Immunoglobulin G Antibodies Against EnvironmentalMoulds in a Norwegian Healthy Population Shows aBimodal Distribution for Aspergillus versicolor

B. Rydjord*, G. Hetlandy & H. G. Wikerz

*Department of Environmental Medicine,

Norwegian Institute of Public Health;

yDepartment of Immunology and Transfusion

Medicine, Ulleval University Hospital, Oslo; and

Department of Microbiology and Immunology and

zHaukeland University Hospital, The Gade

Institute, University of Bergen, Bergen, Norway

Received 31 March 2005; Accepted in revisedform 27 July 2005

Correspondence to: Dr B. Rydjord, Norwegian

Institute of Public Health, P.O. Box 4404

Nydalen, N-0403 Oslo, Norway. E-mail:

britt.rydjord@fhi.no

Abstract

Immunoglobulin G (IgG) antibodies against moulds related to indoor dampnessproblems are used as biomarkers to indicate exposure. In the present study, weevaluated the frequency of mould exposure in an adult healthy population byexamining levels of mould-specific IgG antibodies in Norwegian blood donors.Using enzyme-linked immunosorbent assay, 106 blood donor sera were analyzedfor IgG antibodies to Aspergillus versicolor, Penicillium chrysogenum,Cladosporium herbarum, Stachybotrys chartarum and Fusarium oxysporum. Thelevels of specific IgG antibodies to P. chrysogenum, C. herbarum and S. chartarumcorrelated (r¼ 0.46–0.62). Responses to A. versicolor were considerably strongerthan to the other moulds, and another 996 blood donor sera were analyzed forIgG antibodies to this mould. Women had significantly higher levels of specificIgG antibodies to A. versicolor than men. The concentration of A. versicolor-specific IgG antibodies showed a non-Gaussian, bimodal distribution profile, inwhich 12.5% were defined as positive to exposure. This suggests that significantmould exposure in a healthy population can be calculated from meanþ 1SD.Western blotting analyses showed that antibody responses to A. versicolor werelargely directed against carbohydrate antigens of unknown saccharides.

Introduction

In recent years, the health risk due to dampness and mouldin homes has been the subject of considerable attention.Mould exposure is perceived as a potential risk factor fordevelopment of asthma and allergy [1, 2], as well as for arange of other negative health effects such as chronic respira-tory infections, dry cough, eye irritation, skin symptoms [3,4], atopic dermatitis [5] and hypersensitivity [6]. Negativehealth effects associated with mould exposure have beenfound to depend on the particular species involved [7].

In moisture-damaged buildings, the most common fun-gal genera found are Penicillium, Cladosporium, Aspergillus,Chaetomium, Ulocladium and Stachybotrys, as reported inindoor environmental investigations of schools in Finland[8], public buildings in Denmark [9] and domestic dwell-ings in the UK [10]. Where specified, the most frequentlyoccurring species were P. chrysogenum, A. versicolor andS. chartarum [9]. In a Norwegian study of both waterdamaged and control buildings of schools and homes,the most frequent microfungi were Penicillium,Aspergillus, Cladosporium and Mucor [11].

Unless there is an indoor source, levels of moulds inindoor air reflect the outdoor air [12]. The presence ofmould indoors, as well as outdoors, has a considerableseasonal variation. In Northern Europe, the highest num-ber of spores for most genera in outdoor air are foundduring summer and autumn [12]. Indoor moulds havebeen divided into subgroups according to minimumrequired water activity [13], which describes the wateravailable to microorganisms in the material, and conse-quently, successional colonization at different moisturelevels. Penicillium and Aspergillus belong to the primarycolonizers, Cladosporium and Fusarium to the secondarycolonizers and Stachybotrys belongs to the tertiary coloni-zers, which require the highest water activity for growth[13].

Biomarkers are valuable tools for measurement of inter-actions between biological systems and environmentalagents and can be divided into biomarkers of exposure,effect and susceptibility. The ideal biomarker should besensitive, specific, biologically relevant, practical, inexpen-sive and available [14]. Biomarkers used to measure

doi: 10.1111/j.1365-3083.2005.01672.x............................................................................................................................................................................................................

# 2005 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 62, 281–288 281

exposure to indoor air pollutants are complete bloodcounts, surface receptors of immune cells, and humoralmediators like complement, cytokines and antibodies.Mould-specific immunoglobulin G (IgG) antibodies havebeen used as biomarkers for mould exposure, and the valueof this parameter has been evaluated in several studies [6,15–18]. The IgG levels have been found to be clearlydependent on the extent and pattern of mould exposure,as seen among people exposed to similar environmentalconditions [6]. Obviously, individual factors of theexposed persons affect the response and variety of symp-toms. As reviewed by Husman [4], individual sensitivity,age, smoking, atopic predisposition, previous exposure tomicroorganisms and other irritants and chemicals, as wellas the perception of unpleasant odours and smells, canmodify the adverse effect. The presence of mould-specificIgG antibodies in serum is a sign of exposure rather thandisease, whereas the absence of such antibodies does notexclude symptoms of disease [6, 19, 20]. Studies amongfarmers, cork workers, malt workers, tobacco workers andwood trimmers have suggested that levels of specific IgGantibodies in serum are related to the exposure level tomould spores and to the duration of exposure [15].

IgG antibody molecules are gradually removed from thecirculation with biological half-lives of a few weeks [21]and will disappear gradually when the antigen is elimi-nated from the body. Hence, detectable levels of mould-specific antibodies are probably mostly present amongpeople recently exposed to mould. In spite of this, severalfollow-up studies have shown that the duration of anti-body responses to moulds lasts for a long time. Only smallchanges of the antibody levels have been found within ayear after the mould source was eliminated [6, 22, 23].Thereafter, the antibody levels declined further but werestill higher 5–10 years after retirement compared with notoccupationally exposed populations [23].

There are few references regarding exposure to commonindoor moulds in the general public, and the small popu-lations examined are mostly healthy controls of clinicalstudies. The specificity of the immunological responsesinvolved has also been poorly characterized. Therefore,the level of mould exposure in the Norwegian healthyadult population was evaluated by analyzing blood donorsera for specific IgG antibodies to five different mouldsrelevant to indoor dampness. The immunological responseto A. versicolor was studied more closely, and antigenspecificities and antibody isotypes were examined inselected sera.

Materials and methods

Sera. Sera from 996 blood donors were obtained fromUlleval University Hospital, Department of Immunologyand Transfusion Medicine, Oslo, Norway, during Januaryto October 2000. In general, blood donors were selected

based on ‘Guidelines for the blood transfusion service ofNorway’ set by the Norwegian Board of Health in 1997.The sera were stored at �80 �C until analyzed. There were402 females and 594 males, and the age range was from 20to 70 years (median 43).

Fungal strains and extracts. Commercially cultivatedmould grown on synthetic substrate, harvested, freeze-dried and irradiated (Allergon AB, Angelholm, Sweden),were used as basis for water-soluble mould extracts. Suchextracts were made by adding 10 ml PBS (pH 7.4 ) and25 ml of protease inhibitor cocktail for fungal and yeastextracts (Sigma, St. Louis, MO, USA) to 0.5 g ofA. versicolor, C. herbarum, P. chrysogenum, S. chartarumand F. oxysporum. Each suspension was mixed by gentleshaking for 42 h at 4 �C and centrifuged for 20 min at20,000� g. The supernatant was dialysed with aSpectrapor� membrane (MWCO 3500, Spectrum�

laboratories INC, Rancho Dominguez, CA, USA) for24 h at 4 �C against distilled water, which was changedonce. The solution was filtered through a sterile filter (poresize 0.2 mm) and freeze-dried. Measurements of proteinconcentration in mould extracts were performed usingBicinchoninic acid assay (BCATM Protein Assay Kit,Pierce, Rockford, IL, USA).

Biotinylation of lectins. One milligram of concavalin A(Con A, Amersham Biosciences AB, Uppsala, Sweden),wheat germ agglutinin (Sigma), lentil lectin (Sigma) andpeanut lectin (Sigma), each diluted in 1 ml of 0.1 M

Na2CO3 with 0.5 M of NaCl, pH 8.3, were biotinylatedby incubating with 200 ml of 1 mg/ml biotin-X-NHS(Calbiochem, San Diego, CA, USA) in dimetylsulfoxid(Sigma) for 2 h, then dialysed using Spectrapor� mem-brane (MWCO 3500) overnight against PBS with 0.02%NaN3 (PBS/azid).

Enzyme-linked immunosorbent assay (ELISA). Specificantibodies to A. versicolor, P. chrysogenum, C. herbarum,S. chartarum and F. oxysporum in 106 sera were analyzedusing ELISA. MaxiSorp 96 wells immunoplates (Nunc,Copenhagen, Denmark) were coated with 0.25 mg ofmould extract per well, diluted in 0.05 M carbonate/bicar-bonate buffer of pH 9.6 with 0.02% NaN3, incubated for1 h at room temperature and stored at 4 �C for a max-imum of 10 days. Sera and conjugate were diluted in 0.1 M

of Tris/HCl-buffer pH 7.4 with 1% bovine serum albu-min, 0.05% Tween 20 and 0.02% NaN3. Sera diluted1:600 were incubated for 2 h for detection of antibodiesto P. chrysogenum, C. herbarum, S. chartarum andF. oxysporum, and diluted both 1:600 and 1:6000 for detectionof antibodies to A. versicolor. Alkaline phosphatase (ALP)-conjugated rabbit-antihuman IgG (Dako A/S, Glostrup,Denmark) was diluted 1:500 and incubated for 2 h. Thesubstrate step was performed with 1 mg/ml p-nitrophenylphosphate disodiumhexahydrat (Sigma) in 10% dietanol-aminbuffer, pH 9.8. After 30 min of incubation, the opti-cal density was measured at 405 nm in a MRX Microplate

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# 2005 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 62, 281–288

Reader (Dynatech Laboratories, Chantilly, VA, USA) con-nected to a PC using BIOLINX software (DynatechLaboratories) for instrument operation and calculation.Between each incubation step, the plates were washedfive times using a Dynawasher (Dynatech Laboratories Inc.,Alexandria, VA, USA) with 0.1 M of Tris/HCl-bufferpH 7.4 with 0.05% Tween 20 and 0.02% NaN3. Allincubations were done at room temperature with gentleshaking, and all samples were applied in triplicates with100 ml per well. The background of all solutions, exceptserum, was subtracted before calculating the antibodylevel. A serum sample with high antibody levels to allfive moulds was used as reference in each plate, and eachvalue was set as median of the triplicates.

IgG antibodies to A. versicolor in 996 blood donor serawere analyzed similarly but were optimized with thefollowing modifications; the plates were coated with0.5 mg of A. versicolor extract per well, and sera werediluted 1:6000. As secondary antibody, biotin-conjugatedgoat-antihuman IgG (Bethesda Research Laboratories,Gaitersburg, MD, USA) was diluted 1:10,000 and incu-bated for 2 h, followed by 1-h incubation of streptavidin incomplex with biotinylated ALP (sABC-AP, Dako A/S)diluted in 0.05 M of Tris/HCl buffer and finally substrate.A serum with a high antibody titre to A. versicolor wasselected as standard at 4-fold dilutions from 1:400 to1:1,638,400. The level of specific antibody to A. versicoloris given in arbitrary units (AU) per millilitre, relative to thestandard serum.

Dilution curves of serum with high and low levels ofmould-specific antibodies had the same steep gradient andwere parallel. This parallelism indicated similar avidity ofantibodies in serum with high or low levels of mould-specific antibodies to each of the moulds tested.

Binding of Con A, wheat germ agglutinin, lentil lectinand peanut lectin to mould extracts were analyzed usingMaxisorp immunoplates, with buffer solutions and incu-bation times as described for the ELISA above, but with afew modifications. Plates were coated with 0.5 mg ofextract per well of A. versicolor, P. chrysogenum,C. herbarum, S. chartarum or F. oxysporum, before incuba-tion with biotinylated lectins diluted 4-fold from 2 mg/mlfor 1.5 h, then with sABC-AP for 1 h before addition ofsubstrate. The specificities of the lectins were verified bythe same procedure, except that the plates were coatedwith 0.1 mg of different glycoproteins per well. Then,biotinylated lectins were added at concentrations from0.5 mg/ml to 0.025 mg/ml, followed by sABC-AP andfinally substrate. The glycoproteins used were ovomucoid(Sigma) to bind Con A and lentil lectin, asialoglycophorin(Sigma) to bind peanut lectin and glycophorin (Sigma) tobind wheat germ agglutinin.

SDS-PAGE with Western blotting. All extracts wereevaluated using SDS-PAGE with Pharmacia’s system forhorizontal electrophoresis in a Multiphor II electrophoresis

unit 2117 (Pharmacia LKB Biotechnology AB, Uppsala,Sweden) with precast polyacrylamid gels (ExcelGelSDS gradient 8-18, Amersham Biosciences AB). Eachextract was diluted in sample buffer (0.05 M Tris-acetateof pH 7 with 1% SDS, 0.01% bromophenol blue and0.001% pyronin B), boiled for 3 min, centrifuged at7000� g and applied at 15 mg per lane on sampleapplication pieces (Amersham Biosciences AB) on top ofthe gel. Separation was carried out at a constant current of50 mA and 600 Vmax until the electrophoretic frontreached the anionic buffer strip. The gels were stainedwith Silver staining kit for protein (AmershamBiosciences AB). Extract of A. versicolor was applied at40 mg per lane and electroblotted onto a NitroBindNitrocellulose transfer membrane (MSI Westboro, MA,USA) for 1 h at 200 mA.

A. versicolor blots were incubated with serum samples,lectins or both together. For lectin binding, blots wereblocked in PBS/azid with 0.5% Tween 20 for 1 h. Thenthe blots were incubated with 1 mg/ml of biotinylatedlectin for 2 h in room temperature and overnight at 4 �C,before incubation for 2 h with sABC-AP diluted 1:10,000.Both lectin and avidin were diluted in PBS/azid with 0.1%Tween 20. The blots were developed with Westernlightning CDP-star chemiluminescence reagent(PerkinElmer Life Sciences, Boston, MA, USA) and ana-lyzed using Kodak Image Station 1000 (Eastman Kodakcompany, Rochester, NY, USA). Between each step, theblots were washed 4� 5 min in PBS/azid with 0.1%Tween 20. Serum antibody binding was performed in asimilar way as for the lectin binding. Sera were diluted1:1000, and IgG antibodies bound to the blot weredetected directly with horse radish peroxidase (HRP)-conjugated rabbit-antihuman IgG (Dako A/S) diluted1:1,000 or indirectly for detection of mould-specificIgG isotypes with mouse-antihuman IgG1 (WHOCollaboratory center for human immunoglobulins,Atlanta, GA, USA) diluted 1:3,000 or mouse-antihumanIgG2 (WHO Collaboratory center for human immunoglo-bulins) diluted 1:8000 followed by HRP-conjugated rabbit-antimouse IgG diluted 1:8000. The blots weredeveloped with Western Lightning ChemiluminescenceReagent Plus (PerkinElmer Life Sciences) and visualized onan exposed hyperfilmTM ECLTM (Amersham BiosciencesAB).

Statistics. Statistical analyses were performed with SPSSversion 10.0 or SigmaStat version 2.0, both fromStatistical Analysis Systems for Windows. Normality tests(Kolmogorov-Smirnov) were performed in SigmaStat bothbefore and after log transformation. All normality or equalvariance tests failed, and comparison of groups was per-formed with Mann–Whitney Rank Sum Test for indepen-dent samples. All correlations were tested by Spearman’snonparametric correlation test, giving the correlation coef-ficient r.

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Results

Protein content of mould extracts

By water extraction of freeze-dried mould, less than 5%w/v of the basic material was retrieved in the final extract.Protein measurements of A. versicolor, P. chrysogenum,C. herbarum, S. chartarum and F. oxysporum extractsusing BCA showed a protein concentration of 20, 26,30, 18 and 42%, respectively. The extracts were alsocharacterized using silver-stained SDS-PAGE, whichshowed a broad range of different sized proteins up to100,000 Da for all moulds (Fig. 1). The further character-ization of A. versicolor using SDS-PAGE showed an addi-tional smear in a molecular weight (MW) range of50,000–100,000 Da (Fig. 2, lane A).

IgG antibodies to mould among Norwegian blood donors

Sera from 106 blood donors were tested by ELISA forspecific IgG antibodies to five different moulds: A. versi-color, P. chrysogenum, C. herbarum, S. chartarum andF. oxysporum. As expected, the levels of antibodies betweenC. herbarum, P. chrysogenum and S. chartarum correlated(r¼ 0.46–0.62) (Table 1), indicating simultaneous expo-sure to multiple moulds. Not suprisingsly, being a

secondary colonizer, Cladosporium showed the highestdegree of correlation. The level of antibodies to A. versico-lor and F. oxysporum correlated poorly with all the othermoulds tested (r� 0.35).

The antibody response to A. versicolor was much stron-ger than the antibody response to the other moulds(Fig. 3), and antibodies directed against this mould werestudied in greater depth. To reach a similar level of reac-tivity in ELISA, 10 times less serum was required fordetection of Aspergillus-specific antibodies than antibodiesto the other moulds. Extracts of Aspergillus did not containmore proteins than the other moulds.

Lectin-binding analyses

In ELISA, analyses of lectin-binding capacity to the fivemould extracts (Table 2) showed that Con A boundstrongly to all of them. Wheat germ agglutinin boundextracts of S. chartarum and F. oxysporum to some extent,A. versicolor to a lesser extent and not to P. chrysogenumand C. herbarum. Neither lentil lectin nor peanut lectinbound any of the mould extracts analyzed.

Western blotting showed different binding of lectins tothe A. versicolor extract (Fig. 2, lanes B–E). Con A boundto several protein bands in addition to a smear in the rangeof 30,000–200,000 Da. Wheat germ agglutinin boundA. versicolor extract only to a slight degree and to componentsmigrated longer than those bound by Con A. All four lectinsseemed to bind a common protein band at approximately14,000 Da.

Antigen detection and IgG isotype binding

Selected serum samples with different levels of A. versicolor-specific IgG antibodies, as determined by ELISA, wereanalyzed by Western blotting. In general, both IgG1 andIgG2 antibodies bound to a short smear of separated extractclose to the application point (Fig. 2, lanes F–I), but IgG2

antibodies bound to a smear in broader MW range thanIgG1 antibodies did. Neither IgG1 nor IgG2 antibodiesbound significantly to any distinct bands of separated A.versicolor extract. Both isotypes of IgG antibodies showed anadditional binding to components that did not migratefrom the application point. The intensity of smears forboth isotypes was in accordance with the signal strengthsof specific IgG antibodies found in the different sera usingELISA.

Blotting analyses for all isotypes of IgG antibodies spe-cific to A. versicolor demonstrated a smear of IgG antibo-dies to components bigger than 40,000 Da. There werebindings to additional weak protein bands correspondingto lower molecular masses with some sera (data notshown). Incubation of the blot with Con A before incuba-tion with serum did not prevent the binding of serumantibodies to the mould components (data not shown).

205,000

116,000

97,000

80,00066,000

55,000

45,000

30,000

21,000

14,000

6,500

A B C D E MW std

Figure 1 Silver-stained SDS-PAGE gel loaded with 15 mg per lane of

Aspergillus versicolor (A), Penicillium chrysogenum (B), Cladosporium her-barum (C), Stachybotrys chartarum (D) and Fusarium oxysporum (E).

Molecular weight standard as indicated to the right. Upper arrow to the

left shows the point of application and lower arrow to the left, the

electrophoretic front.

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# 2005 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 62, 281–288

Distribution of IgG antibodies to A. versicolor among

Norwegian blood donors

By ELISA, we analyzed 996 Norwegian blood donors forA. versicolor-specific IgG antibodies. The distribution ofA. versicolor-specific IgG within this population appearedto be bimodal and did not follow a Gaussian distribution,even after transformation (Fig. 4). Most of the sera hadIgG values that were distributed between 0 and 600 AU/ml, forming a peak, with an additional small peak made ofvalues from 60 to 900 AU/ml. Approximately 5% of thesera had values between 900 and 4000 AU/ml. The two

205,000

116,000

97,000

80,000

66,000

55,000

45,000

30,000

21,000

14,000

6,500

A B C D E F G H I MW std

Figure 2 Silver-stained SDS-PAGE gel loaded

with 40 mg of Aspergillus versicolor extract per

lane (A), with complementary Western blot

showing binding of wheat germ agglutinin

(B), lentil lectin (C), concavalin A (D), pea-

nut lectin (E), serum with high (F) and low

(G) levels of Aspergillus-specific IgG1 antibo-

dies, serum with high (H) and low (I) levels of

Aspergillus-specific IgG2 antibodies. Mole-

cular weight standard as indicated to the right.

Upper arrow to the left shows the point of

application and lower arrow to the left, the

electrophoretic front. Bands on lectin blots

appear as light bands and bands on gel and

antibody blots as dark bands.

Table 1 Correlation analysis of mould-specific IgG antibodies to five differ-

ent moulds in 106 sera from healthy blood donors, analyzed using ELISA.

r¼ Spearman’s correlation coefficient

Groups compared r P-value

Aspergillus versicolor versus Penicillium chrysogenum 0.16 0.11

A. versicolor versus Cladosporium herbarum 0.20 0.04

A. versicolor versus Stachybotrys chartarum 0.29 0.003

A. versicolor versus Fusarium oxysporum 0.22 0.03

P. chrysogenum versus C. herbarum 0.59 <0.001

P. chrysogenum versus S. chartarum 0.46 <0.001

P. chrysogenum versus F. oxysporum 0.31 0.001

C. herbarum versus S. chartarum 0.62 <0.001

C. herbarum versus F. oxysporum 0.35 <0.001

S. chartarum versus F. oxysporum 0.18 0.06

F.v.S.c.C.h.P.c.A.v.

OD

val

ues

4

3

2

1

0

Figure 3 Comparison of levels of specific IgG antibodies to five different

moulds: Aspergillus versicolor (A.v.), Penicillium chrysogenum (P.c.),

Cladosporium herbarum (C.h.), Stachybotrys chartarum (S.c.) and

Fusarium oxysporum (F.v.), analyzed using ELISA. As the same amount

of coated extract was used, all sera were diluted 1:600, and all reactions

were stopped simultaneously, the assays used are directly comparable. The

results are given as optical density (OD). The box plots represent median,

25 and 75% percentiles, together with minimum and maximum values

(within 1.5 times of 50% middle box).

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# 2005 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 62, 281–288

main peaks appeared to represent two populations, andvalues from 0 to 600 AU/ml were therefore defined asnegative for A. versicolor-specific IgG antibodies and valuesabove 600 AU/ml were defined as positive. Followingextrapolation of the two peaks down to the baseline, 2%of the values were found in the overlapping area betweenthe two peaks. Of all negative values, 98.5% lay outsidethe positive peak. As much as 12.5% of the blood donorshad A. versicolor-specific antibody values above 600 AU/mland were defined as positive.

The ages of these 996 blood donors ranged from 20to 70 years, but most were between 25 and 58 years.Age did not correlate with the level of mould-specific IgG(r¼�0.14). The levels of A. versicolor-specific IgG weresimilar at different ages but were more variable among theyoungest blood donors (data not shown). All blood typeswere well represented, and blood type did not seem to haveany influence on the level of mould-specific IgG antibodies.

Female blood donors had significantly higher levels ofA. versicolor-specific IgG antibodies (median: 213.5 AU/

ml) than males (median: 143.5 AU/ml) (P< 0.001).According to our cut-off value, 15.9% of the women and10.1% of the men, were positive for IgG anti-A. versicolorantibodies. However, for any of the other four moulds,there was no significant difference between mould-specificIgG antibody levels in men and women (data not shown).

Discussion

Our study shows that exposure to mould is commonamong healthy adults living in the urban southeasternpart of Norway. One of eight had levels of A. versicolor-specific IgG antibodies above cut-off, without havingsymptoms or signs of disease revealed in the blood dona-tion routine questionnaire. This is the first study on mouldexposure in such a large healthy population. Even thoughthe extract contained no more proteins than the otherextracts, the level of IgG antibodies to A. versicolor wasfar higher than to the other moulds tested. This is inaccordance with Finnish studies [24, 25] and could reflect

Table 2 Relative binding capacity of lectins to the five different moulds, as analyzed using enzyme-linked immunosorbent assay. The binding is given

relative to the binding of concavalin A (Con A) to C. herbarum, which was strongest and defined as 1.0.

A. versicolor P. chrysogenum C. herbarum S. chartarum F. oxysporum

Con A 0.9 0.9 1.0 0.7 0.9

Wheat germ agglutinin 0.1 0 0 0.3 0.4

Lentil lectin 0 0 0 0 0

Peanut lectin 0 0 0 0 0

Mean specific IgG concentration (AU/ml)30002400180012006000

Num

ber

of in

divi

dual

s

200

150

100

50

0

Figure 4 Specific IgG antibodies to Aspergillusversicolor among 996 healthy Norwegian adults,

analyzed using ELISA. Individuals with differ-

ent levels of such antibodies distributed bimod-

ally in two main peaks, plus additional spread

high values. A cut-off between the two peaks, at

600 AU/ml, discriminates between individuals

with low exposure and those with considerable

exposure to A. versicolor.

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Aspergillus being more common or has more potent anti-gens than other moulds. Being a primary wall colonizerrequiring low water activity [13], Aspergillus is more ubi-quitous and one of the first moulds to arise duringincreased water availability at water-damaged sites.Aspergillus and Penicillium are found as the two mostfrequent moulds indoors in Scandinavia [9, 11]. Becauseof the induced strong immune reaction, further analyses ofA. versicolor were performed.

A bimodal distribution like the one presented here forspecific antibodies to A. versicolor is usually a result ofmixed subgroups with different means [26]. The mainpeak of serum with low IgG concentrations, and smallerpeak with high concentrations of IgG, plus additional evenhigher values, could reflect exposure from non or barelyexposed to severely exposed individuals, the latter probablydue to indoor mould contamination. As mould is naturallypresent in outdoor air with seasonal variations [12], onewould expect such a low exposure among the majority ofan adult population.

Our bimodal distribution is in contrast with those ofMakkonen [6], who found normal distributions after logtransformation, but of a far smaller population. In thepresent study, neither the specific IgG antibody levels toA. versicolor, P. chrysogenum, C. herbarum, S. chartarumnor F. oxysporum was normally distributed. Nor did wefind any similar bimodal distribution as found for anti-A. versicolor, probably due to large individual differences inthese smaller sample groups. With normal distribution, acut-off is usually made at mean� 2SD. In a bimodaldistribution like ours, it would be natural to set a cut-offbetween the two peaks. This cut-off, giving a positivepopulation of 12.5%, would be equivalent tomeanþ 0.73SD, which defines more people as positivescompared with mean� 2SD. In this case, the overlappingarea between the two peaks was small compared with thedefined exposed or unexposed part, and in the lack ofnormality in a population, we therefore suggestmean� 1SD as a more accurate measurement for exposurein a healthy population. In the present population, thisgives an estimated exposure of approximately 7% toC. herbarum, 15% to S. chartarum, 13% to P. chrysogenumand 17% to F. oxysporum.

Our findings of women having significantly moreAspergillus-specific antibodies than men is in accordancewith a Finnish study of nine indoor-relevant moulds,including A. versicolor [6]. Women also suffer more oftenfrom sick building syndrome than men and seem to reactmore extremely than men on similar environmental con-ditions [27]. The lack of correlation between age and levelsof mould-specific antibodies found in this study are inaccordance with several other studies among adults[6, 28], even though mould-specific IgG antibodies havebeen found to increase in relation to age for the first 6–7years of life and then level-off [25]. Age has previously

been found to be a significant risk factor for sick buildingsyndrome among adult men [27].

Smears of immunogenic antigens, as revealed in ourWestern blotting analyses of IgG1 and IgG2 antibodies,have previously been considered to be of carbohydratenature [29]. As both the length and intensity of smearsin Western blotting corresponded to the concentration ofIgG antibodies seen in ELISA, our analyses indicatedstrongly that the IgG response was directed mainly towardscarbohydrate antigens. This is in accordance with previousstudies, where carbohydrate moieties of S. chartarum com-ponents have been shown to possess antibody-bindingactivity [30], and antibodies to extracellular polysacchar-ides of moulds have been found in relatively high amountsin sera of healthy subjects [28].

By the use of lectins, Western blot binding of Con A,and to a certain degree wheat germ agglutinin, indicatedthe presence of carbohydrate chains containing mannose,glucose and to some degree N-acetyl glucosamin in themoulds. This was expected, as galactomannans have beenregarded as one of the most widely distributed polysac-charides among fungi [31]. Because our blotting analysesshowed that Con A bound other components of the extractthan did the IgG antibodies, and Con A did not seem toprevent serum antibodies from binding to A. versicolorcomponents, the IgG2-reactive antigens were probablyneither a-D-glucose nor a-D-mannose residues. This is incontrast with several studies showing Con A and IgGantibodies binding the same carbohydrate antigens of A.fumigatus [32], A. umbrosus [19] and Saccharopolysporarectivirgula [16 ]. Because over 75,000 species of fungiare known, and several polysaccharides often are associatedwith the cell wall of each species [31], this carbohydrateantigens could be species-dependent. Because of extra-cellular polysaccharide specificity, immunological cross-reactivity occurs only between some closely related genera[28]. Further studies are warranted to identify specificitiesof these antigens.

Acknowledgments

We thank Berit Stensby for technical assistance at extractcharacterization and Dr Jan Kolberg for kindly providinglectins with their complementary sugars and glycoproteins.This work was funded by Norwegian Foundation forHealth and Rehabilitation.

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