Clin. exp. Immunol. (1975) 22, 35-46.
INFLUENCE OF IgG ANTIBODY AND GLYCOPEPTIDE ALLERGENS ON THE CORRELATION BETWEEN THE RADIOALLERGOSORBENT TEST (RAST) AND SKIN TESTING OR BRONCHIAL CHALLENGE WITH ALTERNARIA N. R. LYNCH, P. DUNAND, R. W. NEWCOMB,* H. CHAI AND J. BIGLEY (CARIH) The National Asthma Center, Denver, Colorado, U.S.A. (Received 2 January 1975)
SUMMARY
The radioallergosorbent test (RAST) for alternaria was compared to skin tests and bronchial challenges in children suffering from chronic intractable asthma. In contrast to when such children were tested with a timothy grass pollen extract, the bronchial challenge and skin test results against alternaria did not correlate significantly. When alternaria allergens were coupled to cyanogen bromide-activated microcrystalline cellulose, the RAST correlated with the results of skin testing but not bronchial challenge. It was demonstrated by column immunabsorption that some allergic sera contained sufficient IgG antibody against alternaria to competitively inhibit the RAST. When Sepharose 2B was substituted for cellulose as the insoluble support, the inhibition by IgG antibody was largely overcome and then the RAST correlated with both skin test and bronchial challenge results. Glycopeptides contribute significantly to the allergenicity of alternaria, and when these materials were coupled to a Sepharose 2B conjugate by mild oxidation, the RAST correlated with bronchial challenge, but not skin test, results. It was concluded that in this group of steroid-dependent asthmatic children, the correlation of the RAST with the in vivo challenges was strongly influenced by the presence of IgG antibody in the allergic sera and the chemical nature of the mould allergens investigated.
INTRODUCTION The radioallergosorbent test (RAST), originally developed by Wide, Bennich & Johansson (1967), has been evaluated as a diagnostic tool in the investigation of allergic sensitivity to *
Present address: La Rabida Children's Hospital, Chicago, Illinois, U.S.A. Correspondence: Dr N. R. Lynch, Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, B.P. 8, 94 Villejuif, France.
35
36
N. R. Lynch et al.
allergens from a wide variety of biological origins (Wide, 1973). An incomplete list of the allergens that have been incorporated into this test includes those derived from grass, weed or tree pollens (Lichtenstein et al., 1973; Berg & Johansson, 1974), proteins of insect and animal origin (Sarsfield & Gowland, 1973; Ahlstedt et al., 1974), moulds (Yunginger, Roberts & Gleich, 1974; Hoffman & Haddad, 1974a) and foods (Hoffman & Haddad, 1974b). In general terms, it can be concluded from these studies that, under optimal circumstances, the results of the RAST compare well with more traditional methods of allergic diagnosis, such as skin testing (Hoffman & Haddad, 1974a; Ahlstedt et al., 1974; Berg & Johansson, 1974). The best correlations were found, however, when the allergic patients were strongly sensitive to the allergen being tested; the RAST results in patients whose clinical history or in vivo sensitivity was not definitive were also equivocal (Berg, Bennich & Johansson, 1971; Lichtenstein et al., 1973; Norman, Lichtenstein & Ishizaka, 1973). In addition, the RAST appears to be most reliable when well characterized, or even partially purified, allergen extracts are used (Norman et al., 1973; Hoffman & Haddad, 1974a). The present investigation was undertaken to compare the results of the RAST and skin or bronchial challenges with alternaria in a group of severely asthmatic children. The group of patients examined was drawn from the long-term residents at the Children's Asthma Research Institute and Hospital, Denver, Colorado. These children all suffered chronic intractable asthma and were dependent upon intensive steroid and bronchodilator therapy (Chai & Newcomb, 1973). It was of diagnostic interest to determine the allergic sensitivity of these children to alternaria, but, unfortunately, routine skin and bronchial challenges with extracts of this mould resulted in conflicting and unreliable results. It was hoped that the RAST could supplement, or even replace, these inconvenient and potentially dangerous in vivo challenges. The results of the RAST, and various modifications of this, were therefore compared to those of skin and bronchial challenge in this group of patients, whose allergic sensitivity was not only poorly defined, but who were also being tested with a mould extract that contained a complex mixture of protein, glycoprotein and glycopeptide allergens (Bonilla-Soto, Rose & Arbesman, 1961; Berrens, 1971). MATERIALS AND METHODS Preparation of allergen extracts Twenty grams of dried alternaria (Hollister-Steir Laboratories) was extracted for 14 hr at 40C with 100 ml of 0'1 M Tris (hydroxymethyl) aminoethane (Tris), adjusted to pH 8 0 by the addition of 1 M HCl. After removal of the solid residue by centrifugation, ammonium sulphate was added to a level of 85% saturation. The material precipitated by this procedure was collected by centrifugation and dissolved in 20 ml of 0-1 M Tris-HCl. This preparation was chromatographed on a Sephadex G-25 column (2-5 x 100 cm) in 0-1 M Tris-HCl. The protein peak eluting at the void volume was dialysed against 0 3 M Tris-HCl and applied to a DEAE-cellulose column (1 x 20 cm), equilibrated with the same buffer. The material not bound to this column was concentrated by dialysis (against 0-1 M Tris-HCl) under reduced pressure. This preparation was then applied to a Sephadex G-150 column (2-5 x 100 cm) which had been calibrated for molecular weight determination (Andrews, 1964). The components eluting at volumes corresponding to the molecular weight range of 20-100,000 Daltons were collected. This preparation was effective in eliciting passive cutaneous anaphylactic reactions in sensitized monkey skin at a concentration of 1 ug N/ml.
Factors influencing alternaria RAST
37
A glycopeptide preparation was also obtained from the alternaria extract. The supernatant remaining after precipitation at 85%4 saturation with ammonium sulphate was dialysed against distilled water, then extracted by sequential treatment with hot phenol and alcohol (by the method of Bonilla-Soto, Rose & Arbesman, 1961). Although the yield of this extraction procedure was low (0 1 %), the resulting preparation, which contained 3-5 0 N, was effective in the elicitation of monkey passive cutaneous anaphylactic reactions at carbohydrate concentrations equivalent to 15 jg/ml of hexose. RAST procedure
Microcrystalline cellulose (Avicell) or Sepharose 2B (Pharmacia) were activated with cyanogen bromide by the technique of Yunginger and Gleich (1972). The partially purified alternaria extract was coupled to these supports and the conjugates were resuspended to 1 mg/ml in a 0 1 M Tris-HCl buffer, pH 7-4, which contained 1% 'Tween 20' and 100 normal rabbit serum (RAST buffer). The glycopeptide fraction obtained from alternaria was coupled to a Sepharose 2B-human serum albumin conjugate by modifications of the periodate oxidation methods of Hiramoto et al. (1972) and Sanderson & Wilson (1971). The glycopeptide was subjected to limited oxidation by the addition of 5 x 10-5 M NaIO4 in a 0 01 M acetate buffer, pH 6-3. After 30 min, this preparation was added to the Sepharose 2B-human serum albumin conjugate and incubated for 60 min. This conjugate was then washed by centrifugation and resuspended to 1 mg/ml in RAST buffer. The RAST was performed as originally described by Wide et al. (1967). Fifty microlitres of sera from the asthmatic patients were incubated with 0 5 mg of the allergen-particle conjugates for 14 hr at 40C. The complexes were then washed three times with RAST buffer and incubated for 14 hr with 100 ng N of '25I-labelled (4 /uCi/ug N) anti-IgE antibody. The anti-IgE antibody was isolated from a rabbit antiserum by affinity chromatography on a Sephadex-myeloma IgE column (Lichtenstein et al., 1973) and radiolabelled by the chloramine T method of McConahey & Dixon (1966). After washing, the radioactivity (ct/min) of each complex was measured in a y counter and transformed into a percentage by comparison with an arbitrarily chosen reference allergic serum (Johansson et al., 1971). Immunoabsorption Partially purified IgE and IgG preparations were obtained from the serum of an alternariasensitive asthmatic patient (M.W.) by column immunabsorption, using the technique of Mannik & Stage (1971). Four millilitres of packed Sepharose 4B (Pharmacia) was activated by treatment with CNBr and 20 mg of IgG (the 0 005 M sodium phosphate eluate from DEAE-cellulose) from a monospecific anti-IgE antiserum was coupled to this. An anti-IgG immunabsorbent column was also prepared by coupling 200 mg of the IgG from a monospecific anti-IgG antiserum to 20 ml of Sepharose 4B. The immunabsorbent columns were prepared in borate-buffered saline and, after dialysis against this buffer, 0'5 ml of M.W. serum was applied to the anti-IgE column. The effluent from this column was then applied to the anti-IgG column. After washing, both columns were eluted with 3 M NaSCN. The IgE and IgG preparations that were eluted from these columns by NaSCN were dialysed and concentrated until the concentration of the isolated immunoglobulin was twice its original serum level. More than 90°Y. of the IgE and IgG in the allergic serum was removed by this procedure, although two passages through the anti-IgG column were required to remove all of the IgG from the serum.
38
N. R. Lynch et al.
Immune precipitation The capacity of antibodies in the sera of alternaria-sensitive individuals to precipitate alternaria antigens was examined by Ouchterlony immunodiffusion and quantitative precipitation. Double immunodiffusion was performed in 1.500 agar, prepared in 0-1 M barbital buffer, pH 8-3. Diffusion was allowed to proceed for 48 hr, then the gels were washed, dried and stained with amido black 10B. Quantitative precipitation assays were performed as described by Kabat & Mayer (1961), the alternaria extract being radiolabelled with 1251 to provide an approximate estimate of the amount of antigen in the immune precipitates.
Monkey passive cutaneous anaphylaxis Rhesus monkeys were anaesthetized with 'Sernylan' (Bio-Ceutic Laboratories) and serial dilutions of sera from alternaria-sensitive asthmatic patients were injected intradermally. After 24 hr, 3 ml of 1% Evans blue dye was injected intravenously and both normal and sensitized skin sites were challenged by the intradermal injection of serial dilutions of alternaria extracts. Oedematous reactions at the sensitized skin sites larger than 5 mm diameter were considered positive. Patient population The children described in this report were between 9 and 16 yr of age and were all longterm patients at the Children's Asthma Research Institute and Hospital, Denver, Colorado. The blood collections, skin tests and bronchial challenges performed in this study were part of the established diagnostic protocol of the Institute. All of the patients suffered intractable asthma and were maintained on combined steroid and bronchodilator therapy. The dose of corticosteroid varied widely between individuals, but was in the vicinity of 0 5 mg/kg Serum methyl xanthine levels of 15 jg/ml were maintained as best possible and aerosolized fl-adrenergic agonists were administered to counter acute episodes of wheezing. Many of the patients inhaled 20 mg of disodium cromoglycate q.i.d. None of the patients examined had received immunotherapy with alternaria. The basis for the selection of patients was simply the first seven steroid-dependent, non-hyposensitized patients of each sex to be tested against alternaria for diagnostic purposes over a 20-week period.
Skin test procedure No medications were administered to the children for at least 6, and up to 18, hr before skin testing. Small quantities of a 1:20 w/v alternaria extract (Greer Laboratories) were introduced into the skin of the asthmatic patients by use of a spring-loaded skin puncture device ('Sterneedle', Ormont Drug and Chemical Company). The convenience and reproducibility of this technique was considerably greater than either prick testing or intradermal injection. The perpendicular diameters of the wheal produced in sensitive patients were measured 20 min after injection. As a control test for the skin sensitivity to histamine, 5 pg of this amine was also injected intradermally at the time of the skin test. Bronchial challenge procedure Bronchial challenges against alternaria were performed as described by Tuchinda & Chai (1973), and a brief description of the technique will be presented here. No medications were administered to the children for at least 6 hr before the bronchial challenge. All of the children tested were known to be sensitive to the inhalation of methacholine ('Mecholyl',
Factors influencing alternaria RAST
39
Merck and Company). Thus, prior to the actual challenge with alternaria, control inhalations of methacholine were performed. Increasing amounts of methacholine (up to 2 5 mg) were inhaled until a 20% drop in FEV1 was recorded. After the pulmonary function of the children had returned to baseline (assisted by a saline IPPB if necessary) steadily increasing amounts of alternaria extract were administered by inhalation. Doses of alternaria extract ranging up to 5000 PNU were administered, until a 20% decrease in FEV1 was recorded. RESULTS Evaluation of the RAST The RAST was evaluated by comparison with skin testing and bronchial challenge in fourteen steroid-dependent asthmatic children (9-16 years). Blood was collected from these patients immediately prior to skin testing and bronchial challenge, and the RAST was performed using a partially purified alternaria extract, coupled to microcrystalline cellulose by cyanogen bromide activation. The RAST values (percentage of reference serum) were compared to the results of skin testing and bronchial challenge in two ways. First, the skin test and bronchial challenge results were expressed semiquantitatively and the correlation with RAST values expressed as the Pearson product moment correlation coefficient (r). The amount of alternaria extract (PNU) required to elicit a 20% drop in FEV1 in bronchial challenge and the product of perpendicular wheal diameters in skin tests were taken as semiquantitative estimates of bronchial and skin reactivity to alternaria. As there is an inverse relationship between
*l A
100 _ A
A
A^
80 80
-
0
60 b
0
0
cr_
40
A
t0 20 _ A
0
20
I 40
I 60
I 80
I 100
Skin test score (mm2)
FIG. 1. RAST scores (percentage of reference serum) and skin test wheal sizes (product of perpendicular wheal diameters) for fourteen asthmatic children and four normal children (o) are presented. The RAST was performed with either cellulose-alternaria (e), Sepharose 2Balternaria (a) or Sepharose-glycopeptide (A) conjugates. Skin test scores greater than 25 were considered positive.
N. R. Lynch et al.
40 100
A
A 80 _
[t
0
A
o 00
0
60
0
I-~ ~~~~N 40
A
20
0
1
2
3
4
5
Bronchial challenge score (x 193 PNU) FIG. 2. RAST scores and bronchial challenge scores (number of PNU inhaled to elicit at 20% decrease in FEV1) for fourteen asthmatic children are presented. The RAST was performed with either cellulose-alternaria (o), Sepharose 2B-alternaria (r) or Sepharose-glycopeptide (A) conjugates. Bronchial challenges were considered negative if more than 1500 PNU were inhaled without inducing a 20% drop in FEV1.
bronchial sensitivity and PNU inhaled, the Pearson r coefficient assumed a negative character in comparisons involving this parameter. The second approach to the comparison of RAST values with skin test and bronchial challenge results was to classify the in vivo challenges as either positive or negative and compare the mean RAST values in these two categories. The cut-off values used to define positive and negative responders were a 5 x 5 mm wheal size in skin tests and the inhalation of 1500 PNU in bronchial challenge. Although these values are somewhat arbitrary, they have proven useful in routine testing. Also, the correlations between the RAST and the in vivo tests derived by the first and second methods of comparison were essentially similar, thus supporting the use of these cut-off values. Significant correlation (r = 054, P0 05), or skin test scores (r = 0 14, P>0 05). Of the fourteen asthmatic children tested, four were classified as being positive by both skin test and bronchial challenge, five were negative by both tests, and five were reactive to only one of these challenges. The RAST levels in the sera of the asthmatic individuals who were either positive or negative to skin test or bronchial challenge were also compared (Student's t-test). Significant differences were found when the patients were divided on the basis of their skin (P< 0-05) but not bronchial (P> 0-05) reactivity. The RAST levels in the sera of the patients who were responsive to only one of the in vivo tests were not significantly higher than in patients who were reactive to neither. However, the RAST levels in the latter group were significantly higher (P< 0 0005) than that determined in sera obtained from four non-asthmatic children (Fig. 1).
Factors influencing alternaria RAST
41
To provide a comparison with these results, the responsiveness of a group ofeleven patients against timothy grass pollen extract was examined. The drug therapy received by these patients and the selection procedure employed was essentially the same as for the patients who were tested for their sensitivity to alternaria. However, in these children, and with this grass pollen extract, good correlations were found between skin testing and bronchial challenge (r = -0-89, P
INFLUENCE OF IgG ANTIBODY AND GLYCOPEPTIDE ALLERGENS ON THE CORRELATION BETWEEN THE RADIOALLERGOSORBENT TEST (RAST) AND SKIN TESTING OR BRONCHIAL CHALLENGE WITH ALTERNARIA N. R. LYNCH, P. DUNAND, R. W. NEWCOMB,* H. CHAI AND J. BIGLEY (CARIH) The National Asthma Center, Denver, Colorado, U.S.A. (Received 2 January 1975)
SUMMARY
The radioallergosorbent test (RAST) for alternaria was compared to skin tests and bronchial challenges in children suffering from chronic intractable asthma. In contrast to when such children were tested with a timothy grass pollen extract, the bronchial challenge and skin test results against alternaria did not correlate significantly. When alternaria allergens were coupled to cyanogen bromide-activated microcrystalline cellulose, the RAST correlated with the results of skin testing but not bronchial challenge. It was demonstrated by column immunabsorption that some allergic sera contained sufficient IgG antibody against alternaria to competitively inhibit the RAST. When Sepharose 2B was substituted for cellulose as the insoluble support, the inhibition by IgG antibody was largely overcome and then the RAST correlated with both skin test and bronchial challenge results. Glycopeptides contribute significantly to the allergenicity of alternaria, and when these materials were coupled to a Sepharose 2B conjugate by mild oxidation, the RAST correlated with bronchial challenge, but not skin test, results. It was concluded that in this group of steroid-dependent asthmatic children, the correlation of the RAST with the in vivo challenges was strongly influenced by the presence of IgG antibody in the allergic sera and the chemical nature of the mould allergens investigated.
INTRODUCTION The radioallergosorbent test (RAST), originally developed by Wide, Bennich & Johansson (1967), has been evaluated as a diagnostic tool in the investigation of allergic sensitivity to *
Present address: La Rabida Children's Hospital, Chicago, Illinois, U.S.A. Correspondence: Dr N. R. Lynch, Laboratoire d'Immunopathologie, Institut de Recherches Scientifiques sur le Cancer, B.P. 8, 94 Villejuif, France.
35
36
N. R. Lynch et al.
allergens from a wide variety of biological origins (Wide, 1973). An incomplete list of the allergens that have been incorporated into this test includes those derived from grass, weed or tree pollens (Lichtenstein et al., 1973; Berg & Johansson, 1974), proteins of insect and animal origin (Sarsfield & Gowland, 1973; Ahlstedt et al., 1974), moulds (Yunginger, Roberts & Gleich, 1974; Hoffman & Haddad, 1974a) and foods (Hoffman & Haddad, 1974b). In general terms, it can be concluded from these studies that, under optimal circumstances, the results of the RAST compare well with more traditional methods of allergic diagnosis, such as skin testing (Hoffman & Haddad, 1974a; Ahlstedt et al., 1974; Berg & Johansson, 1974). The best correlations were found, however, when the allergic patients were strongly sensitive to the allergen being tested; the RAST results in patients whose clinical history or in vivo sensitivity was not definitive were also equivocal (Berg, Bennich & Johansson, 1971; Lichtenstein et al., 1973; Norman, Lichtenstein & Ishizaka, 1973). In addition, the RAST appears to be most reliable when well characterized, or even partially purified, allergen extracts are used (Norman et al., 1973; Hoffman & Haddad, 1974a). The present investigation was undertaken to compare the results of the RAST and skin or bronchial challenges with alternaria in a group of severely asthmatic children. The group of patients examined was drawn from the long-term residents at the Children's Asthma Research Institute and Hospital, Denver, Colorado. These children all suffered chronic intractable asthma and were dependent upon intensive steroid and bronchodilator therapy (Chai & Newcomb, 1973). It was of diagnostic interest to determine the allergic sensitivity of these children to alternaria, but, unfortunately, routine skin and bronchial challenges with extracts of this mould resulted in conflicting and unreliable results. It was hoped that the RAST could supplement, or even replace, these inconvenient and potentially dangerous in vivo challenges. The results of the RAST, and various modifications of this, were therefore compared to those of skin and bronchial challenge in this group of patients, whose allergic sensitivity was not only poorly defined, but who were also being tested with a mould extract that contained a complex mixture of protein, glycoprotein and glycopeptide allergens (Bonilla-Soto, Rose & Arbesman, 1961; Berrens, 1971). MATERIALS AND METHODS Preparation of allergen extracts Twenty grams of dried alternaria (Hollister-Steir Laboratories) was extracted for 14 hr at 40C with 100 ml of 0'1 M Tris (hydroxymethyl) aminoethane (Tris), adjusted to pH 8 0 by the addition of 1 M HCl. After removal of the solid residue by centrifugation, ammonium sulphate was added to a level of 85% saturation. The material precipitated by this procedure was collected by centrifugation and dissolved in 20 ml of 0-1 M Tris-HCl. This preparation was chromatographed on a Sephadex G-25 column (2-5 x 100 cm) in 0-1 M Tris-HCl. The protein peak eluting at the void volume was dialysed against 0 3 M Tris-HCl and applied to a DEAE-cellulose column (1 x 20 cm), equilibrated with the same buffer. The material not bound to this column was concentrated by dialysis (against 0-1 M Tris-HCl) under reduced pressure. This preparation was then applied to a Sephadex G-150 column (2-5 x 100 cm) which had been calibrated for molecular weight determination (Andrews, 1964). The components eluting at volumes corresponding to the molecular weight range of 20-100,000 Daltons were collected. This preparation was effective in eliciting passive cutaneous anaphylactic reactions in sensitized monkey skin at a concentration of 1 ug N/ml.
Factors influencing alternaria RAST
37
A glycopeptide preparation was also obtained from the alternaria extract. The supernatant remaining after precipitation at 85%4 saturation with ammonium sulphate was dialysed against distilled water, then extracted by sequential treatment with hot phenol and alcohol (by the method of Bonilla-Soto, Rose & Arbesman, 1961). Although the yield of this extraction procedure was low (0 1 %), the resulting preparation, which contained 3-5 0 N, was effective in the elicitation of monkey passive cutaneous anaphylactic reactions at carbohydrate concentrations equivalent to 15 jg/ml of hexose. RAST procedure
Microcrystalline cellulose (Avicell) or Sepharose 2B (Pharmacia) were activated with cyanogen bromide by the technique of Yunginger and Gleich (1972). The partially purified alternaria extract was coupled to these supports and the conjugates were resuspended to 1 mg/ml in a 0 1 M Tris-HCl buffer, pH 7-4, which contained 1% 'Tween 20' and 100 normal rabbit serum (RAST buffer). The glycopeptide fraction obtained from alternaria was coupled to a Sepharose 2B-human serum albumin conjugate by modifications of the periodate oxidation methods of Hiramoto et al. (1972) and Sanderson & Wilson (1971). The glycopeptide was subjected to limited oxidation by the addition of 5 x 10-5 M NaIO4 in a 0 01 M acetate buffer, pH 6-3. After 30 min, this preparation was added to the Sepharose 2B-human serum albumin conjugate and incubated for 60 min. This conjugate was then washed by centrifugation and resuspended to 1 mg/ml in RAST buffer. The RAST was performed as originally described by Wide et al. (1967). Fifty microlitres of sera from the asthmatic patients were incubated with 0 5 mg of the allergen-particle conjugates for 14 hr at 40C. The complexes were then washed three times with RAST buffer and incubated for 14 hr with 100 ng N of '25I-labelled (4 /uCi/ug N) anti-IgE antibody. The anti-IgE antibody was isolated from a rabbit antiserum by affinity chromatography on a Sephadex-myeloma IgE column (Lichtenstein et al., 1973) and radiolabelled by the chloramine T method of McConahey & Dixon (1966). After washing, the radioactivity (ct/min) of each complex was measured in a y counter and transformed into a percentage by comparison with an arbitrarily chosen reference allergic serum (Johansson et al., 1971). Immunoabsorption Partially purified IgE and IgG preparations were obtained from the serum of an alternariasensitive asthmatic patient (M.W.) by column immunabsorption, using the technique of Mannik & Stage (1971). Four millilitres of packed Sepharose 4B (Pharmacia) was activated by treatment with CNBr and 20 mg of IgG (the 0 005 M sodium phosphate eluate from DEAE-cellulose) from a monospecific anti-IgE antiserum was coupled to this. An anti-IgG immunabsorbent column was also prepared by coupling 200 mg of the IgG from a monospecific anti-IgG antiserum to 20 ml of Sepharose 4B. The immunabsorbent columns were prepared in borate-buffered saline and, after dialysis against this buffer, 0'5 ml of M.W. serum was applied to the anti-IgE column. The effluent from this column was then applied to the anti-IgG column. After washing, both columns were eluted with 3 M NaSCN. The IgE and IgG preparations that were eluted from these columns by NaSCN were dialysed and concentrated until the concentration of the isolated immunoglobulin was twice its original serum level. More than 90°Y. of the IgE and IgG in the allergic serum was removed by this procedure, although two passages through the anti-IgG column were required to remove all of the IgG from the serum.
38
N. R. Lynch et al.
Immune precipitation The capacity of antibodies in the sera of alternaria-sensitive individuals to precipitate alternaria antigens was examined by Ouchterlony immunodiffusion and quantitative precipitation. Double immunodiffusion was performed in 1.500 agar, prepared in 0-1 M barbital buffer, pH 8-3. Diffusion was allowed to proceed for 48 hr, then the gels were washed, dried and stained with amido black 10B. Quantitative precipitation assays were performed as described by Kabat & Mayer (1961), the alternaria extract being radiolabelled with 1251 to provide an approximate estimate of the amount of antigen in the immune precipitates.
Monkey passive cutaneous anaphylaxis Rhesus monkeys were anaesthetized with 'Sernylan' (Bio-Ceutic Laboratories) and serial dilutions of sera from alternaria-sensitive asthmatic patients were injected intradermally. After 24 hr, 3 ml of 1% Evans blue dye was injected intravenously and both normal and sensitized skin sites were challenged by the intradermal injection of serial dilutions of alternaria extracts. Oedematous reactions at the sensitized skin sites larger than 5 mm diameter were considered positive. Patient population The children described in this report were between 9 and 16 yr of age and were all longterm patients at the Children's Asthma Research Institute and Hospital, Denver, Colorado. The blood collections, skin tests and bronchial challenges performed in this study were part of the established diagnostic protocol of the Institute. All of the patients suffered intractable asthma and were maintained on combined steroid and bronchodilator therapy. The dose of corticosteroid varied widely between individuals, but was in the vicinity of 0 5 mg/kg Serum methyl xanthine levels of 15 jg/ml were maintained as best possible and aerosolized fl-adrenergic agonists were administered to counter acute episodes of wheezing. Many of the patients inhaled 20 mg of disodium cromoglycate q.i.d. None of the patients examined had received immunotherapy with alternaria. The basis for the selection of patients was simply the first seven steroid-dependent, non-hyposensitized patients of each sex to be tested against alternaria for diagnostic purposes over a 20-week period.
Skin test procedure No medications were administered to the children for at least 6, and up to 18, hr before skin testing. Small quantities of a 1:20 w/v alternaria extract (Greer Laboratories) were introduced into the skin of the asthmatic patients by use of a spring-loaded skin puncture device ('Sterneedle', Ormont Drug and Chemical Company). The convenience and reproducibility of this technique was considerably greater than either prick testing or intradermal injection. The perpendicular diameters of the wheal produced in sensitive patients were measured 20 min after injection. As a control test for the skin sensitivity to histamine, 5 pg of this amine was also injected intradermally at the time of the skin test. Bronchial challenge procedure Bronchial challenges against alternaria were performed as described by Tuchinda & Chai (1973), and a brief description of the technique will be presented here. No medications were administered to the children for at least 6 hr before the bronchial challenge. All of the children tested were known to be sensitive to the inhalation of methacholine ('Mecholyl',
Factors influencing alternaria RAST
39
Merck and Company). Thus, prior to the actual challenge with alternaria, control inhalations of methacholine were performed. Increasing amounts of methacholine (up to 2 5 mg) were inhaled until a 20% drop in FEV1 was recorded. After the pulmonary function of the children had returned to baseline (assisted by a saline IPPB if necessary) steadily increasing amounts of alternaria extract were administered by inhalation. Doses of alternaria extract ranging up to 5000 PNU were administered, until a 20% decrease in FEV1 was recorded. RESULTS Evaluation of the RAST The RAST was evaluated by comparison with skin testing and bronchial challenge in fourteen steroid-dependent asthmatic children (9-16 years). Blood was collected from these patients immediately prior to skin testing and bronchial challenge, and the RAST was performed using a partially purified alternaria extract, coupled to microcrystalline cellulose by cyanogen bromide activation. The RAST values (percentage of reference serum) were compared to the results of skin testing and bronchial challenge in two ways. First, the skin test and bronchial challenge results were expressed semiquantitatively and the correlation with RAST values expressed as the Pearson product moment correlation coefficient (r). The amount of alternaria extract (PNU) required to elicit a 20% drop in FEV1 in bronchial challenge and the product of perpendicular wheal diameters in skin tests were taken as semiquantitative estimates of bronchial and skin reactivity to alternaria. As there is an inverse relationship between
*l A
100 _ A
A
A^
80 80
-
0
60 b
0
0
cr_
40
A
t0 20 _ A
0
20
I 40
I 60
I 80
I 100
Skin test score (mm2)
FIG. 1. RAST scores (percentage of reference serum) and skin test wheal sizes (product of perpendicular wheal diameters) for fourteen asthmatic children and four normal children (o) are presented. The RAST was performed with either cellulose-alternaria (e), Sepharose 2Balternaria (a) or Sepharose-glycopeptide (A) conjugates. Skin test scores greater than 25 were considered positive.
N. R. Lynch et al.
40 100
A
A 80 _
[t
0
A
o 00
0
60
0
I-~ ~~~~N 40
A
20
0
1
2
3
4
5
Bronchial challenge score (x 193 PNU) FIG. 2. RAST scores and bronchial challenge scores (number of PNU inhaled to elicit at 20% decrease in FEV1) for fourteen asthmatic children are presented. The RAST was performed with either cellulose-alternaria (o), Sepharose 2B-alternaria (r) or Sepharose-glycopeptide (A) conjugates. Bronchial challenges were considered negative if more than 1500 PNU were inhaled without inducing a 20% drop in FEV1.
bronchial sensitivity and PNU inhaled, the Pearson r coefficient assumed a negative character in comparisons involving this parameter. The second approach to the comparison of RAST values with skin test and bronchial challenge results was to classify the in vivo challenges as either positive or negative and compare the mean RAST values in these two categories. The cut-off values used to define positive and negative responders were a 5 x 5 mm wheal size in skin tests and the inhalation of 1500 PNU in bronchial challenge. Although these values are somewhat arbitrary, they have proven useful in routine testing. Also, the correlations between the RAST and the in vivo tests derived by the first and second methods of comparison were essentially similar, thus supporting the use of these cut-off values. Significant correlation (r = 054, P0 05), or skin test scores (r = 0 14, P>0 05). Of the fourteen asthmatic children tested, four were classified as being positive by both skin test and bronchial challenge, five were negative by both tests, and five were reactive to only one of these challenges. The RAST levels in the sera of the asthmatic individuals who were either positive or negative to skin test or bronchial challenge were also compared (Student's t-test). Significant differences were found when the patients were divided on the basis of their skin (P< 0-05) but not bronchial (P> 0-05) reactivity. The RAST levels in the sera of the patients who were responsive to only one of the in vivo tests were not significantly higher than in patients who were reactive to neither. However, the RAST levels in the latter group were significantly higher (P< 0 0005) than that determined in sera obtained from four non-asthmatic children (Fig. 1).
Factors influencing alternaria RAST
41
To provide a comparison with these results, the responsiveness of a group ofeleven patients against timothy grass pollen extract was examined. The drug therapy received by these patients and the selection procedure employed was essentially the same as for the patients who were tested for their sensitivity to alternaria. However, in these children, and with this grass pollen extract, good correlations were found between skin testing and bronchial challenge (r = -0-89, P
