Studies on Aspergillus fumigatus antigens for serodiagnosis

Serodiagnosis

and Immunotherapy

in Infectious Disease ( 1990) 4, 183-191

Studies on Aspergillus fumigatus

B. Banerjee, M. Mukherjee, CSIR Centrr For Biochemicals,

antigens for serodiagnosis

S.V. Gangal and P.U. Sarma*

Pate1 Chest Institute Building, Delhi 110007, India

University Campus.

Culture filtrate (CF) proteinsof a soil strain of Aspergillus fumigatus wereevaluated for their potential usein the serodiagnosis of patientsof allergicbronchopulmonary aspergillosis(ABPA). Analytical fractionation of third-week culture filtrate (III WCF) and third-week mycelium(III W mat) by fast protein liquid chromatography (FPLC) indicatedprotein peaksin the molecularweight rangeof 180,70, 43, 12and 6.5 kDa. The III WCF proteinsshowedvery strong precipitin reactionwith the sera of ABPA patients by double immunodiffusion.The increasedserumIgG and IgE levels(estimatedby microtitre plate ELISA method)correlatedwell with the severity of the disease.The patientswith strong precipitin reaction showedhigh serumIgG levelsagainstA. fumigutus antigens. Aspergillus fumigatus antigens,allergic bronchopulmonaryaspergillosis. fast protein liquid chromatography. ELISA. Keywords:

Introduction Aspergillosis is a disease caused mainly by A. fumigatus which is an important pathogen of the bronchopulmonary system. The clinical spectrum of the disease consists of bronchial asthma, allergic alveolitis, allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma. This fungus is known to produce many antigenic and allergenic components stimulating the production of IgE and IgG antibodies in patients. Detection of these antibodies can be used for the diagnosis of patients with various disorders associated with the fungus. The early diagnosis in these patients is essential to prevent irreversible lung damage. Most of the extracts used in the immunodiagnosis are prepared either from disrupted mycelium of short term (34 days) culture or from a long term (3.-5 weeks) culture filtrate of the fungus grown on a fully synthetic medium. Since the latter are prepared during the autolytic phase of growth, they are believed to contain not only extracellular (metabolic) components but also intracellular (cytoplasmic) components as a result of lysis. Kurup et u/.‘-~ partially characterized glycoprotein antigens from culture filtrate which reacted with IgE and IgG antibodies in sera of patients of ABPA. Harvey et als4reported a major antigen (Ag7) which binds both specific IgE and IgG antibodies in the sera of patients. Several methods such as agar gel double immunodiflusion, complement

* Author

to whom

correspondence

should

be addressed.

183 0888-0786/90/030183 + 09 $03.00/O

(c: 1990Academic PressLimited

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B. Banerjee et al.

fixation, haemagglutination, immunofluorescence, enzyme linked immunosorbent assay. have been used in the detection of antibodies against A. ,firmigatus antigensi -. In this study, preparation and immunological characterization of a potent antigenic extract from culture filtrate of A. &migatus (soil strain) is reported. Analytical fractionation of this preparation was carried out by Fast Protein Liquid Chromatography (FPLC). The antigenicity of the extract was evaluated using hyperimmune sera and sera of patients of allergic bronchopulmonary aspergillosis by immunodiffusion. immunoelectrophoresis and enzyme linked immunosorbent assay (ELISA).

Materials

and methods

Materials General chemicals used were of reagent grade obtained from local companies. Special chemicals such as substrates, enzyme conjugates and other immunochemicals were purchased from M/S Sigma Co. Mould extract. Aspergillus fumigatus (soil strain) was obtained from Indian Type Culture Collection Centre (ITCC), Indian Agricultural Research Institute (IARI), Delhi. This strain was grown as a stationary culture in glucose asparagine medium at 37°C. The culture filtrate (CF) was collected at different stages of growth and was extensively dialysed against deionized water at 4°C and lyophihzed. Dried mycelial proteins were extracted with 0.125 M ammonium bicarbonate solution at the ratio of I:20 (w/v) by continuous stirring at 4°C for 48 h*. The protein content of CF preparation was determined by Bradford method’. Hyperimmune rabbit sera. Hyperimmune serum to (111 WCF) antigens was raised in New Zealand white rabbits. A 10 mg extract of Ill WCF antigens in 2 ml saline was emulsified with Freund’s complete adjuvant and injected subcutaneously at multiple sites. This was repeated for 4 consecutive weeks with Freund’s incomplete adjuvant followed by weekly booster doses until satisfactory titre was obtained as determined by counter immunoelectrophoresis (CIE) test. Human sew. Sera of patients suffering from ABPA were obtained from (a) All India Institute of Medical Sciences, (b) RBTB Hospital, (c) Vallabbhai Pate1 Chest Institute, Delhi, and (d) Chest Clinic of Guntur Medical College Andhra Pradesh, India. The ABPA patients fulfilled the clinical and immunological criteria as described by Rosenberger et al.‘O The control sera were obtained from a blood bank and other voluntary laboratory workers who did not have any respiratory problems. Their sera were found to be negative to precipitin with A. fumigatus antigens. Methods Double immunod$jiision and immunoelectrophoresis. The double immunodiffusion was performed according to the method of Ouchterloney using 1% agarose gel in barbitone buffer, pH 8.6 with 1% sodium citrate and 0.1% sodium azide”. The Ill WCF antigen described above was used at a concentration of 20 mg ml- ‘. After application of the sera. the plates were incubated for 72 h at room temperature. Thereafter, the agarose gels were

Studies on A. fumigatus antigens

185

thoroughly washed in normal saline, dried and stained with Coomassie brilliant blue R-250. Immunoelectrophoresis was performed using 1% agarose in the above mentioned buffer. A volume of 10 ~1of crude A.fumigarus extract (20 mg ml-‘) was used for electrophoresis at 10V cm- ’ for 2 h and incubated overnight with 0.1 ml hyperimmune sera in the trough. Fast protein liquid chromatography ( FPLCj. FPLC was performed on a prepacked Superose 12/6 HR10/30 equipped with pumps, optical unit, U.V. monitor, fraction collector and recorder (Pharmacia Sweden). The eluent contained 0.1 M ammonium bicarbonate, (pH 8.0). The flow rate was 1 ml rnin~-’ and the operating pressure was 2 psi. The whole fungal extract was prefiltered through millipore filter and loaded bq means of tuberculin syringe. The separated zones were detected by U.V. monitor at 280 nm. The absorbance at 280 nm was recorded with a chart speed of 1 cm gap per minute. Enz~~nw lit&d innlzuno.rorhc~nt assays (ELISA j. Indirect ELISA was carried out as described by Voller et al. with somemodifications”. The Laxbro plates (polystyrene flat bottomed) were coated with 0.1 ml of antigen (I I pg ml ‘) in 0.05 M coating buffer. pH 9.6. and incubated overnight at 4°C. The plates were washed three times with washing buffer (0.01 M PBS, pH 7.4, containing 1% BSA and O.OS%Tween 20). Then the plates were incubated with 3% BSA in washing buffer for 1 h at 20°C in order to block the sites of non-specific reactions. The buffer was removed and 0.1 ml of the patient’s sera (diluted to 1:lOO in washing buffer) was added to each well of the microtitre plate. Normal human sera were used ascontrol at the samedilution. The microtitre plates were incubated at 20°C for 3 h followed by washing three times at an interval of I5 min each. The plates were again incubated with 0.1 ml of I:1000 diluted protein A peroxidase per well for 3 h. The plates were washed three times with washing buffer as mentioned above. A volume of 100~1 of substrate (40 mg of 0-phenylenediamine in 50 ml of phosphate citrate buffer. pH 3.6 and 40 PI of 30% hydrogen peroxide) was added to each well and the plates were protected from light for 30min. The reaction was stopped by adding 50 ~1of 5 N HCI and the colour developed was measuredagainst buffer blank at 490 nm.

Results FPLC patterns of crude culture filtrate and mat proteins are shown in Figures I and 2. The major protein peaks of the extracts (III W mat) were in the molecular weight range of 180.70 and 12kDa. The protein peaks of III WCF were in the range of 170.70,43, 12 and lessthan 6 kDa. The protein profiles of antigenic preparations indicated qualitative similarities among different antigenic preparations. However, the concentrations of the individual peaks were noticed to be different. The electrophoretic pattern indicated a polyclonal antibody response with the antigens of III WCF in rabbit. Three strong precipitin arcs with different mobilities are shown in Figure 3. The strong precipitin reaction of theseantigens with the sera of patients by double immunodiffusion is shown in Figure 4. These observations suggest the usefulnessof the antigenic preparation in serodiagnosis. The ELISA results indicated that the level of specific IgE and I,@ antibodies against A.,fitnzigrrrus antigens vary among patients depending on the severity of the disease.The ELISA values were derived after studying 20 patients and 10 normal subjects. Active diseaseshowed two- to three-fold increase in optical density values for A. ,firmigarzr.s specific IgG and IgE antibodies compared to disease at remission. The ratios of

1

I .20

1.05-

0.90

-

E 0.752 4 ‘D 060:: 0.45 t

0

I I

I

I 2

I 4

3 Fraction

Figure 1. FPLC loaded in column of bicarbonate pH 8.0. 29,000, Cytochrome

I 5

/ 7

I 6

I

number

pattern of III WCF of A.firmigatus proteins. A volume, 50 ul of 40 mg ml ’ sample was Superose 12HR10/30, Flow rate was 0.7 ml min ’ and buffer used was 0.1 M ammonium Molecular weight markers were Bovine serum albumin 66,000. Carbonic anhydraseC- 12.000 and Aprotinin-6,000.

24000 1.05 -

p, ,’

0.90

5

:

-

0.75-

0 w ‘j s

0.60-

0.45

-

0

I

2

3 Fraction

4 number

5

6

7

. .. -

Figure 2. FPLC pattern of III W mat proteins of A. fumigatus. A volume, 50 pl of 40 mg ml- ’ sample was loaded in column of Superose 12HR/30, Flow rate was 0.7 ml min’ and buffer used was 0.1 M ammonium bicarbonate pH 8.0.

Studies on A. fumigatusantigens

187

Figure 7. lmmunoelectrophoretic pattern of A. fumigatus III WCF antigens against rabbit hyperimmune serum. A volume, 10 ul of crude extract (20mg ml ‘1 was run at IOV cm ’ for 2 h and incubated overnight with 0.1 ml hyperimmune sera in the trough.

absorbance of normal to patients ranged from I :2 to I:9 as shown in Table 1. These antibodies vary considerably and good correlation exists between high IgG titre and precipitin reaction. The patient’s serum with high IgG titres (OD 1.8 to 1.2) against ,4. fumigutus antigens showed a number of precipitin bands (Figure 5). These results indicated that IgG antibodies were responsible for causing percipitin reaction with .4. ,fumigatus antigens which is one of the most important diagnostic criteria for ABPA.

Figure 4. Double immunodiffusion patterns of ABPA patients against III WCF antigens. Patients (I, 2, 3. 4, 5) and normal (6). A volume, 20 p1 of A. ,fumigalus crude extract (20 mg ml ‘) was added in the central well and IO ~1 of different patients and normal sera were added in the peripheral wells.

I88

B. Banerjee

1. Antigen

Table

Group and subject no. Group

specific immunoglobulin

et al.

levels and precipitin controls ELISA

reactions of patients and norm;

OD values for

kG

I@

I ,440 I.430 I.043 I.430 1‘.146 I.289 1.196 1.880 0.976 I .760

0.560 0.170 0.240 0.330 0.460 0.23 I 0.620 0.560 0.177 0.230

I.359 0.292

0.357 0.175

0.866 0.997 0,570 0.552 0.600 0.661 0.415 0.420 0.480

0.045 0.069 0.075 0.161 0.129 0.240 0.130 0.070 0.060

0.617 0.197

0.108 0.062

0.202

0.250 0.100 0.150 0.080 0.145 0.150 0.090 0.300 0.350

0.030 0.030 0.069 0.040 0.030 0.030 0.010 0.030 0.020 0.030

0.182 0.092

0.031 0.015

Immuno

diffusion test

I

1 2 3 4 5 6 I 8 9 10 Mean SD Group II 1 2 3 4 6 7 8 9 10 Mean SD Controls 1 2 3 4 5 6 I 8 9 IO Mean SD

+ + vc + + ve + ve + + ve + + ve + ve ++ ve + + ve + + ve ++ vc

+ + + + + +

vc ve ve ve ve ve ve ve ve

--

ve vc ve ve ve ve ve vc ve ve

Studies on A. fumigatus

E I26 ? 0 lJJI.4c5 HEn 4 (I) ? I.flw

ii 0 cl :

0.2A stronq precipitjn

189

0 1 i *

0.6-

antigens

0 Weak ptecipitin

O 1 C No precipitin

I D No prccipitin

Figure 5. ELISA titre for specific IgG antibodies of ABPA patients Patients under acute attack. 0 under mild attack, ~3 skin test positive

against A.firmigufus asthma, n controls.

CF antigens.

1;

Discussion A number of reports state that there are significant differences between different isolates of A. ,fumigatus. NCPF strain nos. 2 109 and 2140, IMI strain no. 16030 and ATCC strain no. 42202 (identical with strain no. 507 of Kurup e’t ~1.) were studied in different laboratories’3m’5. In the present study, an indigenous soil strain (ITCC 2605) of .4. ,fumigatus was used. Antigenic extracts of mycelium and culture filtrate proteins were analysed at different stagesof growth by FPLC method. The protein profiles of various antigenic preparations indicated qualitative similarities. However. individual components varied with respect to concentrations. FPLC of III W mat and CF showed a major protein at 180kDa and 170 kDa for mat and culture filtrate proteins, respectively. The antigenic extracts were stained by Periodic Acid Schiff s reagent for glycoproteins. It is observed that the protein with 180 and 170kDa molecular weight is a glycoprotein. The protein profile is similar, with respect to molecular weight range to the results obtained by Calvanico et al.lh and Piechura et a/.” The cell sap antigen reported by Calvanico et ul. had an approximate molecular weight of 180kDa whereas the culture filtrate antigen had a slightly lower molecular weight. This may be due to partial degradation. Rodsaether & Tonder reported a protein antigen of 150kDa molecular weight in

190

B. Banerjee et al.

ruptured mycehum’*. Harvey et al. also reported a glycoprotein of similar molecular weight in culture filtrate extract of long term stationary cultureQ. Piechura et a[. separated a fraction of ruptured mycelium, CS3, which by two-dimensional electrophoresisresolved into multiple constituents in the molecular weight range, 14 to 43 kDa and showed precipitin reaction with sera from patients with aspergilloma and ABPA19. Recently Kurup et al. had reported strong IgE and IgG responsesof ABPA and aspergilloma patients against low molecular weight protein of A.,fumigatu.s~~. The FPLC fractions of A. fumigatus soil strain used in this study also showed protein peaks in the molecular weight range 70, 43, 12 and 6 kDa, which is in accordance with the protein profiles of various strains used in different laboratories. The quantitative differences of components among various fractions make some of the fractions antigenically more relevant for immunodiagnosis of patients as compared to other fractions. The III WCF antigens prepared from the indigenous soil strain gave consistently strong precipitin reactions with sera of patients with different forms of aspergillosislike ABPA and aspergilloma but none with normal controls. The precipitin reactions were more intense in the serum samplesof patients under acute asthmatic attacks than those in the stage of exacerbation or remission. The sameantigenic preparation (III WCF) was used for detecting IgE and IgG antibodies in sera of aspergillosispatients. ELISA with protein A system is observed to be highly sensitive at a low concentration of antigen (2-10 pg ml-‘). The current antigenic preparation gives good discrimination between sera of controls and patients and can be usedin the diagnosis by ELISA for detecting A. fumigates-specific antibodies and precipitating antibodies in patients by double immunodiffusion method. References 1. Kurup VP, ResnickJA, Fink JN. A partially purified glycoprotein antigenfrom Aspergillus fumigatus.Int Arch Allergy Appl Immunol 1986;79: 263-9. 2. Kurup VP, RamasamyM, GreenbergerPA, Fink JN. Isolation and characterization of a relevant Aspergillusfumigatusantigen with IgG and IgE binding activity. In1 Arch Allergy Appl Immunol 1988;86: 17682. 3. Kurup VP, Ting EY, Fink JN. Immunochemicalcharacterization of a major antigenic componentof Aspergillusfumigatusantigens.1983;41: 698-701. 4. Harvey C, Longbottom JL. Characterizationof a major antigeniccomponentof Aspergillus fumigatus.Clin Exp Immunol 1986;65: 206-14. 5. SepulvedaR, Longbottom JL, PepysJ. Enzyme linked immunosorbentassay(ELISA) for IgG and IgE antibodiesto protein and polysaccharideantigensof A. fumigatus.Clin Allergy 1979;9: 359-71. 7. Kauffmann HF, vander Laan S, vander Hyden PJ, vander Heide S, Devris K. Antibody determinationagainstAspergillusfumigatusby meansof the enzymelinked immunosorbent assay.1. Liberation of polystyrene binding componentsduring cultivation. Int arch Allergy Appl Immunol 1985;77: 343-8. 8. Kauffmann HF, Beamont F, Meurs H, DeHeideSV, Devries K. Comparisonof antibody measurements againstAspergillusfumigatusby meansof doublediffusionand enzymelinked immunosorbentassay(ELISA). J Allergy Clin Immunol 1983;72: 255- 61. 9. Bradford MM. Rapid and sensitivemethbd for the quantitation of protein utilizing the principlesof protein dye binding. Anayt Biochem1976;72: 248-54. 10. RosenbergM, Patterson R, Mintzer R, Cooper BJ, Roberts M, Harris KE. Clinical and immunologicalcriteria for the diagnosisof allergic bronchopulmonary aspergillosis.Ann Intern Med 1977;86: 405514. 11. Oucterloney D. Antigen antibody reactionin gels:typesof reactionsin coordinatedsystemof diffusion. Acta Pathol Microbial Stand 1953;32: 231- 7.

Studies

on A, fimigatus

antigens

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12. Voller A. Bidwell D, Bartett A. Microplate ELISA and its applications. In: Malvano R, ed. Immunoenzymatic assay techniques. The Hague: Martinus Nijhoff Publ, 1980: 104-15. 13. Kurup VP Fink JN, Scribner GH. Faik MJ. Antigenic variability of aspergillus fumigatus strains. Microbios 1977; 19: 191~-204. 14. Wallenbeck I, Aukrust L, Einarrsson R. Antigenic variability of different strains of Aspergillus fumigatus. Int Arch Allergy Appl Immunol 1984: 73: 16672. 15. Kurup VP, Fink JN, Barboriak JJ, Scribner G. The detection of circulating antibodies against antigens from three strains of A. fumigatus. Mykosen 1980; 23: 368-72. 16. Calvanico NJ. DuPont BL. Huang CJ, Patterson R. Fink JN. Kurup VP. Antigens of Aspergillus fumigatus I. Purification of a cytoplasmic antigen reacting with sera of patients with aspergillus related disease. Clin Exp Immunol 1981; 45: 667-71. 17. Piechura JE, Huang CJ, Cohen SH et ul. Antigens of Aspergillus fumigatus II. Electrophoretic and clinical studies. Immunology 1983: 49: 657-65. 18. Rodsaether M, Tonder 0. Antibodies to Aspergillus fumigatus. Characterizaton of a haemagglutinogen. Acta Pathol Microbial Stand Sect C 1975; 83: 473-8. 19. Piechura JE. Kurup VP, Fink JN. Calvanico NJ. Antigens of Aspergillus fumigatus III. Comparative immunochemical analysis of clinically relevant aspergilli and related fungal texa. Clin Exp Immunol 1985: 59: 71624. 20. Kurup VP, Grcenberger PA, Fink JN. Antibody responses to low molecular weight antigens of allergic bronchopulmonary aspergillosis. J Clin Microbial 1989: 17: 1312-6.