Eur. J. Epidemiol. 0392-2990
Vol. 8, No. 3
EUROPEAN
JOURNAL
May 1992, p. 356-361
OF
EPIDEMIOLOGY
IDENTIFICATION OF ANTIGENS REACTING WITH ANTI-CANDIDA
GERM TUBE ANTIBODIES
ALBICANS
P. REGULEZ, M.C. ARILLA, J. BIKANDI, G. QUINDOS, R. CISTERNA and J. PONTON 1 Departamento de Microbiologia e Inmunologia - Facultad de Medicina y Odontologia Universidad del Pais Vasco - Apartado 699 - E-48080 Bilbao - Vizcaya - Spain.
Key words: Candida albicans - Germ tube antigens - Anti-germ tube antibodies Anti-Candida albicans germ tube antibodies can be induced in rabbits immunized with different C. albicans extracts. Antigens responsible for the induction of those antibodies have molecular weights of approximately 230-250, 62, 43 and 41 kDa. These antigens are present in the cell wall of both C. albicans morphological forms, although their location seems to be different.
INTRODUCTION
After the observation of the high reactivity of germ tube cell wall antigens when tested by indirect immunofluorescence against highly diluted sera from patients with candidiasis (4), considerable effort has been expended to fred the germ tube-specific antigens. Although monoclonal antibodies are starting to be used to define these antigens (2, 7), most of the work in this area has been done using polyclonal antisera. Germ tube-specific antigens were initially identified by different techniques using anti-Candida albicans germ tube antisera adsorbed with heat-killed yeasts. Sundstrom and Kenny identified two germ tubespecific antigens of molecular weights 200 and 155 kDa (14). Pont6n and Jones (8, 9) described two germ tube-specific antigens of 230-250 and 19 kDa. Two mycelial specific cell wall antigens of 35 and 27 kDa have been also described by Leusch (6). Using monoclonal antibodies, Casanova et al. (2) described two mycelial specific components of 260 and 180 kDa and Ollert and Calderone identified two components of approximate molecular masses of 55 and 60 kDa. (7). However, the complexity of both the antigenic 1 Corresponding author.
extracts and the antisera used have not allowed a complete characterization with respect to their number, structure and function. Identification of such antigens may be of relevance for the serodiagnosis of invasive candidiasis. In fact, an indirect immunofluorescence technique based on the detection of anti-C, albicans germ tube antibodies has been used by Quind6s et al. (10, 11) for the diagnosis of invasive candidiasis. However, Brawner et al. (1) have demonstrated that antigenic expression in C. albicans is a dynamic process which can be influenced by fungal, environmental and nutritional factors. In this study, we have examined the antigens reacting with anti-C, albicans germ tube antibodies, their expression in different media and growth conditions and the induction of anti-C. albicans germ tube antibodies by different extracts of C. albicans.
MATERIALS AND METHODS Organisms and culture conditions - Candida albicans
serotype A [NCPF 3153] was obtained from the National Collection of Pathogenic Fungi (London). It was maintained at 40 C on slants containing 20 g of
356
Vol. 8, 1992
Candida albicans germ tube antigens
glucose, 10 g of yeast extract, and 20 g ofagar per liter. For most experiments, C. albicans blastospores and germ tubes were grown in medium 199 (Sigma Chemical Co., St. Louis, Mo., USA) as previously described (8). In some of the immunofluorescence studies, C. albicans was grown in different media (Glucose-Salts-Biotin (GSB), Glucose-Yeast ExtractPeptone (GYEP), Lee's medium, modified Lee's medium, defined germination medium (GM-2) or 10°/0 fetal calf serum) as described by Brawner et al. (1). Preparation of antigens and antisera - Cell walls of C.
albicans blastospores and germ tubes were extracted in the presence of dithiothreitol (DTT) as reported previously (8). Cell wall extracts from 24-h-old blastospores and 4-h-old germ tubes were labeled DTT-B and DTT-GT, respectively. To obtain culture filtrates, C. albicans blastospores or hyphae were grown in 199 medium for 48 h. The fungal cells were harvested by filtration through a sintered glass filter and the culture medium was then filtered again through 0.45-lam pore filters (Sartorius GmbH, G6ttingen, FRG). The filtrates from blastospores (]3CF) and hyphae (H-CF) were lyophilized to reduce the volume, dialyzed against deionized water for 48 h and lyophilized again. Three types of antisera were prepared: (i). An anti-C, albicans germ tube antiserum was prepared by weekly subcutaneous inoculations of New Zealand White rabbits with 5 x 10~Formalin-killed germ tubes in 0.5 ml of complete Freund adjuvant. For the sake of clarity, it will be referred as the unadsorbed antiserum. (ii) A germ tube-specific antiserum was prepared by adsorption of the unadsorbed antisera with C. albicans blastospores as described previously (8). (iii) For some experiments, an antiserum containing antibodies reacting with the germ tube cell wall surface was obtained by a modification of a previously described method (14). Briefly, the anti-C, albicans germ tube antiserum was first adsorbed with heat-killed blastospores as described above. The adsorbed antiserum was then incubated with an equal volume of a suspension of 1 x 10TM live germ tubes per ml for 2 h at room temperature. Unattached serum proteins were removed by three washes in phosphate-buffered saline (PBS), and attached antibodies were then removed by treatment with 2.5 M NaI in PBS for 1 h at room temperature. Germ tubes were removed by centrifugation, and the supernatant was dialyzed against PBS for 48 h at 4° C. Eluted antibodies were concentrated by using Centricon 10 (Amicon Danvers MA USA) ultrafilter units with a molecular weight cutoff of 10,000. This antiserum will be referred as the GT surface antiserum. For one experiment requiting antiserum depleted of antibodies to germ tubes, the unadsorbed antiserum was either adsorbed 9 times with heat-killed yeasts or 7 times with 40 mg of DTT-B extract. Finally, in some experiments, the rabbits were immunized by biweekly subcutaneous inoculations of the following C. albicans antigens in 0.5 ml of complete Freund adjuvant: (i) 5 x
108 formalin-killed blastospores; (ii) 5 x IlY heat-killed blastospores; (iii) 10 lag of DTT-GT; (iv) 10 lag of DTTB; (v) 10 lag of H-CF; (vi) 10 lag of B-CF. Immunofluorescence - Indirect immunofluorescence
assays (IFA) were carried out as previously described (10). Briefly, 10 lal aliquots of serially diluted sera were applied to the wells of teflon-coated microscope slides to which C. albicans serotype A blastospores and germ tubes had been fixed and incubated for 30 rain at 37° C. After being washed, the slides were incubated with biotin-conjugated, goat anti-rabbit IgG (Sigma) diluted 1:1000 in PBS supplemented with Evans blue (0.001%) and Tween 20 (0.001%). The slides were washed and incubated with fluorescein isothiocyanate-conjugated ExtrAvidin (Sigma) diluted 1:80 for another 30 min under the same conditions. Finally, the slides were washed again, mounted with carbonate-glycerol mounting fluid, and examined with a microscope equipped to detect reflected fluorescence. S D S - P A G E and Western blotting - Sodium dodecyl
sulphate-polyacrilamide gel electrophoresis (SDSPAGE) was performed by the method of Laemmli (5) in a minigel system (Bio-Rad Laboratories, Richmond, Calif., USA). Electrophoresis was carried out in 6 and 130/0 slab gels at 200 V for 45 min. Subsequently, the gels were either stained with Coomassie blue or were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) for 18 h at 60 V according to Towbin et al. (15). After the transfer, the nitrocellulose membranes were blocked in 9% nonfat dry milk in Tris-buffered saline (TBS), washed in TBS and incubated with a 1:32 dilution of the antisera, washed, and incubated with colloidal gold-labeled, affinity-purified goat anti-rabbit IgG (Sigma). Immunoreactive bands were visualized after staining with a gold enhancement kit (Bio-Rad).
RESULTS
Immunoblot analysis using anti-germ tube cell wall antibodies - When tested by IFA, the GT surface
antiserum strongly stained the cell wall surface of 4-hold germ tubes grown in medium 199 (data not shown). Blastospores grown at 37° C were always negative. However, this antiserum stained 2% of blastospores grown at 24° C for 24 h. The cells showing such reactivity were twice the size of non-reactive blastospores. The GT surface antiserum reacted with blots from SDS-PAGE gels in which DTT extracts from both C. albicans blastospores and germ tubes had been run (Figs. 1 and 2). Major bands present in DTT-GT and DTT-B extracts had apparent molecular weights of 230-250, 62, 43 and 41 kDa. The 43 kDa band was not seen when blots were stained with the germ tube-specific antiserum. This antiserum also failed to stain any high molecular weight component in DTT-B extracts.
357
Regulez P. et al.
Eur. J. Epidemiol.
MW (kDa)
MW
(kDa)
97.4
4
66.2
"*
200 45
4 116
31
"*
97.4
-~
66_2
-~
21.5
14.4
"*
I
2
3
i
4
2
3
4
Figure 1. - Immunoblots of 13% SDS-PAGE gels loaded with DTT-GT (lanes 1 and 2) and DTT-B (lanes 3 and 4) extracts. The gel tracks in lanes 1 and 3 were stained with the germ tube surface antiserum. The gel tracks in lanes 2 and 4 were stained with the germ tube-specific antiserum. Molecular weight standards (in thousands) are indicated on the left.
Figure 2. - Irnmunoblots of 6°/o SDS-PAGE gels loaded with DTT-GT (lanes 1 and 2) and DTT-B (lanes 3 and 4) extracts. The gel tracks in lanes 1 and 3 were stained with the germ tube surface antiserum. The gel tracks in lanes 2 and 4 were stained with the germ tube-specific antiserum. Molecular weight standards (in thousands) are indicated on the left.
Sensitivity of C. albicans germ tube antigens to conditions used in SDS-PAGE - To test if conditions
germ tube antibody titer is shown in Table 2. No fluorescence was observed on the germ tube surface after 9 adsorptions. A more efficient blocking of antigerm tube antibodies was observed after incubation of the germ tube specific antiserum with a DTT-B extract.
used in SDS-PAGE were affecting the antigenicity of some o f the components present in the extracts, C. a l b i c a n s germ tubes were incubated for 2 rain at 1000 C with SDS-PAGE treatment buffer and their reactivity was assessed by I F A using both the unadsorbed and the germ tube-specific antisera. The treatment did not affect the reaction with the unadsorbed antiserum, staining the entire surface of both blastospores and germ tubes. However, no fluorescence was observed when the germ tube-specific antiserum was used. Incubation of germ tubes for 2 rain at r o o m temperature with the same buffer did not affect the reactivity of both antisera. The loss of reactivity of germ tubes was also observed after their incubation for 2 rain at 100° C with Tris buffer (Table 1). IFA staining of germ tubes after adsorption of the antiserum - The effect of several consecutive adsorptions with heat-killed blastospores on the anti358
IFA staining of germ tubes grown in different media - To exclude antigenic variability as an explanation for the reactivity observed on the germ tube surface when stained with the germ tube-specific antiserum, a modification of the method of Brawner et al. was followed (1). No fluorescence was observed on blastospores grown at 240 C in GSB or G Y E P for 24 and 48 h. Regardless of growth m e d i u m or length of incubation, germ tubes or hyphae induced at 37 ° C were strongly fluorescent (Fig 3b). Under germinative conditions, around 15% of blastospores grown in GYEP, GM-2 and 10% fetal calf serum for 24 h were weakly fluorescent (Fig. 3c). However, 50% of blastospores grown in Lee's m e d i u m were fluorescent.
Candida albicans germ tube antigens
Vol. 8, 1992
T A B L E 1. - Effect o f different t r e a t m e n t s o n the antigenicity o f C. albicans germ t u b e a n d blastospore cell wall antigens. Treatment
Unadsorbed antiserum
Germ tube specific antiserum
Ba
GTb
B
GT
SDS treatment buffer 100° C, 2 min
+c
+
SDS treatment buffer room temperature, 2 min
+
+
-
Tris buffer 100° C, 2 rain
+
+
-
Tris buffer room temperature, 2 rain
+
+
-
+
Control without treatment
+
+
-
+
d
+
a Fluorescence on the blastospore cell wall. b Fluorescence on the germ tube cell wall. c Positive reaction. Negative reaction.
T A B L E 2. - Effect o f adsorptions o f a n anti-C. germ t u b e a n t i s e r u m with heat-killed blastospores (HK-B) or a dithiothreitol extract from blastospores ( D T T - B ) o n the anti-germ t u b e a n t i b o d y titer.
albicans
N u m b e r o f adsorptions
HK-B
DTT-B
4
512
512
5
128
32
6
64
16
7
64
-
8
16
ND °
9
-
ND
Figure 3. - Phase-contrast (a) and fluorescence photographs (b) of 4-h-old germ tubes stained with the germ tube-specific antiserum. The arrow points to one of the blastospores not visible under fluorescence. Fluorescence of blastospores grown in 10% fetal calf serum at 370 C for 24 h stained with the same antiserum. (Original magnification, X100).
"Not done. 16384 4096
Regardless o f l e n g t h o f i n c u b a t i o n , blastospores g r o w n i n m o d i f i e d Lee's m e d i u m u n d e r germinative c o n d i t i o n s were always negative.
Induction of anti-C, albicans germ tube antibodies in rabbits immunized with C. albicans cell wall antigens The kinetics o f anti-C, albicans cell wall antibodies i n -
a rabbit i m m u n i z e d with formalin-killed germ t u b e s is s h o w n i n figure 4. Anti-C. albicans germ t u b e a n t i b o d i e s were also detected in rabbits i m m u n i z e d with D T T - G T , D T T - B , heat-killed blastospores, formalin-killed blastospores, H - C F a n d B - C F (data n o t shown). 359
1024 256 e~ ¢
Vol. 8, No. 3
EUROPEAN
JOURNAL
May 1992, p. 356-361
OF
EPIDEMIOLOGY
IDENTIFICATION OF ANTIGENS REACTING WITH ANTI-CANDIDA
GERM TUBE ANTIBODIES
ALBICANS
P. REGULEZ, M.C. ARILLA, J. BIKANDI, G. QUINDOS, R. CISTERNA and J. PONTON 1 Departamento de Microbiologia e Inmunologia - Facultad de Medicina y Odontologia Universidad del Pais Vasco - Apartado 699 - E-48080 Bilbao - Vizcaya - Spain.
Key words: Candida albicans - Germ tube antigens - Anti-germ tube antibodies Anti-Candida albicans germ tube antibodies can be induced in rabbits immunized with different C. albicans extracts. Antigens responsible for the induction of those antibodies have molecular weights of approximately 230-250, 62, 43 and 41 kDa. These antigens are present in the cell wall of both C. albicans morphological forms, although their location seems to be different.
INTRODUCTION
After the observation of the high reactivity of germ tube cell wall antigens when tested by indirect immunofluorescence against highly diluted sera from patients with candidiasis (4), considerable effort has been expended to fred the germ tube-specific antigens. Although monoclonal antibodies are starting to be used to define these antigens (2, 7), most of the work in this area has been done using polyclonal antisera. Germ tube-specific antigens were initially identified by different techniques using anti-Candida albicans germ tube antisera adsorbed with heat-killed yeasts. Sundstrom and Kenny identified two germ tubespecific antigens of molecular weights 200 and 155 kDa (14). Pont6n and Jones (8, 9) described two germ tube-specific antigens of 230-250 and 19 kDa. Two mycelial specific cell wall antigens of 35 and 27 kDa have been also described by Leusch (6). Using monoclonal antibodies, Casanova et al. (2) described two mycelial specific components of 260 and 180 kDa and Ollert and Calderone identified two components of approximate molecular masses of 55 and 60 kDa. (7). However, the complexity of both the antigenic 1 Corresponding author.
extracts and the antisera used have not allowed a complete characterization with respect to their number, structure and function. Identification of such antigens may be of relevance for the serodiagnosis of invasive candidiasis. In fact, an indirect immunofluorescence technique based on the detection of anti-C, albicans germ tube antibodies has been used by Quind6s et al. (10, 11) for the diagnosis of invasive candidiasis. However, Brawner et al. (1) have demonstrated that antigenic expression in C. albicans is a dynamic process which can be influenced by fungal, environmental and nutritional factors. In this study, we have examined the antigens reacting with anti-C, albicans germ tube antibodies, their expression in different media and growth conditions and the induction of anti-C. albicans germ tube antibodies by different extracts of C. albicans.
MATERIALS AND METHODS Organisms and culture conditions - Candida albicans
serotype A [NCPF 3153] was obtained from the National Collection of Pathogenic Fungi (London). It was maintained at 40 C on slants containing 20 g of
356
Vol. 8, 1992
Candida albicans germ tube antigens
glucose, 10 g of yeast extract, and 20 g ofagar per liter. For most experiments, C. albicans blastospores and germ tubes were grown in medium 199 (Sigma Chemical Co., St. Louis, Mo., USA) as previously described (8). In some of the immunofluorescence studies, C. albicans was grown in different media (Glucose-Salts-Biotin (GSB), Glucose-Yeast ExtractPeptone (GYEP), Lee's medium, modified Lee's medium, defined germination medium (GM-2) or 10°/0 fetal calf serum) as described by Brawner et al. (1). Preparation of antigens and antisera - Cell walls of C.
albicans blastospores and germ tubes were extracted in the presence of dithiothreitol (DTT) as reported previously (8). Cell wall extracts from 24-h-old blastospores and 4-h-old germ tubes were labeled DTT-B and DTT-GT, respectively. To obtain culture filtrates, C. albicans blastospores or hyphae were grown in 199 medium for 48 h. The fungal cells were harvested by filtration through a sintered glass filter and the culture medium was then filtered again through 0.45-lam pore filters (Sartorius GmbH, G6ttingen, FRG). The filtrates from blastospores (]3CF) and hyphae (H-CF) were lyophilized to reduce the volume, dialyzed against deionized water for 48 h and lyophilized again. Three types of antisera were prepared: (i). An anti-C, albicans germ tube antiserum was prepared by weekly subcutaneous inoculations of New Zealand White rabbits with 5 x 10~Formalin-killed germ tubes in 0.5 ml of complete Freund adjuvant. For the sake of clarity, it will be referred as the unadsorbed antiserum. (ii) A germ tube-specific antiserum was prepared by adsorption of the unadsorbed antisera with C. albicans blastospores as described previously (8). (iii) For some experiments, an antiserum containing antibodies reacting with the germ tube cell wall surface was obtained by a modification of a previously described method (14). Briefly, the anti-C, albicans germ tube antiserum was first adsorbed with heat-killed blastospores as described above. The adsorbed antiserum was then incubated with an equal volume of a suspension of 1 x 10TM live germ tubes per ml for 2 h at room temperature. Unattached serum proteins were removed by three washes in phosphate-buffered saline (PBS), and attached antibodies were then removed by treatment with 2.5 M NaI in PBS for 1 h at room temperature. Germ tubes were removed by centrifugation, and the supernatant was dialyzed against PBS for 48 h at 4° C. Eluted antibodies were concentrated by using Centricon 10 (Amicon Danvers MA USA) ultrafilter units with a molecular weight cutoff of 10,000. This antiserum will be referred as the GT surface antiserum. For one experiment requiting antiserum depleted of antibodies to germ tubes, the unadsorbed antiserum was either adsorbed 9 times with heat-killed yeasts or 7 times with 40 mg of DTT-B extract. Finally, in some experiments, the rabbits were immunized by biweekly subcutaneous inoculations of the following C. albicans antigens in 0.5 ml of complete Freund adjuvant: (i) 5 x
108 formalin-killed blastospores; (ii) 5 x IlY heat-killed blastospores; (iii) 10 lag of DTT-GT; (iv) 10 lag of DTTB; (v) 10 lag of H-CF; (vi) 10 lag of B-CF. Immunofluorescence - Indirect immunofluorescence
assays (IFA) were carried out as previously described (10). Briefly, 10 lal aliquots of serially diluted sera were applied to the wells of teflon-coated microscope slides to which C. albicans serotype A blastospores and germ tubes had been fixed and incubated for 30 rain at 37° C. After being washed, the slides were incubated with biotin-conjugated, goat anti-rabbit IgG (Sigma) diluted 1:1000 in PBS supplemented with Evans blue (0.001%) and Tween 20 (0.001%). The slides were washed and incubated with fluorescein isothiocyanate-conjugated ExtrAvidin (Sigma) diluted 1:80 for another 30 min under the same conditions. Finally, the slides were washed again, mounted with carbonate-glycerol mounting fluid, and examined with a microscope equipped to detect reflected fluorescence. S D S - P A G E and Western blotting - Sodium dodecyl
sulphate-polyacrilamide gel electrophoresis (SDSPAGE) was performed by the method of Laemmli (5) in a minigel system (Bio-Rad Laboratories, Richmond, Calif., USA). Electrophoresis was carried out in 6 and 130/0 slab gels at 200 V for 45 min. Subsequently, the gels were either stained with Coomassie blue or were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) for 18 h at 60 V according to Towbin et al. (15). After the transfer, the nitrocellulose membranes were blocked in 9% nonfat dry milk in Tris-buffered saline (TBS), washed in TBS and incubated with a 1:32 dilution of the antisera, washed, and incubated with colloidal gold-labeled, affinity-purified goat anti-rabbit IgG (Sigma). Immunoreactive bands were visualized after staining with a gold enhancement kit (Bio-Rad).
RESULTS
Immunoblot analysis using anti-germ tube cell wall antibodies - When tested by IFA, the GT surface
antiserum strongly stained the cell wall surface of 4-hold germ tubes grown in medium 199 (data not shown). Blastospores grown at 37° C were always negative. However, this antiserum stained 2% of blastospores grown at 24° C for 24 h. The cells showing such reactivity were twice the size of non-reactive blastospores. The GT surface antiserum reacted with blots from SDS-PAGE gels in which DTT extracts from both C. albicans blastospores and germ tubes had been run (Figs. 1 and 2). Major bands present in DTT-GT and DTT-B extracts had apparent molecular weights of 230-250, 62, 43 and 41 kDa. The 43 kDa band was not seen when blots were stained with the germ tube-specific antiserum. This antiserum also failed to stain any high molecular weight component in DTT-B extracts.
357
Regulez P. et al.
Eur. J. Epidemiol.
MW (kDa)
MW
(kDa)
97.4
4
66.2
"*
200 45
4 116
31
"*
97.4
-~
66_2
-~
21.5
14.4
"*
I
2
3
i
4
2
3
4
Figure 1. - Immunoblots of 13% SDS-PAGE gels loaded with DTT-GT (lanes 1 and 2) and DTT-B (lanes 3 and 4) extracts. The gel tracks in lanes 1 and 3 were stained with the germ tube surface antiserum. The gel tracks in lanes 2 and 4 were stained with the germ tube-specific antiserum. Molecular weight standards (in thousands) are indicated on the left.
Figure 2. - Irnmunoblots of 6°/o SDS-PAGE gels loaded with DTT-GT (lanes 1 and 2) and DTT-B (lanes 3 and 4) extracts. The gel tracks in lanes 1 and 3 were stained with the germ tube surface antiserum. The gel tracks in lanes 2 and 4 were stained with the germ tube-specific antiserum. Molecular weight standards (in thousands) are indicated on the left.
Sensitivity of C. albicans germ tube antigens to conditions used in SDS-PAGE - To test if conditions
germ tube antibody titer is shown in Table 2. No fluorescence was observed on the germ tube surface after 9 adsorptions. A more efficient blocking of antigerm tube antibodies was observed after incubation of the germ tube specific antiserum with a DTT-B extract.
used in SDS-PAGE were affecting the antigenicity of some o f the components present in the extracts, C. a l b i c a n s germ tubes were incubated for 2 rain at 1000 C with SDS-PAGE treatment buffer and their reactivity was assessed by I F A using both the unadsorbed and the germ tube-specific antisera. The treatment did not affect the reaction with the unadsorbed antiserum, staining the entire surface of both blastospores and germ tubes. However, no fluorescence was observed when the germ tube-specific antiserum was used. Incubation of germ tubes for 2 rain at r o o m temperature with the same buffer did not affect the reactivity of both antisera. The loss of reactivity of germ tubes was also observed after their incubation for 2 rain at 100° C with Tris buffer (Table 1). IFA staining of germ tubes after adsorption of the antiserum - The effect of several consecutive adsorptions with heat-killed blastospores on the anti358
IFA staining of germ tubes grown in different media - To exclude antigenic variability as an explanation for the reactivity observed on the germ tube surface when stained with the germ tube-specific antiserum, a modification of the method of Brawner et al. was followed (1). No fluorescence was observed on blastospores grown at 240 C in GSB or G Y E P for 24 and 48 h. Regardless of growth m e d i u m or length of incubation, germ tubes or hyphae induced at 37 ° C were strongly fluorescent (Fig 3b). Under germinative conditions, around 15% of blastospores grown in GYEP, GM-2 and 10% fetal calf serum for 24 h were weakly fluorescent (Fig. 3c). However, 50% of blastospores grown in Lee's m e d i u m were fluorescent.
Candida albicans germ tube antigens
Vol. 8, 1992
T A B L E 1. - Effect o f different t r e a t m e n t s o n the antigenicity o f C. albicans germ t u b e a n d blastospore cell wall antigens. Treatment
Unadsorbed antiserum
Germ tube specific antiserum
Ba
GTb
B
GT
SDS treatment buffer 100° C, 2 min
+c
+
SDS treatment buffer room temperature, 2 min
+
+
-
Tris buffer 100° C, 2 rain
+
+
-
Tris buffer room temperature, 2 rain
+
+
-
+
Control without treatment
+
+
-
+
d
+
a Fluorescence on the blastospore cell wall. b Fluorescence on the germ tube cell wall. c Positive reaction. Negative reaction.
T A B L E 2. - Effect o f adsorptions o f a n anti-C. germ t u b e a n t i s e r u m with heat-killed blastospores (HK-B) or a dithiothreitol extract from blastospores ( D T T - B ) o n the anti-germ t u b e a n t i b o d y titer.
albicans
N u m b e r o f adsorptions
HK-B
DTT-B
4
512
512
5
128
32
6
64
16
7
64
-
8
16
ND °
9
-
ND
Figure 3. - Phase-contrast (a) and fluorescence photographs (b) of 4-h-old germ tubes stained with the germ tube-specific antiserum. The arrow points to one of the blastospores not visible under fluorescence. Fluorescence of blastospores grown in 10% fetal calf serum at 370 C for 24 h stained with the same antiserum. (Original magnification, X100).
"Not done. 16384 4096
Regardless o f l e n g t h o f i n c u b a t i o n , blastospores g r o w n i n m o d i f i e d Lee's m e d i u m u n d e r germinative c o n d i t i o n s were always negative.
Induction of anti-C, albicans germ tube antibodies in rabbits immunized with C. albicans cell wall antigens The kinetics o f anti-C, albicans cell wall antibodies i n -
a rabbit i m m u n i z e d with formalin-killed germ t u b e s is s h o w n i n figure 4. Anti-C. albicans germ t u b e a n t i b o d i e s were also detected in rabbits i m m u n i z e d with D T T - G T , D T T - B , heat-killed blastospores, formalin-killed blastospores, H - C F a n d B - C F (data n o t shown). 359
1024 256 e~ ¢
