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The detection of Aspergillus fumigatus antibodies in sera by immunogold-silver staining
Journal of Microbiological Methods 15 (1992) 113 - 120
ii3
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167- 7012/92/$ 5.00 MIMET 00489
The detection of Aspergillusfumigatus antibodies in sera by immunogold-silver staining Kari E. Reijula, Viswanath P. Kurup and Jordan N. Fink Research Service, VA Medical Center, and the Allergy-lmmunology Division, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA (Received 16 September 1991; revision received 19 December 1991; accepted 20 December 1991)
Summary The demonstration of circulating antibodies against Aspergiilus fumigatus is a useful criterion in the diagnosis of a number of the clinical forms of Aspergillus-induced diseases. Several antibody assays are available for the detection of specific antibodies. Some of these methods are sensitive and specific, but need sophisticated equipment, expensive reagents and cumbersome procedures while others are less sensitive and time consuming. The usefulness of the immunogold-silver assay (IGSA) was evaluated in the present study. The results obtained indicate that IGSA was comparable to other antibody detection assays, but is comparatively simple and less expensive.
Key words: Aspergiilusfumigatus; lmmunogold-silver assay (IGSA); Biotin-avidin linked immunosorbent assay (BALISA); Aspergillosis; Aspergilloma
Introduction
Specific human antibodies against Aspergillus fumigatus have been detected by double immunodiffusion in agar gels (DD), complement fixation, hemagglutination, immunofluorescence (IF), radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) [1 - 6]. Soluble antigens used in these assays have been prepared either from cell or from culture filtrates of A. fumigatus. The standardization and purification of these crude antigen preparations is time consuming, expensive and often difficult [7 - 10]. In order to develop a simple and reliable method to demonstrate IgG and IgE antibodies to A. fumigatus in patient serum, an immunogold assay (IGA) using whole organism as a source of antigen has been recently developed [11]. By this method both specific IgG and IgE antibodies to A. fumigatus have been detected in
Correspondence to: K.E. Reijula. Present address: Oulu Regional Institute of Occupational Health, P.O. Box 451, SF-90101 Oulu, Finland
114 the sera of patients with Aspergillus-induced diseases such as allergic bronchopulmonary aspergillosis (ABPA). Immunogold staining offers several advantages over peroxidase-antiperoxidase or IF-procedures. The technique can be used on slide culture specimens, stained preparations can be stored for future reference, the reaction can be visualized by an ordinary light microscope and no sophisticated equipment is needed. Additionally, the same reagents can be used for both electron and light microscopic studies. However, IGA has been less sensitive in detection of low concentrations of antibody levels or detecting IgE in human sera [11]. In such situations an amplification of the reaction is desirable in order to enhance the sensitivity without sacrificing the specificity. Such amplification of reactivity was utilized by Holgate et al. [12] in tissue sections by means of silver enhancement of colloidal gold reaction. The advantages of immunogold-silver assay (IGSA) over IGA are increased sensitivity, a need for less serum and reagents for the assay, and improved visualization with light microscopy. The present study was therefore designed to evaluate IGSA in detecting anti-A. fumigatus antibodies in human sera. The results indicate that this method is more sensitive than IGA and is comparable to ELISA in offering a reliable and simple technique. Materials and Methods
Human sera Eight serum samples were selected for the study. These included two samples each from ABPA, aspergilloma, invasive aspergillosis and normal subjects. The diagnosis of A. fumigatus-induced diseases was based on standard clinical and immunological criteria [13]. Sera from normal subjects were from laboratory personnel with no apparent evidence of any respiratory diseases. All samples were stored in aliquots at --70
°C and
tha~,~d
f n r ne,~ o c n,~,~AoA
Antigen for the IGSA The young hyphae of A. fumigatus fixed on glass slides were used as antigens in IGSA. Slide cultures of A. fumigatus were prepared by a modified method of Riddel et al. [14] as previously described [5]. Sabouraud's agar blocks (40x 10x 1.5 mm) were aseptically transferred to the center of sterilized slides pre-coated with 0.1 °70 agarose. Spores from 3-day-old cultures of A. fumigatus on Sabouraud's agar were harvested in 5 ml sterile physiological saline. All four sides of the agar block were then inoculated using a freshly prepared spore suspension t.l. .a,~l~ . . . . .~.~,~'~by a ~,,.,~*°-;1",.,.,.,,,,,,,,""**"'~ applicator. Another pre-coated sterile slide was placed on the top of the inoculated .,gar block. The ~:ides were then incubated in Petri dishes with filter paper moistened with sterile distilled water to provide humidity for growth. After 36 h of incubation at 22 °C, when sufficient mycelial growth was detected, the agar blocks separating the slides were removed and the slides were air-dried and fixed in methanol for 30 min. The slides were then stored at 4 °C and could be used for at least 4 weeks without any marked loss of antigenicity.
115
Immunogold-silver staining After fixation, areas showing distinct hyphae of A. fumigatus were marked on the reverse side of the slides using a diamond pencil• Only well differentiated areas with young hyphae were selected on each slide for the IGSA studies. The slides were rinsed for 30 min in phosphate buffered saline (pH 7.4) containing 5% nonfat dry milk (Carnation dry milk), 0.05% (w/v) Tween-20 and 0.1% sodium azide (PBS-Milk-Tween). After washing 3 times for 2 min each with Tris buffer (pH 8.2) containing 0.1% (w/v) BSA and 0.1% sodium azide (TBSA) the slides were drained and transferred into a humid chamber. Rubber rings (6.5 mm internal diameter) were used to retain the reagents in marked areas with consistent ~ontact with hyphae. Sixty #1 of serum dilutions were added into the areas enclosed by the rings. All the dilutions of the sera and reagents were prepared in PBS-Milk-Tween. The slides were first incubated with normal rabbit serum (1" 100) for 30 min at 22 °C. Dilutions of test human sera ranging from 1"10 to 1:10000 were used to evaluate A. fumigatus antibodies. After incubating for 1 h in a humid chamber at 22 °C, the rings were removed, the slides were washed 3 times for 2 min in TBSA and again dried. The air-dried slides were placed into a humid chamber and the rings were repositioned. Sixty #1 of goat antihuman IgG (1-1000, Sigma, St. Louis, MO)or lgE (1" 100, Sigma) were added onto the slides covered by rubber rings. After 1 h incubation at 22 °C, the rubber rings were removed and the slides were washed as above. The slides were then treated with rabbit antigoat lgG (Sigma) conjugated with colloidal gold, at a dilution of 1:200 for 1 h at 22 °C and washed again in TBSA. The slides were first washed in PBS then twice in double distilled water for 3 min each. After draining, the slides were rinsed in 0.2 M citrate buffer prepared freshly by diluting 2 M stock citrate buffer with distilled water. The stock citrate buffer was prepared just before use by dissolving 23.5 g trisodium citrate and 25.5 g citric acid in 100 ml of distilled water. The slides were then treated with freshly prepared silver In nrha,. *,,~ ~ . l , J reaction, .c. n. .h. .l t l n n v i a t . ., -l l~l h l ¢ l.l.l.q. w l i ; ; .1.,.-. Lllqi~ I: I.I I.I l .I U.I I.U .~ U. I U The enhancer was prepared freshly by mixing equal volumes of hydroquinone solution (0.47 g hydroquinone in 7.5 ml of distilled water) and silver solution (0.055 g silver lactate dissolved in 7.5 ml of distilled water) and diluted 1:6 times with distilled water. The enhancer was protected from light by covering with aluminium foil. The slides were allowed to react with the enhancer solution for 4 - 6 min in jars wrapped with aluminium foil. After the silver enhancement the slides were fixed in 1"10 dilution of a fixing solution (Janssen Life Sciences Products, Beerse, Belgium) for 3 min, washed two times for 3 min each in distilled water, dehydrated by passing through changes of 75%, absolute alcohol and xylol, and mounted in Permount (Scientific Products). Appropriate dilutions of serum and reagents were chosen after repeated experiments with positive and negative sera. Colloidal gold was prepared and conjugated to rabbit antigoat IgG (Sigma) according to the procedure described by Geoghegan and Ackerman [15]. The preparations were examined in a Leitz Dialux 20 EB microscope. Following controls were included with each experiment: IGSA was performed by using (1) the slides with antigens only, (2) antigens incubated with IGSA-positive and IGSA-negative sera, (3) antigens treated with goat antihuman IgG with and without human serum, (4) antigens treated with rabbit antigoat IgG conjugated with colloidal gold with and without human serum. ViU~,,,J.
LV
116 A positive reaction was evidenced by binding of gold-silver particles to A. fumigatus hyphae, as demonstrated by black granules on the surface of the hyphae. The results were recorded as negative when the hyphae appeared without any color or granules on the surface of the hyphae. The reaction was graded from ' - ' to ' + + + ' for negative to very strongly positive. The highest dilution which consistently gave at least a ' + ' reaction was recorded as the titer of antibody in serum samples. Grading of the reaction was recorded independently by two observers. A. fumigatus specific IgG and IgE antibodies were also detected by biotin-avidin linked immunosorbent assay (BALISA) [ 16] and DD [l 7, 18].
Specificity of IGSA The specificity of IGSA technique was evaluated by using appropriate negative and positive controls in each determination as well as in absorption tests with relevant antigens. Solutions of Penicillium sp. (10 mg/ml), Candida albicans (100 mg/ml), Faenia rectivirgula (Micropolyspora faeni, 5 mg/ml) and A. fumigatus (10 mg/mi) were incubated (1 h, at 22 °C) with patient sera which consistently showed a ' + + + ' reaction for both IgG and IgE specific antibodies to A. fumigatus in BALISA and IGA. The antigen concentration in serum was adjusted to 1 mg/ml. The incubated serum was used after centrifugation to detect A. fumigatus specific antibodies in sera of the patients by IGSA, and the reaction was recorded. Results and Discussion
The results of the measurement of A. fumigatus antibodies are presented in Table 1, and demonstrate that IGSA can be used to detect A. fumigatus specific IgG and IgE antibodies in human sera. All six sera had precipitins against A. fumigatus by DD whereas the two controls were negative. Specific IgG and IgE antibodies against A. TABLE 1 RESULTS O F I M M U N O G O L D - S I L V E R ASSAY (IGSA) C O M P A R E D W I T H D D A N D BALISA FOR D E T E C T I N G IgG A N D IgE A N T I B O D I E S TO A. F U M I G A T U S Patient no.
1 2 3 4 5 6 7 8
Diagnosis
ABPA ABPA aspergilloma aspergilloma aspergillosis aspergillosis normal normal
IGSA
DD
IgG
IgE
1:10000 b 1:10000 1:1000 1:5000 1:1000 1:1000 -
1:500 1:1000 l:100 l:100 l:100 1:50 -
Optical density read at 490 nm. b Highest serum dilution giving a definite positive reaction. c Nanogram per ml.
a
+ + + + + + -
BALISA
Total IgE c
IgG a
IgE b
1.126 1.304 0.222 0.361 0.372 0.718 0.164 0.063
0.380 0.370 0.033 0.100 0.306 0.394 0.036 0.020
13084 11569 279 96
12 65
117
.eT' o
Fig. 1.
A slide culture of Aspergillusfumigatus showing positive reaction by immunogold-silver assay (IGSA) using ABPA patient serum ( x 250).
fumigatus were detected by BALISA in all patients and in the controls. The titers of IgG and IgE antibodies, however, were higher in the patients' sera. Sera from all patients showed positive reactions to A. fumigatus by IGSA using both IgG and IgE while controls were negative. The IGSA detected A. fumigatus specific IgG (Fig. 1) in all patients' sera; the highest dilution which consistently gave at least a' +' reaction varied from 1:1000 to
t antigoat IgG + gold IgG/Ig
Fig. 2.
Schematic illustration of immunogold-silver staining.
118
Methanol
fixed slide c u l t u r e s
PBS-Milk-Tween
30 min
TBSA w a s h Normal r a b b i t
2 rain x 3
serum 30 min TBSA
Dilutions
of serum 60 min TBSA
Goat a n t i - h u m a n
IgG
(l:1000)/IgE
(i:i00)
60 min
TBSA Rabbit
antigoat
IgG conj.
with c o l l o i d a l
gold
PBS 3 min x 2; H20 3 min x 2 Silver enhancement
Fixing
(see the text)
solution
H20 3 m i n x
Dehydration
4 - 6 min
3 min
2
and m o u n t i n g
Fig. 3. Differentstages of immunogold-silverstaining.
1:10000. All control sera were negative. Specific IgE antibodies to A. fumigatus were detected by IGSA in all patients but not in the controls. In one patient with ABPA a positive reaction was detected at 1:I000 dilution, in two at 1:500 dilution and in all but one at 1:100 dilution of serum. A positive correlation could be demonstrated in the degree of reactivity by IGSA when compared with the results of BALISA. In the absorption tests using soluble antigens of Penicillium sp., Candida albicans and Faenia rectivirgula (Micropolysporafaem) the reactivity and titer of antibody of the patients' serum were not affected when A. fumigatus slide culture antigen was
119 used. On the other hand, a dose-dependent inhibition of antibody reactivity and titer was detected when sera were absorbed using soluble A. fumigatus antigen prior to testing by IGSA. Thus, the antibody against A. fumigatus detected by IGSA is specific and the positive reaction was not a result of a cross-reaction with other fungal antigens. The indirect IF-method has previously been used to detect A. fumigatus antibodies in patients' sera [5]. IGSA, however, offers several advantages over the IF-method. IGSA is more sensitive as the maximum serum dilution titer detected by !GSA was 1:10000 while IF-titer was 1:640. Thus, IGSA-method also uses smaller amounts of test reagents and allows the storage of sections indefinitely. Furthermore, it offers the possibility of a simultaneous evaluation of immunoisotypes and the demonstration of antigen localization on fungal cells by immunoelectronmicroscopy [19]. The IGSA method was found to be superior to DD in detecting A. fumigatus antibodies. IGSA can be carried out more rapidly than DD and it can detect both IgG and IgE antibodies specific to A. fumigatus in titers comparable to ELISA. The present results indicate that IGSA is a reliable and specific method and no crossreactivity could be detected between common fungal antigens. IGSA is simpler than enzyme- or radioimmunoassays (EIA and RIA) and can be performed in a routine clinical laboratory without special equipment. Because of its high sensitivity and the use of whole cells as slide culture antigens this method has an advantage over the EIA and RIA where purified antigens are needed for reliable results. In concordance to the findings of EIA and RIA, in the present study IGSA discriminated patients from controls with regard to specific antibodies against A. fumigatus. In order to shorten the time needed for preparing the Aspergillus slides in the present method we will be testing the usage of lyophilized Aspergillus hyphae in our further studies. In conclusion, IGSA is a simple, and reliable method useful in detecting specific antibodies to A. fumigatus in patient sera. The technique can be performed in a routine clinical laboratory without special equipment. It is faster and more sensitive than DD or IGA, and is at least as sensitive as BALISA.
Acknowledgements The authors are grateful to Dr. Leo Kaufman, CDC, Atlanta, GA, for the generous supply of sera from patients with invasive aspergiUosis. We wish to acknowledge the technical assistance of Laura Castillo, Abe Resnick and Nancy Elms. This investigation is partly supported by a grant from the National Institute of Health (AI-23071), by The Veterans Affairs, and by The Finnish Work Environment Fund, The Foundation for the Study of Allergy, The Finnish Anti-Tuberculosis Association, The Paulo Foundation and Farmer's Social Insurance Institution, Finland.
References 1 We~zel, F. J., Emanuel, D.A. and Gray, R. L. (1972) A simplified hemagglutination test for farmer's lung. Am. J. Clin. Pathol. 57, 206-208. 2 Flaherty, D. K., Barboriak, J., Emanuel, D., Fink, J., Marx, J., Moore, V., Reed, C.E. and Roberts, R. (1974) Multilaboratory comparison of three immunodiffusion methods used for the detection of
120 precipitating antibodies in hypersensitivity pneumonitis. J. Lab. Clin. Med. 84, 298 - 306. 3 Marx, J.J., Motszko, C. and Wenzel, F. J. (1975) Complement fixation test as an aid in the diagnosis of farmer's lung. J. Clin. Microbiol. 1,480- 482. 4 Parrat, D., Nielsen, K. H., Boyd, G. and White, R. G. (1975) The quantitation of antibody in farmer's lung syndrome using a radioimmunoassay. Clin. Exp. Immunol. 20, 217- 221. 5 Kurup, V.P., Barboriak, J.J. and Fink, J. (1977) Indirect immunofluorescent detection of antibodies against thermophilic actinomycetes in patients with hypersensitivity pneumonitis. J. Lab. Clin. Med. 89, 533 - 539. 6 M/intyj/irvi, R.A., Jousilahti, P. and Katila, M.L. (1980) Antibodies to Aspergiilusfumigatus in farmer's lung patients measured by enzyme-linked immunosorbent assay (ELISA). Clin. Allergy 10, 187- 194 7 Kurup, V. E, Resnick, A., Scribner, G.H., Kalbfleisch, J.H. and Fink, J.N. (1984) Comparison of antigens and serological methods in Aspergillusfumigatus antibody detection. Mykosen 27, 4 3 - 50. 8 Kauffman, H.F., Beaumont, F., Meurs, H., De Heide, S.V. and De-Vries, K. (1983) Comparison of antibody measurements against Aspergillusfumigatus by means of double diffusion and enzymelinked immunosorbent assay (ELISA). J. Allergy Clin. Immunol. 72, 255- 261. 9 Longbottom, J. L. (1986) Antigens and allergens of AspergiilusfumigatusII. Their further identification and partial characterization of major allergens (Ag3). J. Allergy Clin. Immunol. 78, 18- 24. 10 Kurup, V. P., Ramaswamy, M., Greenberger, P.A. and Fink, J.N. (1988) Isolation and characterization of a relevant Aspergillusfumigatus antigen with igG- and IgE-binding capacity. Int. Archs. Allergy Appl. Immunol. 86, 176- 182. 11 Gugnani, H.C., Reijuia, K.E., Kurup, V. P. and Fink, J. N. 0989) Detection of IgG and IgE antibodies to Aspergillusfumigatus in human sera by immunogold assay. Mycopathology (in press). 12 Holgate, C.S., Jacson, P., Cowen, P.N. and Bird, C.C. (!983) Immunogold silver staining: new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 31,938- 944. 13 Rosenberg, M., Patterson, R., Murtzer, R., Cooper, B.J., Roberts, M. and Harris, K.E. (1977) Clinical and immunological criteria for diagnosis of allergic bronchopulmonary aspergillosis. Ann. Intern. Med. 80, 405- 414. 14 Riddel, R.W. (1950) Permanent stained alycological preparations obtained by slide culture. Mycologia 42, 265 - 270. 15 Geoghegan, W.D. and Ackerman, G.A. (1977) Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin of colloidal gold for the indirect detection of concanavalin A, wheat germ agglutination and goat antihuman immunoglobulin G on cell surfaces at the electronmicroscopic level: a new method, theory and application. J. Histochem. o, i61 - i65. 16 Kurup, V.P. (1986) Enzyme-linked immunosorbent assay in the detection of specific antibodies against Aspergiilusfumigatus in patients' sera. Zbl. Bakteriol. Hyg. (A) 261, 509- 516. 17 Kurup, V. P., John, K. V., Resnick, A. and Fink, J. N. (1986)'A partially purified glycoprotein antigen from AspergiUusfumigatus. Int. Arch. Allergy Appl. Immunol. 79, 263 - 269. 18 Wadsworth, C. (1962) A microtemplate technique employing a gel chamber compared with other micro- and macrotemplate techniques for immunodiffusion. Int. Arch. Allergy Appl. Immunol. 31, 131 - 137. 19 Romasco, F., Rosenberg, J. and Wybran, J. (1985) An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subset with monoclonal antibodies. Am. J. Clin. Pathol. 84, 307-316.
ii3
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167- 7012/92/$ 5.00 MIMET 00489
The detection of Aspergillusfumigatus antibodies in sera by immunogold-silver staining Kari E. Reijula, Viswanath P. Kurup and Jordan N. Fink Research Service, VA Medical Center, and the Allergy-lmmunology Division, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA (Received 16 September 1991; revision received 19 December 1991; accepted 20 December 1991)
Summary The demonstration of circulating antibodies against Aspergiilus fumigatus is a useful criterion in the diagnosis of a number of the clinical forms of Aspergillus-induced diseases. Several antibody assays are available for the detection of specific antibodies. Some of these methods are sensitive and specific, but need sophisticated equipment, expensive reagents and cumbersome procedures while others are less sensitive and time consuming. The usefulness of the immunogold-silver assay (IGSA) was evaluated in the present study. The results obtained indicate that IGSA was comparable to other antibody detection assays, but is comparatively simple and less expensive.
Key words: Aspergiilusfumigatus; lmmunogold-silver assay (IGSA); Biotin-avidin linked immunosorbent assay (BALISA); Aspergillosis; Aspergilloma
Introduction
Specific human antibodies against Aspergillus fumigatus have been detected by double immunodiffusion in agar gels (DD), complement fixation, hemagglutination, immunofluorescence (IF), radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) [1 - 6]. Soluble antigens used in these assays have been prepared either from cell or from culture filtrates of A. fumigatus. The standardization and purification of these crude antigen preparations is time consuming, expensive and often difficult [7 - 10]. In order to develop a simple and reliable method to demonstrate IgG and IgE antibodies to A. fumigatus in patient serum, an immunogold assay (IGA) using whole organism as a source of antigen has been recently developed [11]. By this method both specific IgG and IgE antibodies to A. fumigatus have been detected in
Correspondence to: K.E. Reijula. Present address: Oulu Regional Institute of Occupational Health, P.O. Box 451, SF-90101 Oulu, Finland
114 the sera of patients with Aspergillus-induced diseases such as allergic bronchopulmonary aspergillosis (ABPA). Immunogold staining offers several advantages over peroxidase-antiperoxidase or IF-procedures. The technique can be used on slide culture specimens, stained preparations can be stored for future reference, the reaction can be visualized by an ordinary light microscope and no sophisticated equipment is needed. Additionally, the same reagents can be used for both electron and light microscopic studies. However, IGA has been less sensitive in detection of low concentrations of antibody levels or detecting IgE in human sera [11]. In such situations an amplification of the reaction is desirable in order to enhance the sensitivity without sacrificing the specificity. Such amplification of reactivity was utilized by Holgate et al. [12] in tissue sections by means of silver enhancement of colloidal gold reaction. The advantages of immunogold-silver assay (IGSA) over IGA are increased sensitivity, a need for less serum and reagents for the assay, and improved visualization with light microscopy. The present study was therefore designed to evaluate IGSA in detecting anti-A. fumigatus antibodies in human sera. The results indicate that this method is more sensitive than IGA and is comparable to ELISA in offering a reliable and simple technique. Materials and Methods
Human sera Eight serum samples were selected for the study. These included two samples each from ABPA, aspergilloma, invasive aspergillosis and normal subjects. The diagnosis of A. fumigatus-induced diseases was based on standard clinical and immunological criteria [13]. Sera from normal subjects were from laboratory personnel with no apparent evidence of any respiratory diseases. All samples were stored in aliquots at --70
°C and
tha~,~d
f n r ne,~ o c n,~,~AoA
Antigen for the IGSA The young hyphae of A. fumigatus fixed on glass slides were used as antigens in IGSA. Slide cultures of A. fumigatus were prepared by a modified method of Riddel et al. [14] as previously described [5]. Sabouraud's agar blocks (40x 10x 1.5 mm) were aseptically transferred to the center of sterilized slides pre-coated with 0.1 °70 agarose. Spores from 3-day-old cultures of A. fumigatus on Sabouraud's agar were harvested in 5 ml sterile physiological saline. All four sides of the agar block were then inoculated using a freshly prepared spore suspension t.l. .a,~l~ . . . . .~.~,~'~by a ~,,.,~*°-;1",.,.,.,,,,,,,,""**"'~ applicator. Another pre-coated sterile slide was placed on the top of the inoculated .,gar block. The ~:ides were then incubated in Petri dishes with filter paper moistened with sterile distilled water to provide humidity for growth. After 36 h of incubation at 22 °C, when sufficient mycelial growth was detected, the agar blocks separating the slides were removed and the slides were air-dried and fixed in methanol for 30 min. The slides were then stored at 4 °C and could be used for at least 4 weeks without any marked loss of antigenicity.
115
Immunogold-silver staining After fixation, areas showing distinct hyphae of A. fumigatus were marked on the reverse side of the slides using a diamond pencil• Only well differentiated areas with young hyphae were selected on each slide for the IGSA studies. The slides were rinsed for 30 min in phosphate buffered saline (pH 7.4) containing 5% nonfat dry milk (Carnation dry milk), 0.05% (w/v) Tween-20 and 0.1% sodium azide (PBS-Milk-Tween). After washing 3 times for 2 min each with Tris buffer (pH 8.2) containing 0.1% (w/v) BSA and 0.1% sodium azide (TBSA) the slides were drained and transferred into a humid chamber. Rubber rings (6.5 mm internal diameter) were used to retain the reagents in marked areas with consistent ~ontact with hyphae. Sixty #1 of serum dilutions were added into the areas enclosed by the rings. All the dilutions of the sera and reagents were prepared in PBS-Milk-Tween. The slides were first incubated with normal rabbit serum (1" 100) for 30 min at 22 °C. Dilutions of test human sera ranging from 1"10 to 1:10000 were used to evaluate A. fumigatus antibodies. After incubating for 1 h in a humid chamber at 22 °C, the rings were removed, the slides were washed 3 times for 2 min in TBSA and again dried. The air-dried slides were placed into a humid chamber and the rings were repositioned. Sixty #1 of goat antihuman IgG (1-1000, Sigma, St. Louis, MO)or lgE (1" 100, Sigma) were added onto the slides covered by rubber rings. After 1 h incubation at 22 °C, the rubber rings were removed and the slides were washed as above. The slides were then treated with rabbit antigoat lgG (Sigma) conjugated with colloidal gold, at a dilution of 1:200 for 1 h at 22 °C and washed again in TBSA. The slides were first washed in PBS then twice in double distilled water for 3 min each. After draining, the slides were rinsed in 0.2 M citrate buffer prepared freshly by diluting 2 M stock citrate buffer with distilled water. The stock citrate buffer was prepared just before use by dissolving 23.5 g trisodium citrate and 25.5 g citric acid in 100 ml of distilled water. The slides were then treated with freshly prepared silver In nrha,. *,,~ ~ . l , J reaction, .c. n. .h. .l t l n n v i a t . ., -l l~l h l ¢ l.l.l.q. w l i ; ; .1.,.-. Lllqi~ I: I.I I.I l .I U.I I.U .~ U. I U The enhancer was prepared freshly by mixing equal volumes of hydroquinone solution (0.47 g hydroquinone in 7.5 ml of distilled water) and silver solution (0.055 g silver lactate dissolved in 7.5 ml of distilled water) and diluted 1:6 times with distilled water. The enhancer was protected from light by covering with aluminium foil. The slides were allowed to react with the enhancer solution for 4 - 6 min in jars wrapped with aluminium foil. After the silver enhancement the slides were fixed in 1"10 dilution of a fixing solution (Janssen Life Sciences Products, Beerse, Belgium) for 3 min, washed two times for 3 min each in distilled water, dehydrated by passing through changes of 75%, absolute alcohol and xylol, and mounted in Permount (Scientific Products). Appropriate dilutions of serum and reagents were chosen after repeated experiments with positive and negative sera. Colloidal gold was prepared and conjugated to rabbit antigoat IgG (Sigma) according to the procedure described by Geoghegan and Ackerman [15]. The preparations were examined in a Leitz Dialux 20 EB microscope. Following controls were included with each experiment: IGSA was performed by using (1) the slides with antigens only, (2) antigens incubated with IGSA-positive and IGSA-negative sera, (3) antigens treated with goat antihuman IgG with and without human serum, (4) antigens treated with rabbit antigoat IgG conjugated with colloidal gold with and without human serum. ViU~,,,J.
LV
116 A positive reaction was evidenced by binding of gold-silver particles to A. fumigatus hyphae, as demonstrated by black granules on the surface of the hyphae. The results were recorded as negative when the hyphae appeared without any color or granules on the surface of the hyphae. The reaction was graded from ' - ' to ' + + + ' for negative to very strongly positive. The highest dilution which consistently gave at least a ' + ' reaction was recorded as the titer of antibody in serum samples. Grading of the reaction was recorded independently by two observers. A. fumigatus specific IgG and IgE antibodies were also detected by biotin-avidin linked immunosorbent assay (BALISA) [ 16] and DD [l 7, 18].
Specificity of IGSA The specificity of IGSA technique was evaluated by using appropriate negative and positive controls in each determination as well as in absorption tests with relevant antigens. Solutions of Penicillium sp. (10 mg/ml), Candida albicans (100 mg/ml), Faenia rectivirgula (Micropolyspora faeni, 5 mg/ml) and A. fumigatus (10 mg/mi) were incubated (1 h, at 22 °C) with patient sera which consistently showed a ' + + + ' reaction for both IgG and IgE specific antibodies to A. fumigatus in BALISA and IGA. The antigen concentration in serum was adjusted to 1 mg/ml. The incubated serum was used after centrifugation to detect A. fumigatus specific antibodies in sera of the patients by IGSA, and the reaction was recorded. Results and Discussion
The results of the measurement of A. fumigatus antibodies are presented in Table 1, and demonstrate that IGSA can be used to detect A. fumigatus specific IgG and IgE antibodies in human sera. All six sera had precipitins against A. fumigatus by DD whereas the two controls were negative. Specific IgG and IgE antibodies against A. TABLE 1 RESULTS O F I M M U N O G O L D - S I L V E R ASSAY (IGSA) C O M P A R E D W I T H D D A N D BALISA FOR D E T E C T I N G IgG A N D IgE A N T I B O D I E S TO A. F U M I G A T U S Patient no.
1 2 3 4 5 6 7 8
Diagnosis
ABPA ABPA aspergilloma aspergilloma aspergillosis aspergillosis normal normal
IGSA
DD
IgG
IgE
1:10000 b 1:10000 1:1000 1:5000 1:1000 1:1000 -
1:500 1:1000 l:100 l:100 l:100 1:50 -
Optical density read at 490 nm. b Highest serum dilution giving a definite positive reaction. c Nanogram per ml.
a
+ + + + + + -
BALISA
Total IgE c
IgG a
IgE b
1.126 1.304 0.222 0.361 0.372 0.718 0.164 0.063
0.380 0.370 0.033 0.100 0.306 0.394 0.036 0.020
13084 11569 279 96
12 65
117
.eT' o
Fig. 1.
A slide culture of Aspergillusfumigatus showing positive reaction by immunogold-silver assay (IGSA) using ABPA patient serum ( x 250).
fumigatus were detected by BALISA in all patients and in the controls. The titers of IgG and IgE antibodies, however, were higher in the patients' sera. Sera from all patients showed positive reactions to A. fumigatus by IGSA using both IgG and IgE while controls were negative. The IGSA detected A. fumigatus specific IgG (Fig. 1) in all patients' sera; the highest dilution which consistently gave at least a' +' reaction varied from 1:1000 to
t antigoat IgG + gold IgG/Ig
Fig. 2.
Schematic illustration of immunogold-silver staining.
118
Methanol
fixed slide c u l t u r e s
PBS-Milk-Tween
30 min
TBSA w a s h Normal r a b b i t
2 rain x 3
serum 30 min TBSA
Dilutions
of serum 60 min TBSA
Goat a n t i - h u m a n
IgG
(l:1000)/IgE
(i:i00)
60 min
TBSA Rabbit
antigoat
IgG conj.
with c o l l o i d a l
gold
PBS 3 min x 2; H20 3 min x 2 Silver enhancement
Fixing
(see the text)
solution
H20 3 m i n x
Dehydration
4 - 6 min
3 min
2
and m o u n t i n g
Fig. 3. Differentstages of immunogold-silverstaining.
1:10000. All control sera were negative. Specific IgE antibodies to A. fumigatus were detected by IGSA in all patients but not in the controls. In one patient with ABPA a positive reaction was detected at 1:I000 dilution, in two at 1:500 dilution and in all but one at 1:100 dilution of serum. A positive correlation could be demonstrated in the degree of reactivity by IGSA when compared with the results of BALISA. In the absorption tests using soluble antigens of Penicillium sp., Candida albicans and Faenia rectivirgula (Micropolysporafaem) the reactivity and titer of antibody of the patients' serum were not affected when A. fumigatus slide culture antigen was
119 used. On the other hand, a dose-dependent inhibition of antibody reactivity and titer was detected when sera were absorbed using soluble A. fumigatus antigen prior to testing by IGSA. Thus, the antibody against A. fumigatus detected by IGSA is specific and the positive reaction was not a result of a cross-reaction with other fungal antigens. The indirect IF-method has previously been used to detect A. fumigatus antibodies in patients' sera [5]. IGSA, however, offers several advantages over the IF-method. IGSA is more sensitive as the maximum serum dilution titer detected by !GSA was 1:10000 while IF-titer was 1:640. Thus, IGSA-method also uses smaller amounts of test reagents and allows the storage of sections indefinitely. Furthermore, it offers the possibility of a simultaneous evaluation of immunoisotypes and the demonstration of antigen localization on fungal cells by immunoelectronmicroscopy [19]. The IGSA method was found to be superior to DD in detecting A. fumigatus antibodies. IGSA can be carried out more rapidly than DD and it can detect both IgG and IgE antibodies specific to A. fumigatus in titers comparable to ELISA. The present results indicate that IGSA is a reliable and specific method and no crossreactivity could be detected between common fungal antigens. IGSA is simpler than enzyme- or radioimmunoassays (EIA and RIA) and can be performed in a routine clinical laboratory without special equipment. Because of its high sensitivity and the use of whole cells as slide culture antigens this method has an advantage over the EIA and RIA where purified antigens are needed for reliable results. In concordance to the findings of EIA and RIA, in the present study IGSA discriminated patients from controls with regard to specific antibodies against A. fumigatus. In order to shorten the time needed for preparing the Aspergillus slides in the present method we will be testing the usage of lyophilized Aspergillus hyphae in our further studies. In conclusion, IGSA is a simple, and reliable method useful in detecting specific antibodies to A. fumigatus in patient sera. The technique can be performed in a routine clinical laboratory without special equipment. It is faster and more sensitive than DD or IGA, and is at least as sensitive as BALISA.
Acknowledgements The authors are grateful to Dr. Leo Kaufman, CDC, Atlanta, GA, for the generous supply of sera from patients with invasive aspergiUosis. We wish to acknowledge the technical assistance of Laura Castillo, Abe Resnick and Nancy Elms. This investigation is partly supported by a grant from the National Institute of Health (AI-23071), by The Veterans Affairs, and by The Finnish Work Environment Fund, The Foundation for the Study of Allergy, The Finnish Anti-Tuberculosis Association, The Paulo Foundation and Farmer's Social Insurance Institution, Finland.
References 1 We~zel, F. J., Emanuel, D.A. and Gray, R. L. (1972) A simplified hemagglutination test for farmer's lung. Am. J. Clin. Pathol. 57, 206-208. 2 Flaherty, D. K., Barboriak, J., Emanuel, D., Fink, J., Marx, J., Moore, V., Reed, C.E. and Roberts, R. (1974) Multilaboratory comparison of three immunodiffusion methods used for the detection of
120 precipitating antibodies in hypersensitivity pneumonitis. J. Lab. Clin. Med. 84, 298 - 306. 3 Marx, J.J., Motszko, C. and Wenzel, F. J. (1975) Complement fixation test as an aid in the diagnosis of farmer's lung. J. Clin. Microbiol. 1,480- 482. 4 Parrat, D., Nielsen, K. H., Boyd, G. and White, R. G. (1975) The quantitation of antibody in farmer's lung syndrome using a radioimmunoassay. Clin. Exp. Immunol. 20, 217- 221. 5 Kurup, V.P., Barboriak, J.J. and Fink, J. (1977) Indirect immunofluorescent detection of antibodies against thermophilic actinomycetes in patients with hypersensitivity pneumonitis. J. Lab. Clin. Med. 89, 533 - 539. 6 M/intyj/irvi, R.A., Jousilahti, P. and Katila, M.L. (1980) Antibodies to Aspergiilusfumigatus in farmer's lung patients measured by enzyme-linked immunosorbent assay (ELISA). Clin. Allergy 10, 187- 194 7 Kurup, V. E, Resnick, A., Scribner, G.H., Kalbfleisch, J.H. and Fink, J.N. (1984) Comparison of antigens and serological methods in Aspergillusfumigatus antibody detection. Mykosen 27, 4 3 - 50. 8 Kauffman, H.F., Beaumont, F., Meurs, H., De Heide, S.V. and De-Vries, K. (1983) Comparison of antibody measurements against Aspergillusfumigatus by means of double diffusion and enzymelinked immunosorbent assay (ELISA). J. Allergy Clin. Immunol. 72, 255- 261. 9 Longbottom, J. L. (1986) Antigens and allergens of AspergiilusfumigatusII. Their further identification and partial characterization of major allergens (Ag3). J. Allergy Clin. Immunol. 78, 18- 24. 10 Kurup, V. P., Ramaswamy, M., Greenberger, P.A. and Fink, J.N. (1988) Isolation and characterization of a relevant Aspergillusfumigatus antigen with igG- and IgE-binding capacity. Int. Archs. Allergy Appl. Immunol. 86, 176- 182. 11 Gugnani, H.C., Reijuia, K.E., Kurup, V. P. and Fink, J. N. 0989) Detection of IgG and IgE antibodies to Aspergillusfumigatus in human sera by immunogold assay. Mycopathology (in press). 12 Holgate, C.S., Jacson, P., Cowen, P.N. and Bird, C.C. (!983) Immunogold silver staining: new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 31,938- 944. 13 Rosenberg, M., Patterson, R., Murtzer, R., Cooper, B.J., Roberts, M. and Harris, K.E. (1977) Clinical and immunological criteria for diagnosis of allergic bronchopulmonary aspergillosis. Ann. Intern. Med. 80, 405- 414. 14 Riddel, R.W. (1950) Permanent stained alycological preparations obtained by slide culture. Mycologia 42, 265 - 270. 15 Geoghegan, W.D. and Ackerman, G.A. (1977) Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin of colloidal gold for the indirect detection of concanavalin A, wheat germ agglutination and goat antihuman immunoglobulin G on cell surfaces at the electronmicroscopic level: a new method, theory and application. J. Histochem. o, i61 - i65. 16 Kurup, V.P. (1986) Enzyme-linked immunosorbent assay in the detection of specific antibodies against Aspergiilusfumigatus in patients' sera. Zbl. Bakteriol. Hyg. (A) 261, 509- 516. 17 Kurup, V. P., John, K. V., Resnick, A. and Fink, J. N. (1986)'A partially purified glycoprotein antigen from AspergiUusfumigatus. Int. Arch. Allergy Appl. Immunol. 79, 263 - 269. 18 Wadsworth, C. (1962) A microtemplate technique employing a gel chamber compared with other micro- and macrotemplate techniques for immunodiffusion. Int. Arch. Allergy Appl. Immunol. 31, 131 - 137. 19 Romasco, F., Rosenberg, J. and Wybran, J. (1985) An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subset with monoclonal antibodies. Am. J. Clin. Pathol. 84, 307-316.