Detection of allergy to nuts by the radioallergosorbent test Donald Gerald

N. Gillespie, M.D., Shiginori J. Gleich, M.D. Kochester,

Nakajima,

M.D.,

and

M&n.

The diagnosis of food allergy is often di&u,lt to make by conoentional mean.% IIistories are frequently ambiguous, and skin testing is of dubious reliability because of the number of false-positive and false-negative reactions. We hate evaluated the radioallergosorbent test (RAST) for the in vitro measurement of the specific IgE antibodies to nuts, including Brazil nut, almond, walnut, pecan, Gashe%, and the legume, peanut. Serums were obtained from 18 patients with a history of nut allergy and IgE level and speoifio IgX antibodies were measured. Thirteen of the 18 patients had significantly elevated IgE antibody (greater than tzc;ice control) to one or more of the allergens. Prawn&-Kiistner tests on selected serums in general corroborated the results of the in vitro studies. Five patients had RAST elevations to B or more wuts. As a group RAST-positive patients had elevated mean serum IgE levels and m.ore severe clinical symptoms (p < 0.01). The speoifioity ati cross-reactivity of IgE antibodies to different nut antigens w(w’ investigated by RAST inhibition zcith serums from 5 patients having high levels of IgE antibody. In 4 patients no crossreactivity betccpen Brazil nut and peanlct was found. In contrast, several nut extracts inhibited the reaction of pecan allergen vith IgE antibodies. These results indicate that specific IgB antibodies can be measured by RAST in patients with nut allergy and the cross-reactivity of nut antigens can be investigated. RAST u*ould appear to be most useful in confirming the diagnosis of nut hypersensitil:ity in chilikn or in highly allergic patients in ,whom skin testing poses a risk of anaph,ylaxis.

The diagnosis of allergy to foods has long been a vexing problem in medical practice. The physician is usually dependent on the history as the results of skin-testing are frequently equivocal.‘, 2 Also, no reliable laboratory test is available. Presently the best approach to the diagnosis of food allergy consists of obtaining a careful history followed by elimination diets and challenge with the suspected foods. Since the review in 1949 by Ingclfinger, Lowell, and Franklin3 of gastrointestinal allergy, many in vitro laboratory tests have been described*+ but none has been widely accepted. IIaddad and KorotzerlO have reported a specific method for detecting reaginic antibodies to foods using the rat mast cell degranulation technique, but this remains a difficult experimental procedure, The introduction of the radioallcrgosorbent test (R.AST) by Wide, BenFrom the Departments of Pediatrics, Medicine, Immunology, and Microbiology, the Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Graduate School of Medicine. Rupported in part by Grant No. AI-11483 from the National Institute of Allergy and Tnfectious Diseases! Sational Institutes of Health, United States Public Health Service, and by a contract with the Food and Drug Administration, 223-73-1164. Received for publication Nov. 18, 1974. Accepted for publication March 13, 1975. Reprint requests to: Gerald J. Gleich, M.D., Mayo Clinic and Foundation, Rochester, Minn. 55901. Vol.

57, No.

4, pp.

304-309

VOLUME NUMBER

Allergy

57 4

to nuts

303

nich, and Johansson’l provided a tool for the in vitro detection of specific IgE antibodies to allergens. Clinical studies have shown this procedure to be a reliable index of allergy to various allergens. ml5 In this study we have used the RAST for the detection of IgE antibodies to nuts. MATERIALS Patients

AND

METHODS

We studied 18 patients who responded to an advertisement requesting serum from individuals with a history of sensitivity to nuts. Clinical histories ranged from vague gastrointestinal discomfort to severe anaphylactic reactions. The severities of the clinical reactions were graded by assigning a score of 1 to each of the following signs and symptoms: urticaria, respiratory distress (laryngospasm or asthma), angioedema, hypotension, and unconsciousness. The serums from 4 patients were tested for their capacity to sensitize passively normal skin by the method of Prausnitz-Kiistner (P-K test) in 2 recipients.1 None of the serums contained the Australian antigen. In the performance of the test 0.1 ml of serum was injected intradermally and 48 hr later the sites were challenged by injection of 0.004 ml of a 1: 100 concentration of allergen.

Measurement IgE described

of IgE protein

was measured previously.16

in the serum

of all 18 patients

by double-antibody

radioimmunoassay

as

Allergens Peanut, almond, Brazil nut, black walnut, English walnut, cashew, and pecan extracts, all in 50T0 glycerin, were purchased from Hollister-Stier (H.S.) Laboratories (Spokane, Wash.) .* Peanut and almond extracts were prepared in our laboratory after defatting with acetone and diethyl ether. These defatted nuts as well as undefatted raw and roasted peanuts were ground and extracted with distilled water at 4’ C over a 24-72 hr period. The undefatted extracts were centrifuged at 17,000 rpm for 30 min and the clear layer between the top fatty and bottom particulate layers was removed. Final concentrations of peanut allergens based on dry weight measurements were 16 mg/ml for the raw peanut extract, 30 mg/ml for the roasted peanut extract, and 38 mg/ml for the defatted raw peanut extract.

Performance

of the RAST

Eleven different nut antigen preparations were coupled to microcrystalline cellulose. Then 3 ml of peanut extract containing approximately 100 mg dry weight or 3 ml of the various nut extracts (1 :lO), respectively, were added to 500.mg samples of microcrystalline cellulose activated by reaction with cyanogen bromide as described elsewhere.17 After mixing overnight at 4” C the solid-phase antigens were washed with cold 0.2M H,PO,-0.04M NaOH-0.16M NaCl (pH 8.1) until the 280 nm absorbance of the supernatant solutions was less than 0.03. The washed solid-phase allergens were suspended in O.lM pH 7.4 K,HPO,-KH,PO, buffer containing 4% fetal calf serum and 0.1% NaN, and stored at 4” C. The performance of the RAST has been described previously .ls, 1s Briefly, solid-phase allergens are incubated with serum, washed, incubated with radiolabeled antibody to IgE, washed again, and finally counted in a gamma scintillation counter. The radioactivity associated with the solid-phase antigen is a measure of the specific IgE antibody level and in this report the result is expressed as counts bound divided by total counts x 100. The coeficient of variation of RAST in these studies was 19.5%.

“In

this paper legume.

we will

refer

to the peanut

as a nut,

although

we recognize

that

the peanut

is a

VOLUME NUMBER

57 4

TABLE

I. Characteristics

Pt.

Allergy

,of

Associated allergies

History of nut allergy

SW

31

13

M F

3

6

M

4

53

F

Peanut Peanut and many nuts AR, A, E Peanut and many nuts Peanut AR, A A, AR Many nuts A, AR, E Many nuts A, AR, E Brazil nut,

14

A, AR? A, E, U

ii 7

::

iti M

8

40

F

AR, E

9 10

43 34

M M

AR AR

1:

33 24

F

AR, E

13 14

23 28

F F

0” A, AR

15 16

40

ii

Clinical reaction score

Clinical reaction

II. RAST

Time since reaction (yr)

3f

2,300 7,600

:

Ang, U, A

3+

1,600

2

Ang, TT, syncope, pruritus A% U Ang, U, TT, A An , U, TT, iarrhea, syncope Ang, A, TT $giTU. A ,

5+

473

2

i,’

3,220 1,760 268

-? 8

3,120

25

341 1,560

6 2

1,089 134 209 229

7

3t

Brazil nut and many nuts Many nuts Brazil nut, walnut, pecan Many nuts Ang, U, dyspnea Walnut, pecan Ang, U Many nuts Mouth ulcers Peanut, pecan, U, A walnut, cashew Many nuts Ang, U, TT English wa 1nut Nausea Peanut TT, heartburn Many nuts Pruritus

5’ 3+

! 26

Ang to angioedema,

results Solid-phase

Patient

Serum W (rig/ml)’

U, syncope Ang, TT, pruritus

!ZR AR ii 30 4”; E A, AR 0: no other allergic symptoms. *The 94% confidence interval is 6-780 ng/ml.ls tA refers to asthma, AR to allergic rhinitis, E to eczema, U to urticaria, and TT to throat tightness. SHistory not obtained. $Now able to ingest peanuts in small quantities.

TABLE

305

patients

Age

:

to nuts

Peanut

Almond

Brazil

nut

antigen

Black walnut

English walnut

Pecan

Cashew

:

24.4* 16.7

031

1

0.71 0.60

-

-

0.80

:

3”:::

01 86

1.17

-

0.84 -

1-

63 -

;

ii

-

i?

ii -

11.8 028 4.21 1.20

4.01 q -

2.34 -

= -

2>3 -

1;

-

-

0.87 6.71

-

II

1

1

t: 5.54 2.37 3.22 68 1.26 =13 OS8 1 Nonatopic O.SOfo.O6$ 0.36f0.03 0.29 i 0.04 0.28 f 0.03 0.38yO.08 0.50f0.06 0.34yO.03 controls *Results are expressed as counts bound to solid-phase allergen divided by total counts x 100. Positive tests were at least twice the control value. t Negative ( < twice control value). E ? 2 SEM.

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307

relation between a high serum IgE value and a high clinical reaction score ( rR = i-0.66; p < 0.01). The specificity of the reactions between IgE antibodies and nut allergens was investigated by inhibition experiments and the results are shown in Table III and Fig. 1. No significant cross-reactivity between peanut and Brazil nut was found in the 5 patients tested. This was true in patients with a positive test to both peanuts and Brazil nuts (Patients 3 and 6) as well as in patients with a positive test to only 1 nut antigen (Patients 4 and 10). Pecan was the only nut extract tested that significantly inhibited the reaction in the peanut RAST. Significant inhibition of the pecan RAST was produced by peanut, Brazil nut, black walnut, and pecan itself. The serums of two patients (Nos. 3 and 10) were further tested with a variety of foods, as shown in Fig. 1. These results confirmed the absence of cross-reactivity between peanut and Brazil nut and also showed no major inhibition of the peanut and Brazil nut R,AST by English walnut or cashew. In Patient 3, who was highly sensitive to peanut, no inhibition by pea extract was found. Passive-transfer tests were performed on serums from 4 patients, 2 with (Nos. 3 and 4) and 2 without (Nos. 15 and 17) elevated levels of IgE antibodies to nuts. In the case of Patient 3, who was sensitive to a variety of nuts, P-K tests were positive to all of those implicated by RAST. In addition, this patient had a positive test to pea extract, a food which he is able to ingest. Patient 4 had a strongly positive test to peanuts and a weakly positive test (7-mm wheal anti 23mm erythema) to Brazil nut. Concerning the 2 RAST-negative serums, Patient 17 had negative P-K tests with Brazil nut and peanut whereas Patient 15 had a positive P-K test with peanut (12-mm wheal and 3%mm erythema) and a weakly positive test with Brazil nut (7-mm wheal and 2%mm erythema) . DISCUSSION In this study we determined whether RAST could be employed for the detection of IgE-mediated food allergy. For this purpose we selected for study nut allergy because this form of sensitivity is particularly florid. Our results support the view that in many patients immediate-type nut allergy is mediated by specific IgE antibodies. As a group the nut-sensitive patients in our study had a high frequency of atopic diseasesand most had elevated total serum IgE levels. When examined individually, however, it is apparent that one need not have a serum IgE value above the normal range to have an elevated level of specific IgE antibodies against nut antigens and to be clinically sensitive. Our results deal only with IgE antibodies and they do not exclude a role for IgG reagins.20 The results of the inhibition experiments on the serums from patients sensitive to different nut antigens support the view that there is little, if any, antigenie cross-reactivity between peanuts and Brazil nuts. Moreover, in Patient 3, it is of interest that another legume-pea-does not inhibit the peanut RAST to a significant degree. Since Vaughan’szl biologic classification of foods, crossreactivity of antigens within the same biologic family has been suspected. In Patient 3 this doesnot appear to be the case. Again in this instance the laboratory

308

Gillespie,

Nakajima,

and

J. ALLERGY

Gleich

CLIN.

IMMUNOL. APRIL 1976

test confirms the medical history in that Patient 3 is able to tolerate peas but is exquisitely sensitive to peanuts. The RAST has proved to be a useful test for the investigation of respiratory allergies. I3 Our results support the view that the RAST will aid in the detection of reaginic-mediated food allergies in man and they corroborate the reporm of Hoffman and Haddad*? dealing with a variety of food allergens, including nuts, as well as the report of Schur, Hyde, and Wypych2” dealing with egg-white sensitivity. We thank

Mr.

Richard

T. Jones

for

assisting

in certain

of these

experiments.

REFERENCES 1 Sheldon, J. M., Lovel, R. G., and Mathews, K. P.: A manual of clinical allergy, ed. 2, Philadelphia, 1967, W. B. Saunders Co., pp. 196-225. 2 Ancoma, C. R., and Schumacher, T. C.: The use of raw foods as skin testing material in allergic disorders, Calif. Med. 73: 476, 1950. 3 Ingelfinger, F. J., Lowell, F. C., and Franklin, W.: Gastrointestinal allergy, N. Engl. J. Med: 241: 303, 1949. 4 Coca, A. F.: Familial non-reaginic food allergy, Springfield, Ill., 1943, Charles C Thomas, Publisher, p. 96. 5 Vi~ughn, W. T.: Leukopenic index as a diagnostic method in the study of food allergy, J. Lab. Clin. Med. 21: 1278, 1936. 6 Shioda, H., and Meshima, T.: The significance of mast cells in nasal smears from patients with food allergy, J. ALLERGY 37: 321, 1966. 7 Popa, V., Teculescu, D., Stanescu, D., and Garnilescu, N.: The value of inhalation tests in perennial bronchial asthma, J. ALLERGY 37: 321, 1966. 8 Bullock, J. I)., Bodenbender, M. T., Kantnas, S. B., and Miller, C. E.: Skin window as a diagnostic tool in food allergy, Ann. Allergy 26: 17i, 1968. 9 Bryan, W. T.: Cytotoxic reactions in the diagnosis of food allergy, Otolaryngol. Clin. North Am. 4: 523, 1971. hypersensitivity reactions to food antigens, 10 Haddad, Z. H., and Korotzer, J. L.: Immediate J. ALLERGY CLIN. IMMUNOL. 49: 210, 1972. 11 Wide, L., Bennich, H., and Johansson, S. G. 0.: Diagnosis of allergy by an in vitro test for allergen antibodies, Lancet 2: 1105, 1967. 12 Berg, T., Bennich, H., and Johansson, 5. G. 0.: In vitro diagnosis of atopic allergy. I. A comparison between provocation tests and the radioallergosorbent test, Int. Arch. Allergy Appl. Immunol. 40: 770, 1971. 13 Aas, K., and Johansson, S. G. 0.: The radioallergosorbent test in the in vitro diagnosis of multiple reaginic allergy, J. ALLERGY CLIN. IMMUNOL. 48: 134, 1971. 14 Lichtenstein, L. M., Ishizaka, Ii., and Norman, P. S.: IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 52: 472, 1973. 15 Yunginger, J. W., and Gleich, G. J.: Seasonal changes in IgE antibodies and their relationship to IgG antibodies during immunotherapy for ragweed hay fever, J. Clin. Invest. 52: 1268, 1973. 16 Gleich, G. J., Averbeck, A. F., and Swedlund, H. A.: Measurement of IgE in normal and allergic serum by radioimmunoassay, J. Lab. Clin. Med. 77: 690, 1971. 17 Yunginger, J. W., and Gleich, G. J. : Comparison of the protein-binding capacities of cyanogen bromide-activated polysaccharides, J. ALLERGY 50: 109, 1972. 18 Yunginger, J. W., and Gleich, G. J.: Seasonal changes in IgE antibodies and their dationship to IgG antibodies during immunotherapy for ragweed hay fever, J. Ch. Invest,.

52: 1268, 1973.

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19 Gleich, G. J., and Jones, R. T.: Measurement of IgE antibodies by the radioallergosorbent test. I. Technical considerations in the performance of the test, J. hILERcY CLIK. Ilrnrr’xor,. 55: 334, 1975. 20 Parish, 1%‘. E. : Skin sensitizing non-TgE antibodies: Association between human IgG S-TS J., editors: Progress in immunology, Amsterdam, and IgG,, in Brent, L., and Holborow, North-Holland Publishing Co., vol. 4, p. 19, 1974. 21 Vaughan, 15’. T.: Food allergens. I. A genetic classification with results of group testing. J. ALLEKCY 1: 385, 1930. 22 Hoffman, D. R., and Haddad, Z. II.: Diagnosis of IgE-mediated reactions to food antigens by radioimmunoassay,J. ALLERGY CLIN. IMMUNOL.~~: 165,1974. sensitivity and atopic eczema, J. 23 Schur, S., Hyde, J. S., and Wypych, J. 1.: Egg-white

ALLERGY CLIN. IMMUNOL. 54: 174,1974.