIFE APPL. MICROBIOL.

TABLE 3. Cross-reactions between antigens from Histoplasma capsulatum and sera from
blastomycosis, coccidioidomycosis, cryptococcosis, and tuberculosis
Mycelial-phase antigens" Yeast-phase antigens
Diagnostic status Serum no.
SAFA CF PPTN LA SAFA CF

Blastomycosis RB 46 | - - 23 1:32
B2------
B3 19 - 40 1:8
B4 59 - - 1:8 39 1:16
B5 30 - - - 24 -
B6------
Coccidioidomycosis C2 - - - - - -
C2 94-----
C3 144 - - - 28 -
C4 11-----
C5 52 - - - - 1:8
C6 19 - - - 22 -
Cryptococcosis Dl 41 - - - - -
D2 - - - - 51 1:32
D3 12 - - - - -
D4------
D5 21 - - - - -
D6 11 - - 1:32 - -
Tuberculosis Ti 7 _ 1:8 23 -
T2 - - - - 19 -
T3 9-----
T4------
T5 7 - - - 7 -
T6------

a Numbers in SAFA column are fluorometer dial readings. Complement fixation (CF) and latex
agglutination (LA) values are serum titers; PPTN = precipitin tests.
contrast, only one of the six cryptococcosis sera
reacted with the yeast-phase SAFA antigen, and
this serum also reacted strongly in the CF test
(titer 1: 32).
Results in Table 3 also show that none of the
six tuberculosis sera gave reactions considered
positive in CF or SAFA tests with mycelial-phase
antigens. One serum (T-1) had a latex agglutina-
tion titer of 1: 8 and a SAFA yeast-phase FDR of
23, whereas another had a SAFA yeast-phase
FDR of 19. The remaining four sera were consid-
ered negative.
DiscussIoN
Purification of histoplasmin by gel filtration
made it possible to dry this soluble antigen on
filter paper discs without loss of serological activ-
ity for use in an immunofluorescence test. Ap-
parently the presence of high concentrations of
medium or metabolic constituents in the crude
histoplasmin caused a loss of the antigenic princi-
ple when antigen was dried (Toussaint, unpub-
lished data). The elution pattern of crude histo-
plasmin passed over Sephadex G-200 revealed two
minor peaks of protein material showing SAFA,
complement-fixing, and precipitating-antigen ac-
tivity, and one large peak of lower molecular
weight material that had a greenish-brown color
but was serologically inert (Fig. 1).
Yeast-phase polysaccharide obtained by frag-
mentation, deproteinization, and concentration
was a soluble product that could be further puri-
fied and characterized by gel filtration. The elu-
tion of antigen reactive in the immunofluorescence
test among the earliest fractions collected (Fig. 2)
suggested a molecular weight approaching
200,000.
Chemical determinations of the purified histo-
plasmin used as SAFA antigen revealed the pres-
ence of protein, carbohydrate, lipid, and some
nucleic acid (Table 1). The fractions containing
the antigen (tubes 8 to 19, Fig. 1) possessed both
complement-fixing and precipitating activity in
addition to immunofluorescence activity. On the
other hand, the deproteinized polysaccharide-
lipid antigen extracted from H. capsulatum did
not fix complement and showed no precipitin
activity (Fig. 2). Perhaps removal of nitrogenous
material from this antigen was responsible for the
failure to fix complement as has been suggested
VOL. 15, 1967 DETECTION OF H. CAPSULATUM ANTIBODIES
in the case of antigens from other pathogenic
fungi (13). Both mycleial- and yeast-phase anti-
gens were stable to heating (70 C for 30 min),
and discs impregnated with these antigens could
be kept at 4 C for several months without loss of
activity.
Preliminary evaluation of the SAFA test for
histoplasmosis by use of mycelial- and yeast-phase
antigens suggested adequate sensitivity for sero-
diagnosis, and in some instances the procedure
appeared to be superior to the standard tests.
This was evidenced by the frequent ability of a
single antigen, e.g., purified histoplasmin, to de-
tect antibody when standard histoplasmin CF
tests are negative (Table 2). A few of the sera
from healthy individuals tested by the SAFA pro-
cedure gave positive reactions with both mycelial-
and yeast-phase antigens. Since the "normal"
sera were obtained from residents of Washington,
D.C., and surrounding states, an area moderately
endemic for histoplasmosis, it is likely that at
least some may have had contact with H. capsula-
tum or its products. Thus, it would appear that
the presence of a detectable reaction in the SAFA
test does not necessarily indicate active histoplas-
mosis.
Results of Table 3 indicated that sera from
proven cases of blastomycosis, coccidioidomyco-
sis, cryptococcosis, and perhaps tuberculosis react
in SAFA test with antigens from H. capsulatum.
The occurrence of such cross-reactions with anti-
gens from Histoplasma is well known (2). Al-
though the SAFA procedure may be somewhat
less specific for differentiation between coccidioid-
omycosis and histoplasmosis than the CF, pre-
cipitin, or agglutination tests, this deficiency
appeared to be compensated for by the greater
sensitivity of the immunofluorescence test. In
view of its relative technical simplicity and the
ability to obtain results on the day of testing, the
SAFA procedure seems to be worthy of special
consideration for use in laboratories not equipped
to perform complement fixation tests for histo-
plasmosis. It appears to be more suitable than
either the tube latex agglutination or precipitin
tests as a screening procedure to identify cases
which require additional immunological, histo-
logical, and cultural studies. In addition, the
SAFA procedure can be used to obtain results
from sera that cannot be tested by complement
fixation because of anticomplementary activity,
or from sera that are not suitable for use in the
agglutination test. Experience gained by the test-
ing of more than 1,000 serum specimens sub-
mitted for routine fungal serological studies have
supported these conclusions.
355
Improvement of specificity of the SAFA tests
for histoplasmosis conceivably may be achieved
by further chemical purification of the antigens,
e.g., use of additional steps of column chromatog-
raphy or chemical precipitations. However, efforts
along these lines necessarily would be restricted
to relatively mild procedures since experience has
shown that extensive chemical treatment may re-
duce rather than improve specificity of antigens.
For example, Pine et al. (7) reported that antigens
extracted from yeast-phase H. capsulatum by a
variety of chemical procedures were less specific
than the unaltered, whole-cell preparations. Per-
haps a more rewarding approach toward improv-
ing specificity of SAFA antigens might utilize
genetically homogeneous cultures of carefully
selected strains of fungi.
ACKNOWLEDGMENT
We wish to thank Robert L. Taylor for supplying
the standard complement-fixing antigens used in these
studies. We also thank Louis Barno for his excellent
technical assistance and Andre J. Toussaint for his
advice concerning the SAFA procedure.
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