The radioallergosorbent test in the diagnosis of canine atopic disease R. E. W. Halliwell, Philadelphia,
Ph.D., Vet.M.B.,*
and G. A. Kunkle,
D.V.M.
Pa.
The radioallergosorbent test (RAST) was adapted for use in the dog employing purified anti-canine IgE. The agreement between positive RAST and positive skin test ranged from 82% for ragweed to only 12.57 of or dan de1ion. Agreement between negative tests in the case of all allergens was 100%. Further developmental studies of canine RAST are warranted in view of the value of the dog as an experimental model of atopic disease.
The dog is the only subhuman specieswhich suffers from a clearly defined atopic disease. Unlike man, however, the dog manifests its atopic state almost exclusively by pruritus,’ and although a variety of dermatitic changes may result from self-inflicted trauma, it is questionableif there is any primary eruptive process involving the skin. Hayfever-like signs are sometimesnoted, but spontaneousallergic asthma is exceedingly rare. The latter, however, is readily induced upon nebulization with high concentrations of allergen in suitably sensitive individuals.23 3 This species difference in the clinical signs may reflect nothing other than the establishedvariation in the relative density of tissue mast cells in the target organs of the two species.4 The canine diseaseis mediatedby a homologue of IgE which sharescommon antigenic determinantsand many other physicochemical characteristics with the human counterpart.5-7 The immunoglobulin has been purified and extensively characterized. One notable feature is that levels of IgE in the dog are very much higher than those in man-in one report the mean serum level in 50 random normal North American dogs was 198.9 pg/mL8 This high level is probably a reflection of the multiple internal and external parasitism from which virtually all dogs suffer. From the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania. Supportedby a grant from the Tarrant County (Texas) Veterinary Medical Association through the Morris Animal Foundation. Received for publication Nov. 4, 1977. Accepted for publication July 3, 1978. Reprint requeststo: Dr. R. E. W. Halliwell, College of Veterinary Medicine, Box J-126, JHMHC, University of Florida, Gainesville, FL 32610. *Present address: College of Veterinary Medicine, Box J-126, JHMHC, University of Florida, Gainesville, FL 32610. Vol. 62, No. 4, pp. 236-242
The diagnosis and therapy of canine atopic disease represent an important part of veterinary practice. Additionally, the atopic dog has a unique place as an experimental model. Detection of reaginic antibodies in this specieshas hitherto been performed by means of Prausnitz-Kiistner tests, but this approachhas limitations in the dog as the titer of a given serum may vary widely when assessedin different recipient animals. In one study, serum from an animal hypersensitive to ragweed was titered in ten normal dogs with results that varied from negative at l! 10 to positive to a dilution of 1/5,12O.s Repeat testing in the same animal, however, usually gave concordant results. The introduction of the radioallergosorbent test (RAST) in man representeda novel approach to the diagnosisof atopic allergy,‘O and the adaptationof the test for the dog not only has important implications for veterinary medicine but also is a prerequisite for the further development and use of this speciesas an experimental model. Its developmentfor the dog followed the samegeneral lines as did the test in man. One important modification, however, was necessitated by the fact that no canine IgE myeloma has yet been found. The immunoabsorbentfor preparation of purified anti-canine IgE was therefore made by coupling appropriate chromatographicfractions of serum high in IgE to the solid-phase adsorbent. MATERIALS AND METHODS Antiserum to canine IgE This was prepared as described in an earlier publication,J by immunizing rabbits with canine IgE/anti-canine IgE agar precipitin bands and absorption of the resulting antiserum with canine IgGSab.5 Each batch of antiserum was rigorously checked for specificity as described earlier.6 The more important criteria used were the ability of the antisera to remove P-K reactivity from reaginic serum and to induce
0091-6749/78/100236+07$00.70/0
@ 1978 The C. V. Mosby
Co.
VOLUME NUMBER
Radioallergosorbent
62 4
reversed cutaneous anaphylaxis upon intradermal injection in high dilutions. No protein other than IgE was detectable by immunoelectmphotesis in a wide range of canine sera.
lntradermal
skin tests
Hair was clipped from the lateral chest area and 0.05 ml of each allergenic extract (Greer Laboratories, Lenoir, N. C.) was injected intradermally. A diluent negative control and a positive control of l/100,000 w/v histamine phosphate (Eli Lilly, Indianapolis, Ind.) were routinely included. Extracts were used at 1,000 PNU with the exception of house dust, which often has irritant properties in the dog and was used at 250 PNU. The diameter of the wheal was measured after fifteen min and graded as follows: Negative less than 3 mm greater than control + 1 3-5 mm greater than control +2 5-8 mm greater than control + 3 8- 15 mm greater than control f4 over 15 mm greater than control The skin tests were not regarded as valid unless the positive control was at least +2. The usual reason for inhibition was recent administration of corticosteroids, which often markedly inhibits the wheal and to a lesser extent the accompanying erythema. I
Purification anti-canine
and radiolabeling IgE
of
Twelve 7-ml samples of a mixture of canine sera high in IgE were fractionated by filtration through 3 in-series 4 x 60 cm columns of Sephacryl S200 (Pharmacia Fine Chemicals, Piscataway, N. J.). Fractions containing the highest levels of IgE which were those immediately prior to the 7S peak and from the first third of the ascending part of that peak were pooled and concentrated. The protein content of the pool was measured by absorbance at 280 nm using canine Cohn Fr II (Miles Research Products, Elkhart, Ind.) as the standard. The IgE content was then quantitated by a double-antibody overlay modification of the single radial immunodiffusion technique employing previously described standards.6 The IgE content of the pooled fractions quantitated in this way amounted to 21% of the total protein. Eighty-five mg of this protein in 5 ml were coupled to 12 ml of cyanogen bromide-activated Sepharose 4B (Pharmacia) by the method of Axen, Porath, and Emback.” Subsequent analysis showed that 90% of the protein was absorbed onto the Sepharose. An IgG fraction of rabbit anti-canine IgE was prepared by precipitation with 33.3% saturated ammonium sulfate followed by exclusion from diethylaminoethyl cellulose (DE 52, Whatmann, Maidstone, Kent, England) in 0.035 M phosphate buffer, pH 8. Aliquots containing 20 mg of protein were applied to the immunoabsorbent column and purified antibody was eluted by lowering the pH to 2.5 with glycine-HCI buffer. Three milligrams of purified antibody were then radioiodinated with 500 /*c of carrier-free Na12”I (New England Nuclear, Boston, Mass.) using chloramine T.‘* The specific activity of the labeled anti-IgE was 109 pc/mg.
Performance
test in canine atopic disease
237
of RAST
Allergen-coupled cellulose discs (Phabedas; Pharmacia Fine Chemicals, Piscataway, N. J.) were rehydrated, rinsed three times in distilled water, and suspended in RAST buffer which was 0.05 M phosphate-buffered saline, pH 7.9, with addition of 0.3% human serum albumin (Sigma, St. Louis, MO.), 0.05% sodium azide, and 0.05% Tween 20 (Fisher, Fair Lawn, N. J.). Undiluted patient’s serum (0.05 ml) was added to the disc and incubated for 3 hr at room temperature. The discs were then washed three times with RAST buffer and incubated overnight at room temperature with 0.05 ml of 0.03 mglml solution of labeled purified antibody. The radioactivity derived from this quantity of labeled antibody added amounted to 59,612 cpm. Previous studies using high-titer reaginic serum and sera from nonallergic dogs with high serum IgE showed this amount of antibody to be optimally discriminating. After three further washes with RAST buffer, the radioactivity associated with the discs was measured using a Packard auto-gamma counter. Tests were performed in duplicate, and the value recorded was the mean of the two tests. The agreement between duplicates improved as greater familiarity with the techniques employed in the procedure was acquired. In the tests reported in this study, 10% of duplicates varied by less than 10% in cpm, and 82% differed by less than 20%. Variation in excess of 20% implied a technical error and the test was repeated. Routinely included as negative controls were discs whose first incubation was with RAST buffer (blank), and also discs incubated with serum from a skin test-negative dog with an IgE level of 660 pg/ml (high IgE control). Controls employed to demonstrate the specificity of the reaction included the addition of an excess of fluid-phase allergen in the first stage of the procedure and an excess of unlabeled anti-IgE in the second stage. Both of these manipulations reduced the counts obtained from strongly positive sera to around those resulting from the high IgE control. Counts were not significantly affected by the addition of unlabeled anti-IgG prior to the labeled anti-IgE in the second stage. The tests recorded in this report were performed with the same batch of discs and of labeled anti-IgE over a threeweek period. Two positive control sera were incorporated in each test and appropriate adjustments were made for the decay in the label. A cpm twice that of the high IgE control was defined as a positive RAST and a borderline was I .5 times that same control.
RESULTS Results of ragweed
RAST
RAST was performed on sera from 20 atopic dogs using discs to which short ragweed antigen was coupled. Seventeen of the dogs had shown positive skin tests to a mixture of tall and short ragweed antigen. RAST was performed on 6 sera obtained from beagles in England which is free of ragweed. Results
238
Halliwell
J. ALLERGY
and Kunkle
.=BO A=WT
CLIN. IMMUNOL. OCTOBER 1978
.
kf M moo .
. .
.
. .
1000 A
A A
1000
A
A
.
.
04:
A
A
A
AA
:
:* 101 Yii,L,“S177 ns
FIG. 1. Correlation between results of skin tests to tall and short ragweed mix and RAST for short ragweed.
FIG. 2. Seasonal atopic dogs.
from these latter 6 dogs were closely grouped (246 to 389 cpm with a mean of 3 12). Sera from the three American atopic dogs who showed strong sensitivities to many allergens but were skin test-negative to ragweed gave somewhat higher values at 379, 416, and 473 cpm, respectively. Sera from the 17 ragweedallergic dogs gave a range of 324 to 4,120 cpm with a mean of 1,261. There was no consistent tendency for dogs with the stronger skin tests to give higher values (Fig. 1). RAST using tall ragweed antigen attached to the discs failed to discriminate between allergic and nonallergic dogs. Assuming a positive RAST as two times the high IgE control, there was an 81% agreement between positive skin tests and positive RAST in that sera from 13 of the 17 skin test-positive dogs were positive by RAST (Table I). Sera from the 9 dogs who had given negative skin tests to ragweed, i.e., the 6 normal English dogs and the 3 American atopic dogs, all were negative by RAST. If a positive RAST were defined as 1.5 times the high IgE control, the percent agreement between positive skin tests and positive RAST increased to 89%, but that between negative tests was reduced to 90% (Table I). In the case of 2 atopic dogs, RAST was performed on sera taken at various times over a two-year period (Fig. 2). The results from Dog BD showed an apparent seasonal variation in the second year, as was the case with Dog WT in the first season. The trend for WT was less marked during the second year, and indeed two of the samples gave only borderline values, one of which, surprisingly enough, was taken just after the ragweed pollination season.
were categorized into two groups, namely, those with a good history of house dust allergy (i.e., perennial pruritus which could not be ascribed to any other etiology) and those with a poor history whose pruritus was predominantly seasonal. Serum from the majority of the good history group gave positive P-K tests at dilutions in excess of 1I1 00. RAST performed with crude house dust antigen discs failed to discriminate between sera from dogs with negative skin tests and those with positive skin tests in either group. The tests were therefore repeated using house dust mite antigen discs (Dermatophagoides farinae). This procedure proved far more discriminating (Fig. 3). Counts resulting from tests on serum from the good history group ranged from 369-2,778 cpm with a mean of 858 and the data for the poor history group ranged from 307-465 cpm with a mean of 362. The latter were close to the results derived from sera from the 4 atopics that had negative skin tests which ranged from 289-436 cpm with a mean of 379. Five of 12 in the good history group thus had a positive RAST yet all 5 in the poor history group were negative. The 4 atopics with negative skin tests were likewise negative by RAST. The good history group thus gave a 42% agreement between positive skin test and positive RAST, which increased to 66% if borderline RAST results were considered positive (Table I). Comparable data for the poor history group were 0% and 20%.
Results of house dust RAST Twenty-one atopic dogs were skin-tested for house dust and 17 of these gave positive results. These 17
variations
Results of timothy
in ragweed
RAST values
in 2
grass RAST
Eighteen atopic dogs were skin-tested with mixed grass antigen (7 grasses including timothy) and RAST was performed on their sera with timothy grass antigen discs. Sera from the 14 dogs with positive skin tests gave counts that ranged from 302-2,710 cpm with a mean of 1,044 and those from dogs with nega-
VOLUME NUMBER
Radioallergosorbent test in canine atopic disease 239
62 4
TABLE I. Percentage
agreement
between
results of skin tests and RAST
No.
Positive RAST 2X control
tested
Ragweed
25
Positive* Negative?
82 100 42
House dust mite (good history)
12
Positive
House dust mite (poor history)
9
Timothy grass
18
Dandelion
II
Lamb’s-quarters
12
English plantain
12
Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative
0 100 64 100 12.5 100 27 100 39 100
Positive RAST 1.5X control
88 90 66 20 100 71 100 28 loo 45 100 50 loo
*Percentage of dogs with a positive skin test whose sera were positive by RAST. tpercentage of dogs with a negative skin test whose sera were negative by RAST.
tive skin tests ranged from 377-498 cpm with a mean of 441 (Fig. 4). Sera from 9 of the dogs with positive skin tests were accordingly positive by RAST and the agreement between positive tests was thus 64% (Table I). This increased to 71% if doubtfuls were considered positive. Sera from the 4 dogs with negative skin tests were negative by RAST. Results of RAST for dandelion, lamb’+quarters, and English plantain Twelve atopic dogs were skin-tested with these three antigens and RAST was performed on their serum. The results are depicted in Fig. 4. The values for sera from animals which gave positive skin tests to dandelion varied from 280 cpm to 1,649 cpm with a mean of 598 using dandelion antigen discs. Corresponding data for animals with negative skin tests ranged from 319 to 420 cpm with a mean of 368. Sera from dogs which gave positive skin tests to English plantain gave counts ranging from 310 to 2,322 cpm with a mean of 813 when that antigen was coupled to the discs. Sera from animals with negative skin tests ranged from 329 to 388 cpm with a mean of 359. In the case of lamb’s-quarters, values for sera from animals with positive skin tests varied from 341 to 3,120 cpm with a mean of 732, and the single negative animal gave a value of 388 cpm. Examination of the data shows that RAST was much less sensitive for these antigens in that sera from skin test-positive dogs were rarely positive by RAST. The agreement between positive skin test and positive
RAST was 12.5% for dandelion, 27% for lamb’squarters, and 39% for English plantain. Again, false-positives were not encountered, and sera from animals with negative skin tests were always negative by RAST. If borderlines were considered positive, the agreement between positive skin test and positive RAST increased to 28%, 45%, and 50%, respectively, yet all sera from dogs with negative skin tests were still negative by RAST. DISCUSSION In this study, sera from animals with negative skin tests gave results that were clustered close to both the blank and the high IgE control in the case of all allergens. This permitted the adoption of a cpm value of 2X the high IgE control as the definition of a positive RAST. When considered in conjunction with the skin test, this was highly discriminating in the case of negative tests. In none of these studies was the RAST value for serum from an animal with a negative skin test less than 25% below the adopted cutoff point, and in the case of most allergens it was around 40% below. One and one-half times the high IgE control was adopted as borderline. If this value had been adopted as the cutoff point for a positive RAST, an appreciable increase in the percent agreement between positive skin tests and positive RAST would have resulted (Table I), but in the case of ragweed it reduced the percent agreement between negative skin tests and negative RAST to 90%. In addition, sera from a number of the skin test-negative animals came very close to the cutoff point. If was therefore felt safer to retain the description of borderline for those sera that gave
240
Halliwell
ATOPICS
and Kunkle
ATOPICS
iK’NP&!ST SK’NpOFsT ooo HlSlu w II w)R HlsToR
J. ALLERGY
BLANK
n
ATOPICS SK’NNAY
. C
P M 2000
. . 1000
.
. . . :***
.
J
FIG. 3. Correlations between results of skin tests to house dust extract and RAST for house dust mite ID. farinae).
cpm values between 1.5 and 2 times the high IgE control. The value of RAST as a diagnostic tool in human allergic disease is controversial. Comparisons between positive skin tests and positive RAST have not always shown excellent agreement, although the negative tests are invariably in better agreement.13,l4 This latter finding was strikingly manifest in the canine study reported here. Despite the very much higher levels of IgE in the canine, false-positive results were not encountered, and the agreement between negative skin tests and negative RAST in the caseof all allergens was 100%. RAST and skin testsare, of course, measuringdifferent things, one being an indication of cell-bound antigen-specific IgE and the other being an indication of the amount of the sameantibody that is circulating. No conclusive data exist on the factors controlling the ratio of cell-bound to circulating IgE. Studies in the canine on the relative distribution of total (not antigen-specific) IgE have shown a poor correlation betweenthe level of serum IgE and that bound to skin assessedby either reversed cutaneousanaphylaxis or by the percentageof mastcells with IgE demonstrable by immunofluorescence.l5 In the light of these considerations, it would indeed be surprising if results of skin tests and RAST were invariably in good agreement. It is at first sight surprising that in studies in man correlations between RAST and provocation tests have been somewhat closer than those of skin tests and provocation, I4as both of the latter are indications of cell-bound IgE. However, in the light of present
CLIN. IMMUNOL. OCTOBER 1979
stateof knowledge of the dynamics of total IgE, there is no reasonto believe that the distribution of antibody in the respiratory mucosa and the skin must, of necessity, be closely parallel. Provocation tests were not performed on thesecanine patients, but the results of house dust RAST merit consideration. Before the testswere performed, a group of set-awere singled out for use in the development of the test from animals whose history of house dust allergy was particularly convincing. The averagestrength of the positive skin tests in this group to 250 PNU of house dust did not differ appreciably from that of the group that had a poor history and who manifested their disease predominantly in the pollen season-yet the difference in their RAST results using mite extract coupled to the discs was striking, namely, a 42% agreementbetween positive skin test and positive RAST for the good history group and a zero agreementfor the poor history group. The fact that the skin testswere performed with whole dust extract rather than mite extract, which was not available in a form suitable for intradermal use at that time, may appear to limit the significance of thesedatabut probably detractsfrom it only minimally. The widely held contention in allergy practice in man that the house dust mite is the major allergen in housedust is supportedby recent studiesin the dog.16Skin tests were performed on 50 suspected atopic dogs using three IO-fold dilutions of a 250PNU extract of house dust (Greer Laboratories, Lenoir, N. C.) and the same strengths of D. furinae extract (Hollister-Stier Laboratories, Spokane, Wash.). Thirty-eight of these gave positive tests to 250 PNU of both extracts, and 12 gave negative tests to both. Positive testswere more often encounteredto higher dilutions of mite extract than to dilutions of whole house dust extract. This is interpretable as strong evidence that D. farinae is the major allergen responsible for house dust allergy in atopic dogs. The marked difference in positive RAST and skin test agreement between the different allergens is noteworthy. At two extremes, there was an 82% agreement between positive skin tests and positive RAST in the caseof ragweed, and only 12.5% in the case of dandelion. Detailed analysis of the variables in the performanceof RAST in man have been published17*ia and are plannedfor the canine system. One factor which was shown to be important was the amount of allergen attached to the solid phase-the greater the amount, the better was the discrimination. la Thus, the presenceof more specific allergen, rather than total protein, on the disc could well account for the noted variation in test agreementwith the different allergens. Further consideration of the house dust results supports this concept. RAST tests
VOLUME NUMBER
62 4
Radioallergosorbent
ANTIGEN
BLANK
HIGH IgE C ONTROL
atopic disease
241
ENGLISH PLANTAIN
TIMOTHY GRASS ATOPICS ,KIN TES’ POS
test in canine
AlOPICS SIKIN TEST NEG.
KrofKs
Ph.D., Vet.M.B.,*
and G. A. Kunkle,
D.V.M.
Pa.
The radioallergosorbent test (RAST) was adapted for use in the dog employing purified anti-canine IgE. The agreement between positive RAST and positive skin test ranged from 82% for ragweed to only 12.57 of or dan de1ion. Agreement between negative tests in the case of all allergens was 100%. Further developmental studies of canine RAST are warranted in view of the value of the dog as an experimental model of atopic disease.
The dog is the only subhuman specieswhich suffers from a clearly defined atopic disease. Unlike man, however, the dog manifests its atopic state almost exclusively by pruritus,’ and although a variety of dermatitic changes may result from self-inflicted trauma, it is questionableif there is any primary eruptive process involving the skin. Hayfever-like signs are sometimesnoted, but spontaneousallergic asthma is exceedingly rare. The latter, however, is readily induced upon nebulization with high concentrations of allergen in suitably sensitive individuals.23 3 This species difference in the clinical signs may reflect nothing other than the establishedvariation in the relative density of tissue mast cells in the target organs of the two species.4 The canine diseaseis mediatedby a homologue of IgE which sharescommon antigenic determinantsand many other physicochemical characteristics with the human counterpart.5-7 The immunoglobulin has been purified and extensively characterized. One notable feature is that levels of IgE in the dog are very much higher than those in man-in one report the mean serum level in 50 random normal North American dogs was 198.9 pg/mL8 This high level is probably a reflection of the multiple internal and external parasitism from which virtually all dogs suffer. From the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania. Supportedby a grant from the Tarrant County (Texas) Veterinary Medical Association through the Morris Animal Foundation. Received for publication Nov. 4, 1977. Accepted for publication July 3, 1978. Reprint requeststo: Dr. R. E. W. Halliwell, College of Veterinary Medicine, Box J-126, JHMHC, University of Florida, Gainesville, FL 32610. *Present address: College of Veterinary Medicine, Box J-126, JHMHC, University of Florida, Gainesville, FL 32610. Vol. 62, No. 4, pp. 236-242
The diagnosis and therapy of canine atopic disease represent an important part of veterinary practice. Additionally, the atopic dog has a unique place as an experimental model. Detection of reaginic antibodies in this specieshas hitherto been performed by means of Prausnitz-Kiistner tests, but this approachhas limitations in the dog as the titer of a given serum may vary widely when assessedin different recipient animals. In one study, serum from an animal hypersensitive to ragweed was titered in ten normal dogs with results that varied from negative at l! 10 to positive to a dilution of 1/5,12O.s Repeat testing in the same animal, however, usually gave concordant results. The introduction of the radioallergosorbent test (RAST) in man representeda novel approach to the diagnosisof atopic allergy,‘O and the adaptationof the test for the dog not only has important implications for veterinary medicine but also is a prerequisite for the further development and use of this speciesas an experimental model. Its developmentfor the dog followed the samegeneral lines as did the test in man. One important modification, however, was necessitated by the fact that no canine IgE myeloma has yet been found. The immunoabsorbentfor preparation of purified anti-canine IgE was therefore made by coupling appropriate chromatographicfractions of serum high in IgE to the solid-phase adsorbent. MATERIALS AND METHODS Antiserum to canine IgE This was prepared as described in an earlier publication,J by immunizing rabbits with canine IgE/anti-canine IgE agar precipitin bands and absorption of the resulting antiserum with canine IgGSab.5 Each batch of antiserum was rigorously checked for specificity as described earlier.6 The more important criteria used were the ability of the antisera to remove P-K reactivity from reaginic serum and to induce
0091-6749/78/100236+07$00.70/0
@ 1978 The C. V. Mosby
Co.
VOLUME NUMBER
Radioallergosorbent
62 4
reversed cutaneous anaphylaxis upon intradermal injection in high dilutions. No protein other than IgE was detectable by immunoelectmphotesis in a wide range of canine sera.
lntradermal
skin tests
Hair was clipped from the lateral chest area and 0.05 ml of each allergenic extract (Greer Laboratories, Lenoir, N. C.) was injected intradermally. A diluent negative control and a positive control of l/100,000 w/v histamine phosphate (Eli Lilly, Indianapolis, Ind.) were routinely included. Extracts were used at 1,000 PNU with the exception of house dust, which often has irritant properties in the dog and was used at 250 PNU. The diameter of the wheal was measured after fifteen min and graded as follows: Negative less than 3 mm greater than control + 1 3-5 mm greater than control +2 5-8 mm greater than control + 3 8- 15 mm greater than control f4 over 15 mm greater than control The skin tests were not regarded as valid unless the positive control was at least +2. The usual reason for inhibition was recent administration of corticosteroids, which often markedly inhibits the wheal and to a lesser extent the accompanying erythema. I
Purification anti-canine
and radiolabeling IgE
of
Twelve 7-ml samples of a mixture of canine sera high in IgE were fractionated by filtration through 3 in-series 4 x 60 cm columns of Sephacryl S200 (Pharmacia Fine Chemicals, Piscataway, N. J.). Fractions containing the highest levels of IgE which were those immediately prior to the 7S peak and from the first third of the ascending part of that peak were pooled and concentrated. The protein content of the pool was measured by absorbance at 280 nm using canine Cohn Fr II (Miles Research Products, Elkhart, Ind.) as the standard. The IgE content was then quantitated by a double-antibody overlay modification of the single radial immunodiffusion technique employing previously described standards.6 The IgE content of the pooled fractions quantitated in this way amounted to 21% of the total protein. Eighty-five mg of this protein in 5 ml were coupled to 12 ml of cyanogen bromide-activated Sepharose 4B (Pharmacia) by the method of Axen, Porath, and Emback.” Subsequent analysis showed that 90% of the protein was absorbed onto the Sepharose. An IgG fraction of rabbit anti-canine IgE was prepared by precipitation with 33.3% saturated ammonium sulfate followed by exclusion from diethylaminoethyl cellulose (DE 52, Whatmann, Maidstone, Kent, England) in 0.035 M phosphate buffer, pH 8. Aliquots containing 20 mg of protein were applied to the immunoabsorbent column and purified antibody was eluted by lowering the pH to 2.5 with glycine-HCI buffer. Three milligrams of purified antibody were then radioiodinated with 500 /*c of carrier-free Na12”I (New England Nuclear, Boston, Mass.) using chloramine T.‘* The specific activity of the labeled anti-IgE was 109 pc/mg.
Performance
test in canine atopic disease
237
of RAST
Allergen-coupled cellulose discs (Phabedas; Pharmacia Fine Chemicals, Piscataway, N. J.) were rehydrated, rinsed three times in distilled water, and suspended in RAST buffer which was 0.05 M phosphate-buffered saline, pH 7.9, with addition of 0.3% human serum albumin (Sigma, St. Louis, MO.), 0.05% sodium azide, and 0.05% Tween 20 (Fisher, Fair Lawn, N. J.). Undiluted patient’s serum (0.05 ml) was added to the disc and incubated for 3 hr at room temperature. The discs were then washed three times with RAST buffer and incubated overnight at room temperature with 0.05 ml of 0.03 mglml solution of labeled purified antibody. The radioactivity derived from this quantity of labeled antibody added amounted to 59,612 cpm. Previous studies using high-titer reaginic serum and sera from nonallergic dogs with high serum IgE showed this amount of antibody to be optimally discriminating. After three further washes with RAST buffer, the radioactivity associated with the discs was measured using a Packard auto-gamma counter. Tests were performed in duplicate, and the value recorded was the mean of the two tests. The agreement between duplicates improved as greater familiarity with the techniques employed in the procedure was acquired. In the tests reported in this study, 10% of duplicates varied by less than 10% in cpm, and 82% differed by less than 20%. Variation in excess of 20% implied a technical error and the test was repeated. Routinely included as negative controls were discs whose first incubation was with RAST buffer (blank), and also discs incubated with serum from a skin test-negative dog with an IgE level of 660 pg/ml (high IgE control). Controls employed to demonstrate the specificity of the reaction included the addition of an excess of fluid-phase allergen in the first stage of the procedure and an excess of unlabeled anti-IgE in the second stage. Both of these manipulations reduced the counts obtained from strongly positive sera to around those resulting from the high IgE control. Counts were not significantly affected by the addition of unlabeled anti-IgG prior to the labeled anti-IgE in the second stage. The tests recorded in this report were performed with the same batch of discs and of labeled anti-IgE over a threeweek period. Two positive control sera were incorporated in each test and appropriate adjustments were made for the decay in the label. A cpm twice that of the high IgE control was defined as a positive RAST and a borderline was I .5 times that same control.
RESULTS Results of ragweed
RAST
RAST was performed on sera from 20 atopic dogs using discs to which short ragweed antigen was coupled. Seventeen of the dogs had shown positive skin tests to a mixture of tall and short ragweed antigen. RAST was performed on 6 sera obtained from beagles in England which is free of ragweed. Results
238
Halliwell
J. ALLERGY
and Kunkle
.=BO A=WT
CLIN. IMMUNOL. OCTOBER 1978
.
kf M moo .
. .
.
. .
1000 A
A A
1000
A
A
.
.
04:
A
A
A
AA
:
:* 101 Yii,L,“S177 ns
FIG. 1. Correlation between results of skin tests to tall and short ragweed mix and RAST for short ragweed.
FIG. 2. Seasonal atopic dogs.
from these latter 6 dogs were closely grouped (246 to 389 cpm with a mean of 3 12). Sera from the three American atopic dogs who showed strong sensitivities to many allergens but were skin test-negative to ragweed gave somewhat higher values at 379, 416, and 473 cpm, respectively. Sera from the 17 ragweedallergic dogs gave a range of 324 to 4,120 cpm with a mean of 1,261. There was no consistent tendency for dogs with the stronger skin tests to give higher values (Fig. 1). RAST using tall ragweed antigen attached to the discs failed to discriminate between allergic and nonallergic dogs. Assuming a positive RAST as two times the high IgE control, there was an 81% agreement between positive skin tests and positive RAST in that sera from 13 of the 17 skin test-positive dogs were positive by RAST (Table I). Sera from the 9 dogs who had given negative skin tests to ragweed, i.e., the 6 normal English dogs and the 3 American atopic dogs, all were negative by RAST. If a positive RAST were defined as 1.5 times the high IgE control, the percent agreement between positive skin tests and positive RAST increased to 89%, but that between negative tests was reduced to 90% (Table I). In the case of 2 atopic dogs, RAST was performed on sera taken at various times over a two-year period (Fig. 2). The results from Dog BD showed an apparent seasonal variation in the second year, as was the case with Dog WT in the first season. The trend for WT was less marked during the second year, and indeed two of the samples gave only borderline values, one of which, surprisingly enough, was taken just after the ragweed pollination season.
were categorized into two groups, namely, those with a good history of house dust allergy (i.e., perennial pruritus which could not be ascribed to any other etiology) and those with a poor history whose pruritus was predominantly seasonal. Serum from the majority of the good history group gave positive P-K tests at dilutions in excess of 1I1 00. RAST performed with crude house dust antigen discs failed to discriminate between sera from dogs with negative skin tests and those with positive skin tests in either group. The tests were therefore repeated using house dust mite antigen discs (Dermatophagoides farinae). This procedure proved far more discriminating (Fig. 3). Counts resulting from tests on serum from the good history group ranged from 369-2,778 cpm with a mean of 858 and the data for the poor history group ranged from 307-465 cpm with a mean of 362. The latter were close to the results derived from sera from the 4 atopics that had negative skin tests which ranged from 289-436 cpm with a mean of 379. Five of 12 in the good history group thus had a positive RAST yet all 5 in the poor history group were negative. The 4 atopics with negative skin tests were likewise negative by RAST. The good history group thus gave a 42% agreement between positive skin test and positive RAST, which increased to 66% if borderline RAST results were considered positive (Table I). Comparable data for the poor history group were 0% and 20%.
Results of house dust RAST Twenty-one atopic dogs were skin-tested for house dust and 17 of these gave positive results. These 17
variations
Results of timothy
in ragweed
RAST values
in 2
grass RAST
Eighteen atopic dogs were skin-tested with mixed grass antigen (7 grasses including timothy) and RAST was performed on their sera with timothy grass antigen discs. Sera from the 14 dogs with positive skin tests gave counts that ranged from 302-2,710 cpm with a mean of 1,044 and those from dogs with nega-
VOLUME NUMBER
Radioallergosorbent test in canine atopic disease 239
62 4
TABLE I. Percentage
agreement
between
results of skin tests and RAST
No.
Positive RAST 2X control
tested
Ragweed
25
Positive* Negative?
82 100 42
House dust mite (good history)
12
Positive
House dust mite (poor history)
9
Timothy grass
18
Dandelion
II
Lamb’s-quarters
12
English plantain
12
Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative
0 100 64 100 12.5 100 27 100 39 100
Positive RAST 1.5X control
88 90 66 20 100 71 100 28 loo 45 100 50 loo
*Percentage of dogs with a positive skin test whose sera were positive by RAST. tpercentage of dogs with a negative skin test whose sera were negative by RAST.
tive skin tests ranged from 377-498 cpm with a mean of 441 (Fig. 4). Sera from 9 of the dogs with positive skin tests were accordingly positive by RAST and the agreement between positive tests was thus 64% (Table I). This increased to 71% if doubtfuls were considered positive. Sera from the 4 dogs with negative skin tests were negative by RAST. Results of RAST for dandelion, lamb’+quarters, and English plantain Twelve atopic dogs were skin-tested with these three antigens and RAST was performed on their serum. The results are depicted in Fig. 4. The values for sera from animals which gave positive skin tests to dandelion varied from 280 cpm to 1,649 cpm with a mean of 598 using dandelion antigen discs. Corresponding data for animals with negative skin tests ranged from 319 to 420 cpm with a mean of 368. Sera from dogs which gave positive skin tests to English plantain gave counts ranging from 310 to 2,322 cpm with a mean of 813 when that antigen was coupled to the discs. Sera from animals with negative skin tests ranged from 329 to 388 cpm with a mean of 359. In the case of lamb’s-quarters, values for sera from animals with positive skin tests varied from 341 to 3,120 cpm with a mean of 732, and the single negative animal gave a value of 388 cpm. Examination of the data shows that RAST was much less sensitive for these antigens in that sera from skin test-positive dogs were rarely positive by RAST. The agreement between positive skin test and positive
RAST was 12.5% for dandelion, 27% for lamb’squarters, and 39% for English plantain. Again, false-positives were not encountered, and sera from animals with negative skin tests were always negative by RAST. If borderlines were considered positive, the agreement between positive skin test and positive RAST increased to 28%, 45%, and 50%, respectively, yet all sera from dogs with negative skin tests were still negative by RAST. DISCUSSION In this study, sera from animals with negative skin tests gave results that were clustered close to both the blank and the high IgE control in the case of all allergens. This permitted the adoption of a cpm value of 2X the high IgE control as the definition of a positive RAST. When considered in conjunction with the skin test, this was highly discriminating in the case of negative tests. In none of these studies was the RAST value for serum from an animal with a negative skin test less than 25% below the adopted cutoff point, and in the case of most allergens it was around 40% below. One and one-half times the high IgE control was adopted as borderline. If this value had been adopted as the cutoff point for a positive RAST, an appreciable increase in the percent agreement between positive skin tests and positive RAST would have resulted (Table I), but in the case of ragweed it reduced the percent agreement between negative skin tests and negative RAST to 90%. In addition, sera from a number of the skin test-negative animals came very close to the cutoff point. If was therefore felt safer to retain the description of borderline for those sera that gave
240
Halliwell
ATOPICS
and Kunkle
ATOPICS
iK’NP&!ST SK’NpOFsT ooo HlSlu w II w)R HlsToR
J. ALLERGY
BLANK
n
ATOPICS SK’NNAY
. C
P M 2000
. . 1000
.
. . . :***
.
J
FIG. 3. Correlations between results of skin tests to house dust extract and RAST for house dust mite ID. farinae).
cpm values between 1.5 and 2 times the high IgE control. The value of RAST as a diagnostic tool in human allergic disease is controversial. Comparisons between positive skin tests and positive RAST have not always shown excellent agreement, although the negative tests are invariably in better agreement.13,l4 This latter finding was strikingly manifest in the canine study reported here. Despite the very much higher levels of IgE in the canine, false-positive results were not encountered, and the agreement between negative skin tests and negative RAST in the caseof all allergens was 100%. RAST and skin testsare, of course, measuringdifferent things, one being an indication of cell-bound antigen-specific IgE and the other being an indication of the amount of the sameantibody that is circulating. No conclusive data exist on the factors controlling the ratio of cell-bound to circulating IgE. Studies in the canine on the relative distribution of total (not antigen-specific) IgE have shown a poor correlation betweenthe level of serum IgE and that bound to skin assessedby either reversed cutaneousanaphylaxis or by the percentageof mastcells with IgE demonstrable by immunofluorescence.l5 In the light of these considerations, it would indeed be surprising if results of skin tests and RAST were invariably in good agreement. It is at first sight surprising that in studies in man correlations between RAST and provocation tests have been somewhat closer than those of skin tests and provocation, I4as both of the latter are indications of cell-bound IgE. However, in the light of present
CLIN. IMMUNOL. OCTOBER 1979
stateof knowledge of the dynamics of total IgE, there is no reasonto believe that the distribution of antibody in the respiratory mucosa and the skin must, of necessity, be closely parallel. Provocation tests were not performed on thesecanine patients, but the results of house dust RAST merit consideration. Before the testswere performed, a group of set-awere singled out for use in the development of the test from animals whose history of house dust allergy was particularly convincing. The averagestrength of the positive skin tests in this group to 250 PNU of house dust did not differ appreciably from that of the group that had a poor history and who manifested their disease predominantly in the pollen season-yet the difference in their RAST results using mite extract coupled to the discs was striking, namely, a 42% agreementbetween positive skin test and positive RAST for the good history group and a zero agreementfor the poor history group. The fact that the skin testswere performed with whole dust extract rather than mite extract, which was not available in a form suitable for intradermal use at that time, may appear to limit the significance of thesedatabut probably detractsfrom it only minimally. The widely held contention in allergy practice in man that the house dust mite is the major allergen in housedust is supportedby recent studiesin the dog.16Skin tests were performed on 50 suspected atopic dogs using three IO-fold dilutions of a 250PNU extract of house dust (Greer Laboratories, Lenoir, N. C.) and the same strengths of D. furinae extract (Hollister-Stier Laboratories, Spokane, Wash.). Thirty-eight of these gave positive tests to 250 PNU of both extracts, and 12 gave negative tests to both. Positive testswere more often encounteredto higher dilutions of mite extract than to dilutions of whole house dust extract. This is interpretable as strong evidence that D. farinae is the major allergen responsible for house dust allergy in atopic dogs. The marked difference in positive RAST and skin test agreement between the different allergens is noteworthy. At two extremes, there was an 82% agreement between positive skin tests and positive RAST in the caseof ragweed, and only 12.5% in the case of dandelion. Detailed analysis of the variables in the performanceof RAST in man have been published17*ia and are plannedfor the canine system. One factor which was shown to be important was the amount of allergen attached to the solid phase-the greater the amount, the better was the discrimination. la Thus, the presenceof more specific allergen, rather than total protein, on the disc could well account for the noted variation in test agreementwith the different allergens. Further consideration of the house dust results supports this concept. RAST tests
VOLUME NUMBER
62 4
Radioallergosorbent
ANTIGEN
BLANK
HIGH IgE C ONTROL
atopic disease
241
ENGLISH PLANTAIN
TIMOTHY GRASS ATOPICS ,KIN TES’ POS
test in canine
AlOPICS SIKIN TEST NEG.
KrofKs
