Characterization of Antigens from Aspergillus Fumigatus I. Preparation of Antigens from Organisms Grown in Completely Synthetic Medium1

SANG J. KIM and SOTIROS D. CHAPARAS

SUMMARY Aspergillus fumigatus was found to grow well in completely synthetic asparagine glycerol medium. Four-day-old mycelia were totally disrupted by vigorous mechanical shaking with 0.45-/an glass beads. Such mechanically produced extracts contained at least 52 precipitating antigens. T h e number of protein and carbohydrate components was maximal in 4-day-old organisms and decreased in older cultures. Conversely, the number of components in culture nitrates increased with longer incubation periods, but was only approximately one-half that produced by the 4-day-old mycelial extracts.

Introduction Infection with t h e different species of the genus Aspergillus may result in various clinical forms of aspergillosis, d e p e n d i n g o n t h e i m m u n e status of the host. D e m o n s t r a t i o n of t h e organism in resected lesions may b e diagnostic, b u t cultural diagnosis is difficult because of the ubiquity of aspergilli in the e n v i r o n m e n t of laboratories, a n d because the organism may be found in t h e lungs of those with u n r e l a t e d chronic l u n g conditions. N u m e r o u s i m m u n o l o g i c tests have been used to d e t e r m i n e the presence of a n t i b o d y or reactivity of cells to antigens of aspergilli (1-15). T h e results of serodiagnostic tests in p a t i e n t s w i t h various forms of aspergillosis have b e e n conflicting because of variations in sensitivity, t h e qualitative n a t u r e of test systems, a n d the widespread use of u n s t a n d a r d i z e d antigen preparations.

(Received in original form February 6, 1978 and in revised form June 15,1978) 1 From the Division of Bacterial Products, Bureau of Biologies, U.S. Food and Drug Administration, 8800 Rockville Pike, Bethesda, Md. 20014.

I n this first p a p e r , we studied t h e conditions necessary for o p t i m a l growth of Aspergillus fumigatus a n d m e t h o d s for its efficient d i s r u p t i o n . T h e minimally d e n a t u r e d antigens so p r o d u c e d will serve as a source for fractionation procedures aimed at i m p r o v i n g the reactivity a n d specificity of diagnostic reagents for aspergillosis, a n d for the creation of a large lot of a U.S. S t a n d a r d in the control of potency of reagents to be used for in vivo a n d in vitro tests. Materials and Methods Strain and the inoculum. Aspergillus fumigatus NIH 5233 (ATCC 13073) was obtained from the Medical Mycology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Conidia from a 3-day-old culture on malt extract agar slants were suspended in the spore preservation medium (0.2 M sodium glutamate, 0.1 M sucrose, 0.02 M semi-carbazide), and the suspension was passed through sterile cotton to remove hyphal fragments and long spore chains. T h e suspension was diluted to contain approximately 10 million spores per ml. This spore suspension was stored in aliquots of 1.0 ml at —70° C until used to inoculate the medium. Media. Three chemically denned media were used in order to select the most suitable medium for antigen production: asparagine synthetic medium (16),

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Czapek-Dox medium, and Dorset-Henley medium (17). Growth of the fungus at 37° C and at initial p H of 7.0 was compared on an incubator shaker (Gyrotory model G-25, New Brunswick Scientific Co., N.J.). A large production lot of mycelium was prepared by growing A. fumigatus in a 100-liter fermenter containing 50 liters of asparagine synthetic medium at an initial pH of 5.0. The yield of mycelial mass after 4 days of incubation was approximately 5 kg wet weight. T h e mycelial mat was aliquoted and frozen until it was mechanically disrupted. Antigen preparations. T h e culture filtrate was separated from mycelia by passage through Whatman No. 42 paper and concentrated by vacuum dialysis using Visking cellulose membrane (1-cm diameter) that had been thoroughly soaked and rinsed in distilled water. Concentration was accomplished in 3liter Erlenmeyer flasks, half-filled with distilled water, that were connected to a vacuum of 27 pounds per in2. T h e concentrated culture filtrate was then centrifuged at 10,062 g for 30 min; the supernatant was dialyzed against 5 changes of 200 volumes of deionized water at 4° C during a 48-hour period and was then freeze-dried. Antigens from mycelium. T h e mycelium separated from the culture filtrate was washed once with water and resuspended with 2 to 3 parts water; the mixture was then briefly homogenized with a blender. Approximately 25 ml of the mycelial slurry were transferred to a 60-ml bottle with 22 ml of 0.45-mm glass beads. This mixture was then homogenized on a Braun mechanical cell homogenizer for 5 min with intermittent chilling with C 0 2 . T h e mycelial homogenate was centrifuged at 37,000 g for 45 min, and the supernatant was passed through a 0.20-^m membrane filter (Nalgene filter unit). The clear supernatant was further dialyzed against 2 changes of deionized water and freeze-dried. T h e freeze-dried material was reconstituted in phosphate-buffered 0.85 per cent saline (pH 7.2) with various bases (weight/vol), depending on the purpose of the immunologic study. T h e protein content of all antigen preparations was determined by the method of Lowry and associates (18). Carbohydrate content was determined by the resorcinol method (19). Analytical poly aery I amide gel electrophoresis. Polyacrylamide gel electrophoresis was performed using disc electrophoresis apparatus (model 1200, Canalco, Rockville, Md.) as described previously (20). The separating gel contained 7 per cent (weight/ vol) acrylamide at p H 8.9, and the stacking gel contained 2.5 per cent (weight/vol) acrylamide at p H 6.7. Gels were stained for proteins with Coomassie brilliant blue; for polysaccharides and glycoproteins, by the periodic acid-Schiff (PAS) reaction. Immunization of animals. Mycelial homogenate, disrupted with a Braun mechanical cell homogenizer, was freeze-dried and reconstituted in sterile saline to contain 40 mg per ml. T h e suspension was emulsified with Arlacel A (Atlas Chemicals) and Drakeol 6VR (Penreco) at the ratio of 100:35:65 by volume.

New Zealand white female rabbits weighing approximately 3 kg were given subcutaneous injections of 0.1 ml of the emulsion in each footpad and in the inguinal areas. Booster injections were given every 2 weeks in the inguinal area until sera gave maximal precipitation reactions in Ouchterlony tests. Two goats were also sensitized similarly, but injections were given only in the inguinal area. Serologic tests. Double diffusion tests were performed according to the method of Crowle (21) in 1 per cent Noble agar containing 1 per cent sodium azide on 2.5- by 7.5-cm glass slides. Fused-rocket immunoelectrophoresis was performed according to a modification of the method of Svendsen (22) in 1 per cent agarose containing B2buffer, pH 8.6 (i.s. 0.075). On a precoated glass slide (6.5 by 10 cm), 8 ml of warm agar were poured and allowed to gel. Overlapping 2- by 8-mm oval antigen wells separated by approximately 1.5 mm were cut into agar. A layer of agar 3 to 4 mm above the wells was removed and replaced with a similar Jayer that contained 30 per cent antiserum. Antigens placed in the wells were allowed to diffuse for 1 hour before electrophoresis at 2.9 V per cm and 20 mA for 4.5 hours. Two-dimensional immunoelectrophoresis was performed according to the method of Weeke (23). Antigens were electrophoresed in the first dimension for 90 min, and in the second dimension for 4.5 hours into agar containing 30 per cent antiserum as described previously. Results O p t i m a l harvest of A. fumigatus in which exp o n e n t i a l growth usually c o n t i n u e d for 9 days (figure 1) was o b t a i n e d from cultures grown o n asparagine synthetic m e d i u m . T h e p H of t h e culture filtrate decreased to 3.3 by the seventh day of i n c u b a t i o n a n d increased gradually to 7.2 by 6 weeks. G r o w t h in Czapek-Dox m e d i u m was c o m p a r a b l e to t h a t in asparagine synthetic med i u m , b u t growth in Dorset-Henley m e d i u m was poor. T h e yield of mycelial extract of organisms grown in asparagine synthetic m e d i u m could b e affected by factors such as the ratio of water to mycelia in the B r a u n homogenizer bottle, the size of glass beads, a n d t h e h o m o g e n i z a t i o n time. W h e n glass beads 1.0 or 0.25 m m in d i a m e t e r were used, or w h e n the r a t i o of water to m a t was n o t o p t i m a l , d i s r u p t i o n was incomplete. Complete d i s r u p t i o n was confirmed by microscopic observation, which showed only mycelial debris with n o intact mycelia (figure 2C). D i s r u p t i o n was complete w i t h i n 5 m i n w h e n 25 m l of a 70 p e r cent slurry of mycelia in water (vol/vol) were m i x e d with 22 m l of 0.45-mm beads a n d shaken in a B r a u n homogenizer. Sonication at

CHARACTERIZATION OF ANTIGENS FROM ASPERGILLUS

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ASM: Asparagine synthetic medium CDM: Czapek -Dox medium DHM: Dorset Henley medium

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Characterization of antigens from Aspergillus fumigatus. I. Preparation of antigens from organisms grown in completely synthetic medium.

Characterization of Antigens from Aspergillus Fumigatus I. Preparation of Antigens from Organisms Grown in Completely Synthetic Medium1 SANG J. KIM a...
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