Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillus fumigatus in sera of patients with different lung diseases

Journal of Immunological Methods, 75 (1984) 117-128 Elsevier

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JIM03296

Radioallergosorbent Test (RAST) for Measurement of IgG Antibodies to Aspergillusfumigatus in Sera of Patients with Different Lung Diseases Mahmoud Dewair and Xaver Baur Department of Pneumology, Medical Clinic I, Klinikum Grosshadern, University of Munich, Marchioninistr. 15, D-8000 Munich 70, F.R.G. (Received 9 May 1984, accepted 23 August 1984)

A radioallergosorbent test (RAST) for measuring human anti-Aspergillus fumigatus (Af) antibodies of the IgG class is described. The use of 125I-labelled animal antibodies against human IgG is compared with the use of 12SI-labelled protein A. Under optimal conditions the radioactivity binding ratio between pooled patients' serum and pooled healthy persons' serum is 8-11.5. The immunoblotting technique was used to investigate so-called non-specific binding. The results obtained show that most if not all human sera contain anti-Af antibodies of the IgG type. The difference between pathological and normal immunological response to Af antigens seems to be in the antibody titres rather than in the presence or absence of antibodies to these antigens. Key words: I g G - R A S T - P A - R A S T - IgG antibody to Aspergillus fumigatus - immunoblotting

Introduction

The fungus Aspergillus fumigatus (Af) is known to be associated with several pulmonary diseases such as bronchial asthma (Henderson, 1968; Pepys, 1973), allergic bronchopulmonary aspergillosis (APBA) (Longbottom and Pepys, 1964), aspergilloma (McCarthy and Pepys, 1973) and farmer's lung (Roberts and Moore, 1977; Schatz et al., 1977). One important factor in diagnosis of these diseases is the quantitative measurement i n patients' sera of precipitating (IgG) antibodies to components of Af extracts (Wang et al., 1978). Enzyme-linked immunosorbent assays (ELISA) for measurement of anti-Af antibodies have been described (M~ntyj~rvi et al., 1980; Greenberg and Patterson, 1982; Sch6nheyder and Anderson, 1983). Alternatively the radioallergosorbent test (RAST) (Johansson et al., 1972), commonly used to measure IgE antibodies, may be adapted to measure antibodies of the IgG class. One attractive advantage of radioactively labelled rather than enzyme-labelled tracers is that the endproduct measured -- the radioactivity bound -- is more linearly related to the amounts of the radioactively labelled tracer 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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bound. Both 125-I-labelled animal immunoglobulins to human IgG ( I . a . IgG) (Shimizu et al., 1978) and lzSI-labelled protein A (IPA) (Delespesse et al., 1979; Hamilton et al., 1979) were used to determine antigen-specific IgG antibodies in human sera, both of these reagents being now commercially available. Both laboratory prepared and purchased IPA are much cheaper than I • a. IgG, while the use of IPA has been shown to make the assay more sensitive and specific than I • a • IgG (Hamilton et al., 1979). IPA may however be criticized because it does not bind to human IgG 3 (Van Loghem et al., 1982). We have used both I - a • IgG and IPA in a RAST adapted to measure human IgG antibodies specific to Af proteins and the results obtained with the 2 tracers were statistically compared. One important problem in such assays is the non-specific binding or 'noise' which may make it difficult to define the borderline between positive and negative serum samples. We have attempted to increase the information available about this non-specific binding by use of the immunoblotting technique. The results indicate that this non-specific binding is in fact mostly due to the presence in almost all serum samples, including those from healthy donors, of IgG-anti-Af antibodies. The use of this finding to locate the borderline between pathological and normal serum titres of IgG-anti-Af antibodies is discussed.

Materials and Methods

The mould mat of Aspergillus fumigatus (Af) culture, containing both mycelia and spores was a generous gift from Allergon (Engelholm). lzSI-labelled protein A (IPA) of specific activity about 9 /~Ci//~g and 125I-labelled, affinity-purified, goat immunoglobulin to human IgG (I. a. IgG) of specific activity about 5 /~Ci/#g were from New England Nuclear (Dreieich). Essentially gamma-globulin-free bovine serum albumin (BSA) was from Sigma (Munich), Tween 20 from Merck (Darmstadt), and horse serum (HS) from Serva (Heidelberg). Reagents and apparatus for polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose (NC) membranes were from Bio-Rad (Munich). RAST paper discs were cut from Munktell filter papers type 00 (Hamburg).

Human sera Seventy-two serum samples were obtained from patients suffering from asthma (A, n = 26), farmer's lung (F1, n = 24), allergic bronchopulmonary aspergillosis (ABPA, n = 7), aspergilloma (Asp, n = 5) and from cystic fibrosis and sarcoidosis (n = 10). Control serum samples were from 24 healthy persons free from respiratory symptoms and showing normal lung function parameters.

Preparation of Aspergillus fumigatus (Af) extract The extraction buffer consisted of 0.05 M sodium phosphate, pH 7.8, 0.1% sodium azide, 0.1% potassium cyanide, 0.15 M sodium chloride and 0.0025 M phenylmethane sulphonyl fluoride. One hundred grams of Af mould mat were homogenized in 1 litre of the extraction buffer using an Ultra-Turrax homogenizer

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(Ika-Werk, Breisgau) 5 times at 2 min intervals, keeping the mixture at 4°C. The mixture was then centrifuged for 1 h at 10,000 × g. The clear supernatant was dialyzed against extraction buffer, then against water. It was then 90% saturated with ammonium sulphate, stirred for 18 h and the precipitated Af protein was collected by centrifugation, dissolved in and dialyzed against water.

Binding of A f protein to paper discs Af proteins were bound to cyanogen bromide-activated paper discs as described by Ceska et al. (1972) 2.5, 5, 7.5 or 10/,g Af protein to each paper disc. Measurement of IgG antibodies to A f protein 125I-labelled protein A-RAST (PA RAST) and 125I-labelled goat immunoglobulin to human IgG-RAST (IGG RAST) were used. In order to find the optimal conditions for these RASTs several factors were investigated: (a) the amount of Af protein per paper disc introduced into the coupling buffer; (b) the kind and amounts of ballast proteins and Tween 20 included in dilution and wash buffers; (c) the dilution factor of human sera; (d) the incubation time with human sera and with the radioactive tracers and the amounts of the latter. In these investigations conditions were sought which gave the greatest difference between pooled patients' serum and the pooled reference control serum (RCS). The pooled patients' serum was prepared by adding together equal amounts of sera taken from aspergilloma and aspergillosis patients, whereas RCS was pooled from 24 sera obtained from healthy persons. Based on results of these preliminary investigations the optimal conditions for carrying out I G G and PA RASTs were found to be as follows. IGG R A S T Paper discs were prepared by adding 5/xg Af protein per CNBr-activated paper disc in the coupling buffer. Before use, the paper discs were rinsed in freshly prepared PBST-HS (50 mM N a 2 H P O a / N a H 2 P O 4, 0.15 M NaC1, 0.04% sodium azide, 1% Tween 20, 2% horse serum, pH 7.5). Each disc was incubated for 18 h at room temperature in 100 ~1 of human serum diluted 1:400 in PBST-HS. After incubation the disc was washed 3 times, each time by incubation for 45 min in 2 ml of PBST (50 mM Na2HPO4/NaHzPO4, 0.15 M NaC1, 0.04% sodium azide, 1% Tween 20, pH 7.5). The paper disc was then incubated for 2 h at room temperature in 50/,1 of 125I-labelled goat immunoglobulin to human IgG (I- a- IgG), each disc receiving about 30,000 cpm, corresponding to about 5 ng I. a. IgG. After incubation, the radioactive solution was aspirated and the paper disc washed as above. The radioactivity bound was counted with an efficiency of 70-80%. PA-RAST The same Af paper discs were used as for I G G RAST. Before use, the discs were rinsed in freshly prepared PBST-BSA-1 (50 mM N a 2 H P O 4 / N a H 2 P O 4, 0.15 M

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NaC1, 0.04% sodium azide, 1% Tween 20, 1% BSA (w/v), p H 7.5). Each paper disc was incubated 18 h at room temperature in 100/zl human serum diluted 1 : 1600 in PBST-BSA-1. After this first incubation the disc was washed by 3 incubations for 45 min each in 2 ml PBST-BSA-2 (the same as PBST-BSA-1 except that the concentration of BSA was reduced to 0.25% (w/v)). Each disc was then incubated at room temperature for 2 h in 100/~1 of 125I-labelled protein A (IPA) diluted in PBST-BSA-1. Each disc received 75,000-100,000 cpm, corresponding to 7-10 ng IPA. The radioactive solution was aspirated and the discs were washed as above, and finally the bound radioactivity was counted.

IGG R A S T / PA-RAST unit One such unit is taken to be equal to the amount of radioactivity b o u n d / p a p e r disc after incubation with RCS and I • a. IgG or RCS and IPA. The non-specifically bound radioactivity measured when no serum was included in the first incubation was subtracted. Thus the RAST result for each serum tested was evaluated relative to that for the reference control serum.

Polyacrylamide gel electrophoresis Slab gels made of 7-20% exponential polyacrylamide concentration gradient were used. The electrophoresis was run in the discontinuous buffer described by Laemmli (1970), in the presence of 0.1% sodium dodecyl sulphate.

Immunoblotting Immunoblotting was carried out as described by Towbin et al. (1979) except that Tween 20 was included in the blocking, washing and diluting buffers (Batteiger et al., 1982). Briefly, Af protein at a concentration of 3 m g / m l in sample buffer was applied to the whole surface of the slab gel with a 1-well comb. After electrophoresis, the resolved polypeptides were electrophoretically transferred to a nitrocellulose (NC) sheet, which was then incubated for 1 h at 40°C in PBS containing 0.1% Tween 20. Longitudinal strips, 0.5 cm wide were cut from the NC sheet. Each strip was incubated for 18 h in 4 ml containing 50 /~1 of human serum. The strip was then washed several times and incubated again for 1 h in 4 ml containing about 30 ng of I . a - I g G (about 150,000 cpm). Finally N C strips were washed, air-dried and autoradiographed. Protein concentrations were determined by the method of Lowry (1951) with the protocol described by Peterson (1977).

Counter current immunoelectrophoresis This was carried out as described by Gocke and Calderon (1970), with different dilutions of ABPA sera and different amounts of Af proteins. The plates were stained with Coomassie brilliant blue R 250.

Results

In order to include as many as possible Aspergillus fumigatus antigens, whole organisms were extracted. The Af protein obtained could be resolved into at least 12

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polypeptides of molecular weights ranging from 14 to more than 100 kDa (Fig. 1). The average amount of Af protein bound per paper disc was found to be 670 ng. These were discs prepared by addition of 5 /~g Af protein/disc in the coupling buffer. Higher input of Af protein (10/~g/disc) increased the amount b o u n d / d i s c by about 10-15% but had no advantage over 5/~g/disc. Under the conditions described in Methods, signalto noise ratios of 8 and 11.5 were achieved for I G G and PA RASTs respectively; these were the ratios of RAST values for pooled patients' serum and for pooled reference control serum. Some individual patients' sera gave higher ratios of 9-11 and 11-13 in I G G and PA RASTs respectively (Fig. 2 and Fig. 3). The amount of radioactivity bound by RCS was always comparable with the mean amount bound by the 24 control serum samples. This mean plus 2 SD was always less than twice the RCS signal (2 × RCS). For routine analyses RCS is always tested in parallel and the amount of radioactivity bound by RCS taken as one

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arbitrary R A S T unit (RU). F r o m Figs. 2 a n d 3 it can be seen that the majority of patients' sera (which were expected to have significant I g G - a n t i - A f a n t i b o d y titres) gave R A S T values greater than 2 R U ; these were sera from farmer's lung, ABPA, a n d aspergilloma patients. R A S T values for most asthma patients a n d for all control healthy donors lay below the 2 R U level. We therefore used the 2 R U threshold to distinguish between R A S T positive patients' sera on one h a n d a n d R A S T - n e g a t i v e patients' or control sera on the other. Both I G G a n d PA R A S T are fairly reproducible. Interassay coefficients of variation were 17% a n d 15% (n = 6) for I G G R A S T a n d PA R A S T respectively. Intra-assay coefficients of variation were less than 10% f o r - b o t h R A S T s (n = 7). S t u d e n t ' s t-test showed excellent correlation between I G G and PA R A S T results (Fig. 4). Moreover, with the 2 R U threshold almost all the I G G R A S T positive sera were also PA R A S T positive. Both I G G a n d PA R A S T are more sensitive than the s e m i q u a n t i t a t i v e c o u n t e r current i m m u n o e l e c t r o p h o r e s i s test, in which only 5 of the 7 A B P A sera gave precipitin lines. Some radioactivity is always b o u n d by paper discs i n c u b a t e d in control sera or in 15-

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124 p a t i e n t s ' sera not expected to have IgG a n t i - A f antibodies. This could be e x p l a i n e d either by non-specific b i n d i n g or by the presence in all h u m a n sera tested of varying a m o u n t s of a n t i - A f antibodies. In o r d e r to d e c i d e between these 2 possibilities we investigated 12 c o n t r o l a n d 15 p a t i e n t s ' sera b y i m m u n o b l o t t i n g . This technique s h o u l d be m o r e sensitive and specific since it allows detection of a n t i b o d i e s to i n d i v i d u a l p o l y p e p t i d e s rather than to the whole mixture of A f proteins, as is the case in R A S T . T h e sera tested by i m m u n o b l o t t i n g were selected from the whole range of I G G R A S T values, at intervals small (0.1 0.3 R U ) at the m i n i m u m end of the range a n d increasing t o w a r d s the m a x i m u m end. As can Joe seen in Fig. 5 for the c o n t r o l sera a n d Fig. 6 for the p a t i e n t s ' sera, all the sera p r o m o t e d b i n d i n g of the r a d i o a c t i v e tracer to discrete p o l y p e p t i d e b a n d s transferred from the gel to the N C m e m b r a n e . N o n - s p e c i f i c b i n d i n g would have been expected to give a c o n t i n u o u s s m e a r along the whole length of the nitrocellulose strip. The i m m u n o b l o t s showed an interesting qualitative difference between R A S T positive a n d R A S T negative samples. Sera with I G G R A S T values of 3 RU or more p r o d u c e d b i n d i n g of r a d i o a c t i v e

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125

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Fig. 6. Results of immunoblotting analysis of 15 patients' sera according to IGG RAST values. tracer to almost all the resolvable Af polypeptides, whereas those with I G G RAST values less than 3 R U appeared to contain I g G antibodies to fewer Af polypeptides. It is noteworthy that the number of radioactive bands in the immunoblots produced by some patients' sera was greater than the number of Af polypeptides seen on the original acrylamide gel. This may be due to reactions with strongly antigenic Af polypeptides present in amounts too small to be detected by the protein dye.

Discussion

The involvement of the Aspergillus fungus in several lung diseases makes it desirable to have a sensitive and reproducible method for quantitative measurement of I g G anti-Af antibodies in human sera. Qualitative assays such as double immunodiffusion are not always capable of distinguishing between normal and pathological responses to such ubiquitous antigens, owing to the existence in most human sera of I g G anti-Af antibodies. The difference between pathological and normal states is in the titres of these antibodies, i.e., in the magnitude of the immune response, lzsI-labelled protein A (IPA) and lzsI-labelled immunoglobulin to human I g G ( I . a . IgG) have been used in RAST or RAST-like assays for the measurement of specific IgG in human sera (Shimizu et al., 1978; Hamilton et al., 1979; Delespesse et al., 1979). Published RAST protocols for measuring anti-Af antibody titres give rather low signal to noise ratios. Optimal RAST conditions seem to vary from one antigen to another and complex mixtures of antigens may need special

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optimization of conditions. We have, therefore, investigated the different factors which affect measurement of anti-Af antibodies by RAST. It was necessary to test different amounts of Af protein for the preparation of paper disc-immobilized antigens. Also, although horse serum (Kemeny et al., 1980) and bovine serum albumin were found adequate as ballast proteins for I G G and PA RASTs, optimal concentrations of these ballast components in different wash and dilution buffers had to be found by systematic variation. Other factors investigated were the optimal periods of first and second incubation of Af paper discs with human serum and radioactive tracer, the optimal amounts of radioactive tracers and their ages, the effect of the detergent Tween 20 and its optimal concentration in wash and dilution buffers and the dilution factor of human sera. The most suitable combinations of RAST conditions were those described in Methods. Under these conditions RAST value ratios of pooled patients' serum to pooled control serum (RCS) of 8 and 11.5 were achieved by I G G and PA RAST respectively. For some individual patients' sera these ratios were as high as 9 11 and 11-13. Recently Djurup et al. (1983) published a solid-phase radioimmunoassay for human IgG antibodies to pollen antigens which used antigens bound to nylon balls. In this assay a ratio of 8-12 between radioactivity bound by high-titre serum and that bound by normal human sera was achieved. For routine analyses we use IPA, i.e., PA RAST, firstly because IPA is much cheaper than I - a- IgG, and secondly PA RAST is rather more sensitive than I G G RAST. A third reason is that IPA may be used over a longer period than I • a. IgG; one batch of IPA may be kept for up to 3 - 4 months and can still be used without significant loss of sensitivity or significant increase in background binding. The sensitivity and specificity obtainable with I • a- I g G decline significantly after 5 - 6 weeks of storage, perhaps because the biological activity of I - a . IgG is susceptible to radiological damage. The fact that PA has only very little affinity to IgG 3 (Van Loghem et al., 1982) led us to compare PA RAST with I G G RAST using a relatively large number of serum samples. This comparison gave a highly significant correlation between the 2 RASTs, results for almost all serum samples agreeing between the 2 tests. Others have used high-titre patients' sera as references for evaluation of RAST or RAST-like assay results (Shimizu et al., 1978; Delespesse et al., 1979; Djurup et al., 1983). We chose to use a pooled reference control serum (RCS) rather than a high-titre patient serum for the following reasons: (a) the RCS represents the average normal immunological response to Af antigens and the RAST value for a patient's serum can thus be directly compared with this average normal value; (b) serum samples from healthy donors are available in greater quantities than high-titre patients' sera, while the latter are subject to ageing and vary considerably in titre; (c) differences between strongly positive sera used by different investigators are likely to be greater than differences between sets of serum samples taken from asymptomatic donors. However, if a highly positive serum is preferred as a reference, the same technique may be followed and the RAST values obtained compared with the reference. In any

127 case, parallel measurement of RAST values for a group of asymptomatic controls or their pooled sera, is necessary. In our hands the RCS gave RAST values comparable with or slightly higher than the mean RAST value of the 24 separate serum samples which it contained. We use the RCS RAST value as an arbitrary unit, 2 such units being more than the mean plus 2 SD of RAST values given by the 24 control sera, so that a RAST value of 2 units is significantly (95% confidence) higher and may be used to distinguish pathological from normal anti-Af titres. In the PA RAST only 1 of the 24 farmer's lung, 1 of the 7 ABPA, and none of the 5 aspergilloma sera gave values less than 2 RUs. All serum samples, including healthy donors sera, tested by PA or I G G RAST gave binding of significant amounts of radioactivity to the RAST paper discs. Immunoblotting showed that all the sera tested contain IgG antibodies to discrete Af polypeptides, and that these contribute at least in part to the so-called non-specific binding in this and perhaps in similar assays is very likely. However, immunoblots of control and low titres sera differed significantly from those of high titre, mostly patients' sera. Low titre sera bound to only few, often different, Af polypeptides. On the other hand high titre sera recognized all electrophoretically resolvable Af polypeptides. The binding of radioactive tracer was specific, since it corresponded to the positions of Af proteins. The continuous dark smear in the upper region of 1 immunoblot (serum 8, Fig. 5) is due to binding by this serum to material in Af extract which diffuses into the upper region of polyacrylamide gel upon electrophoresis. Even in this case binding is most likely specific since the lower two thirds of the immunoblot remained clear. The total IgG concentration does not seem to affect significantly the specificity of the assay. The mean IgG concentration of the 24 control sera used here was not significantly different from that of the patients' sera but all control sera gave RAST values far below those of farmer's lung, ABPA and aspergilloma sera.

Acknowledgement This work was supported by the Deutsche Forschungsgemeinschaft. The skilful technical assistance of Ms. Barbara Jarosch is acknowledged.

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128 Johansson, S.G.O., H.H. Bennich and T. Berg, 1972, Prog. Clin. Immunol. 1,157. Kemeny, D.M., M.H. Lessof and A.K. Trull, 1980, Clin. Allergy 10, 413. Laemmli, U.K., 1970, Nature (London) 227, 680. Longbottom, J.L. and J. Pepys, 1964, J. Pathol. Bacteriol. 88, 141. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. McCarthy, D.S. and J. Pepys, 1973, Clin. Allergy 3, 57. M~ntyj~vi, R.A., P. Jousilahti and M.L. Katila, 1980, Clin. Allergy 10, 187. Pepys, J., 1973, Clin. Allergy 3, 1. Peterson, G.L., 1977, Anal. Biochem. 83, 346. Roberts, R.C. and V.L. Moore, 1977, Am. Rev. Resp. Dis. 116, 1975. Schatz, M., R. Peterson and J. Fink, 1977, J. Allergy Clin. lmmunol. 60, 27. Sch6nheyder, H. and P. Anderson, 1983, Int. Arch. Allergy Appl. Immunol. 70, 108. Shimizu, M., K. Wicher, R.E. Reisman and C.E. Arbesman, 1978, J. Immunol. Methods 19, 317. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Van Loghem, E., B. Frangione, B. Recht and E.C. Franklin, 1982, Scand. J. Immunol. 15, 275. Wang, J., R. Patterson, M. Rosenberg, M. Roberts and B. Cooper, 1978, Am. Rev. Resp. Dis. 117, 917.