The structuring of an allergy index based on IgE-mediated skin sensitivity to common environmental allergens

The structuring of an allergy index based on IgE-mediated skin sensitivity to common environmental allergens*

Linda R. Freidhoff, M.S., David G. Marsh, Ph.D., Deborah A. Meyers, Ph.D., and Rabia Hussain, Ph.D., Baltimore, Md.

We computed skin-test sensitivity levels in 485 adults puncture-tested with eight standardized, high-quality inhalant allergens tested at single concentrations. In order to quantitate the *‘average” IgE-mediated skin sensitivity of each subject, we used both nonparametric and parametric statistical methods to generate two “allergy indices” (Allergy Index I and Allergy Index II) based on sensitivity end-point data from the subpopulations of individuals positive to six of the eight allergens. For the 192 skin test-positive subjects, Allergy Index I and Allergy Index II were signijicantly correlated with each other (r, = 0.98, p < 0.001) and with the number of positive skin-test reactions (rS = 0.9, p < 0.001) as well as with log[total serum IgE] (r z 0.4, p < O.OI). In IO2 ragweed-positive subjects, log[spec@ IgE to ragweed] was significantly correlated with ragweed-specific “ragweed indices I ana’ II” (r x 0.6, p < O.OI). Furthermore, the average daily symptom scores reported by 14 ragweed-positive subjects during the ragweed pollination season were significantly correlated with ragweed indices I and II (p < 0.05). We propose the use of Allergy Index II in epidemiologic and genetic studies of allergic phenotypes as well as in clinical decisions for diagnosis and immunotherapeutic intervention. (J ALLERGYCLINIMMUNOL 72:274-287, 1983.)

Skin testing with unstandardized allergenic extracts has been used as a diagnostic tool in many investiga- tions of the prevalence of atopic sensitivity in populations. ‘-I3 Apart from two recent studies,lZ* i3 arbitrary cutoff points have been used to discriminate between positive and negative reactions,‘, 4, .i, *, lo or among degrees of positive response.2-4s 7, *, lo We believe that skin testing is the simplest and, potentially, the most meaningful and objective technique for allergy diagnosis. However, much could be done to improve both the quality of the allergens and the methods of collection and evaluation of the data. Powerful im- munochemical techniques such as CIE are now avail-

From the Division of Clinical Immunology, The Johns Hopkins University School of Medicine at the Good Samaritan Hospital, Baltimore, Md.

Supported by National Institutes of Health Program Project Grant AI- 13370.

Received for publication Oct. 21, 1982. Accepted for publication March 28, 1983. Reprint requests: Linda R. Freidhoff, The Good Samaritan Hospi-

tal, 5601 Loch Raven Blvd., Baltimore, MD 21239. *Publication No. 499 of the O’Neill Research Laboratories, The

Good Samaritan Hospital, Baltimore, Md.

able for quality control of allergen extracts. Also, a more appropriate method of quantitating IgE-mediated skin sensitivity toward an array of environmental allergens would be to allow for a continuum of response rather than the use of discrete cutoff points. This approach would permit a more rigorous statistical analysis of epidemiologic and genetic factors that determine the expression of atopic disease and would enable the clinician to make more rational decisions for both diagnosis and treatment.

Using the skin-puncture techniquei I5 with a panel of standardized, high-quality inhalant allergens, we computed the levels of skin-test sensitivity (“sensitivity end points”) toward these allergens in 485 adults. Each person’s “allergy indices” were then derived by both nonparametric and parametric statistical approaches (allergy indices I and II, respectively). In order to compare allergy indices I and II and to validate the concept of an allergy index as a reliable measure of skin sensitivity, (1) the allergy indices for the group of 192 skin-test positive individuals were investigated in relation to total serum IgE levels, (2) in a subset of these subjects, the relationship between their allergy indices and the severity

274

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A hhreviations used CIE: Crossed immunoelectrophoresis

CRlE: Crossed radioimmunoelectrophoresis FDA: Food and Drug Administration HSA: Human serum albumin IUIS: International Union of Immunological

Societies r: Correlation coefficient (linear

regression analysis) r,: Correlation coefficient (Spearman

Rank Order Test) RAST: Radioallergosorbent test RIST: Radioimmunosorbent test AgE: Antigen E RID: Radial immunodiffusion

DEAE: Diethylaminoethyl PNU: Protein nitrogen units

of allergic symptoms was studied, and (3) specific indices for ragweed were compared with ragweed- specific IgE antibody levels and seasonal symptoms toward ragweed pollen.

MATERIALS AND METHODS Subject selection

We studied a total of 485 Caucasian subjects (316 M, 169 F) with ages ranging from 20 to 64 yr [mean 40 + 11 (S.D.)]. All were employees of the Westinghouse Electric Corporation in northern Baltimore County, Md., who were found by a genetic-epidemiologic study of human immune responsiveness to allergens, described in detail elsewhere.14 They comprised the following subgroups: (1) a stratified random sample of 320 subjects, equally distributed with respect to sex and age (40 persons of each sex for each of the age ranges 20-30, 31-40, 41-50, and 51-60) and (2) an additional 165 self-reported allergic subjects (156 M, 9 F; mean age 40 ? 9). The total sample included the 361 Caucasian subjects reported previously.14 One hundred ninety-two (155 M. 37 F; age 20 to 58 yr, mean 37 2 10) subjects were skin test-positive to at least one allergen in our puncture test panel (Tables I and II).

Symptom-medication diaries

Twenty-six (23 M, 3 F) of the 192 skin test-positive subjects were part of a separate 12 mo longitudinal study of allergic responsiveness (unpublished). Fourteen of these subjects were positive to our short ragweed extract. Throughout the year they kept weekly symptom diaries, used to record the number of days per week that the subject had symptoms of hay fever and/or asthma as well as the number of antihistamine and/or steroid tablets taken per week. Two subjects were dropped because they had taken large quantities of steroids and it was difficult to weigh the effect of these drugs against that of the antihistamines taken by other study subjects. Two further subjects were dropped

Allergy index 275

because they had taken very large quantities of antihistamines. Using the daily diary developed by Norman et al. ,IB the subjects recorded various types of allergic symptoms and the type and strength of any medications taken during the ragweed pollination season. We then obtained mean daily symptom-medication scores based on the duration of symptoms and the use of relevant medications such as antihistamines. ”

Antigens

All antigens were dialyzed, lyophilized preparations, stored at -20” C in sealed containers. Consistent lots were used throughout the study. The extracts were dialyzed in order to enable comparisons based on “nondialyzable solids” to be made between extracts and to remove any hygro- scopic materials.

Pollen extructs. Short ragweed (Ambrosia elatior) and timothy grass (Phleum prutense) pollens were obtained from Greer Laboratories, Inc., Lenoir, N.C., and perennial rye grass (Lo&m perenne) from Bencard Allergy Unit, Betchworth, Surrey, U.K., within 2 mo after collection. The dried pollens. stored at -20” C in sealed containers until used, were known to possess a high degree of allergenic activity on the basis of previous studies.* All the selected pollens (100 to 200 gm) were defatted by succes- sive extraction with 8 X 1 L of peroxide-free diethyl ether and dried under vacuum. The defatted pollens were then extracted with stirring for 20 to 24 hr at 4” C with 0.125M NH4HC03 at pH 7.8 (1: 10, w/v), dialyzed in size 18 Visk- ing tubing (Visking Corp., Chicago, Ill.) against 5 x 72 L of 0.002M NH,HCO,, and finally against 2 x 72 L of deionized water over a period of 4 days and lyophilized.” In order to compare the allergenic activity of a rapidly released, basic antigen fraction of ragweed with that of a more conventional extract (see refs. 18 and 19), a second batch of defatted short ragweed pollen was extracted for 16 min at 23” CYR and dialyzed and lyophilized similarly to the other pollen extracts.

Other extracts. Pilot tests were performed with several commercial aqueous extracts of cat and dog danders, the molds Alternaria, Aspergillus, Cladosporium. Helminthos- porium, and Penicillium as well as house dust and house dust mites (Dermatophagoides pteronyssinus). All extracts (1 : 10, w/v) were dialyzed and lyophilized similarly to the pollen extracts and were tested by the puncture method (see below) at concentrations of 0.01, 0.1, I .O, and 3.0 mg/ml in groups of 15 to 25 persons considered to be clinically allergic respectively to cat, dog, molds, and house dust (i.e., they reported symptoms after exposure to the relevant allergen), as well as 15 nonallergic controls. On the basis of these studies, we eliminated all mold extracts except Alter-

*Some preliminary studies were also performed using extracts of oak and “mixed tree” (equal quantities of ash, birch, beech, hickory, maple, and sycamore) pollens. However, these extracts were eliminated in order to limit the number of skin tests. Of the few people who were sensitive to these extracts, all were also sensitive to ragweed and/or grass pollen extracts.

276 Freidhoff et al J. ALLERGY CLIN IMMUNOL. SEPTEMBER 1983

TABLE I. Crude allergenic extracts used for skin testing

Antigens Concentration

(mglml) Sites*

Pollens Short ragweed (Ambrosia

fllatior) 74 hr 16 min

Perennial rye grass (Lo/km perenne)

Timothy grass (Phleum pratense)

Animal danders Cat Dog

Other Alternaria spp. House dust

Histamine (positive control)” Diluent (negative control)“

0.3 1.15 0.3 11 0.3 2,14

0.3

3.0 3.0

1.0 3.0 0.5

3.13

4 5

6 9,12

IO

.<Sixteen skin-test sites were distributed evenly along both sides of the volar surface of the right forearm, the other arm being re- served for tests of highly purified allergens (not discussed here). Sites 1 through 8 were positioned consecutively on the radial side of the arm, with site 1 closest to the thumb. Sites 9 through 16 were positioned consecutively on the ulnar side of the arm, with site 16 closest to the elbow. Site 9 was located ap- proximately 12 mm farther from the wrist area than site 1. Sites 8 and 16 were used for controls. In 124 random subjects re- plicate tests of histamine (site 12) and timothy (site 13) were replaced by additional allergens (not discussed here).

RHistamine concentration was expressed as concentration of histamine base in phosphate-saline-phenol (without HSA).

“Phosphate-saline-phenol-HSA (see text).

nuria. Few (< 10%) of the allergic people responded to the other mold extracts and we wished to limit the number of skin tests. Mite extract, although more potent in several subjects than house dust extract, was eliminated because the house dust extract covered a broader range of allergenic specificities. Of people responding to mite and/or house dust, 2118 were mite positive but dust negative and 7118 were mite negative and dust positive; the remainder were sensitive to both antigens.

Preparation and storage of antigen solutions. On the basis of the pilot studies discussed above, the most allergen- ically reactive antigens were selected. Antigen concentrations were chosen that were sufficiently high to pick up all clinically atopic people, but insufficient to induce extremely large local reactions or systemic symptoms in highly atopic subjects. The use of high concentrations of house dust extract was also limited by the possibility of toxic reactions. The antigen solutions were prepared in the following buffer: NaCl (5.00 gm), Na,HPO, . 7H20 (1.07 gm), KH2P04 (0.36 gm), phenol (4.00 gm), and HSA (0.30 gm) plus deionized water to 1 L. The solutions were sterilized and

stored in small aliquots at -70” C until used. After thawing and dilution as necessary in the same buffer, they were maintained at 4” C (including during the skin test sessions) for a maximum of 4 mo. Antigenic analysis of AgE content of the ragweed solutions and of group I content of the grass solutions showed no detectable loss in comparison with the lyophilized controls. Also. extensive comparison of the skin-test activities of freshly thawed solutions vs. materials, stored at 4” C for 5 mo, revealed no detectable loss in potency. HSA was used to stabilize the antigen solutions”’ but was not considered necessary for the histamine control (500 pgiml histamine base). which was freshly obtained from the Johns Hopkins Pharmacy as needed.

Antigenic analysis

Radial immunodiffi.~ion.21 Ragweed AgE, Ra3, Ra5, and Ra6 were analyzed in the two ragweed extracts and group I and group II in the rye and timothy grass extracts by using monospecific antisera and highly purified antigen standards (cf. refs. 2 1 to 25). Analyses of Cat- 1 and cat albumin in the cat extract were kindly performed by Dr. John Ohman, Veterans Administration Hospital, Boston.2” Cat-l was also analyzed in our laboratory by using antigen standards and an anti-Cat-1 serum supplied by Dr. Harold Baer, FDA.

In order to compare the extracts used for this study against available FDA, IUIS, and other reference preparations, we also performed relevant RID analyses on these reference materials.

Crossed immunoelectrophoresis. Analyses were made both of the extracts used for the Westinghouse study and of the selected reference materials (where available). The methods used were analogous to those of Lowenstein” and Lowenstein and Marsh.‘H Antisera were raised in nine rabbits toward short ragweed (24 hr), in 11 rabbits toward short ragweed (16 min), and in three goats toward rye grass extract. Immunoglobulin G fractions (2.5 to 6 times concentrated) were obtained by ammonium sulfate precipitation and DEAE-cellulose chromatography of each of the serum po01s.*~ Immunoglobulin G fractions (5 times concentrated) from antisera raised in groups of three rabbits toward timothy grass, Alternaria, cat, and dog extracts were prepared by ALK and Dako Laboratories, Copenhagen, Den- mark, and were kindly made available by Dr. Henning Lowenstein, University of Copenhagen. The precise condi- tions used in the CIE experiments are included in the legends to Figs. 1 to 3.

Puncture skin testing

Each subject was skin-tested with eight crude allergen extracts plus histamine and diluent controls (Table I). The skin test procedure’” was a modification of a semiquantita- tive puncture method developed by Santilli et al. l5 and con- sisted of applying drops of the allergen solutions to the forearm, followed by pricking through the drops with a bifurcated smallpox vaccination needle (Wyeth Laborato- ries, Marietta, Pa.) designed to introduce a small, repro- ducible amount of antigen. Any positive reaction was recorded for subsequent quantification by tracing around the

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TABLE II. Antigen contents of Westinghouse skin test extracts and of reference materials

Ragweed extracts

Short ragweed, W 24 hr 16 min

FDA (BOB) standards EIRa,’ E2Ra, ’ E3Ra.’ EhRa’

IUIS reference” Am.e.76”

AgE Wwd

34.5 k 0.7 1.18 k 0.04

8.4 ? 0.3 27.5 k 1.8 33.6 k 1.7 18.8 + 1.2

36.3 + 1.5 23.9 k 1.8

Ra3

hh3)

3.10 k 0.08 3.32 k 0.28

5.03 +- 0.44 5.43 2 0.21 5.66 k 0.13 4.65 t 0.36 4.73 2 0.39 2.28 2 0.08

Ra5

hh4)

3.92 + 0.03 5.01 ? 0.41

2.54 L 0.18 2.47 k 0.23 4.28 t 0.47 2.71 +- 0.17 2.68 ? 0.25 2.52 k 0.20

Ra6

hh4)

8.53 k 0.56 16.2 -t 0.8

10.7 + 0.7 9.38 5 0.64 10.2 L 1.1 10.8 + 0.3 9.2 t 0.5

N.D.

Grass extracts

Rye grass, W Timothy grass, W’ FDA (BOB) rye standards

E2Ry E3Ry E8Ry

IUIS ref. (timothy)’

Group I

how)

96.0 k 5.2 58.1 k 8.3

40.5 k 1.2 29.2 t 1.9 65.3 k 2.5 51.8 k 6.7

Group II

Ww)

48.7 -+ 4.8 10.5 2 1.6

30.3 + 3.0 31.0 * 2.0 42.6 k 1.7 5.37 * 0.56

Cat extracts Cat-l

(pdw)

Cat albumin

kt~w41

Cat, W" 89 329 Cat 149 (Ohman std.)” 105 326

W = materials used in the Westinghouse study; Am.e. = Ambrosia elazior. Values are means and standard deviations of at least six determinations. ‘Original determinations were made with 1: 10, w/v, extracts. The cited antigen contents are based on recovery rates of 12.0 mg/ml non-

dialyzable solids for a 1 : 10, w/v, concentration of short ragweed pollenlY and 10.7 and 7.5 mg/ml nondialyzable solids for rye and timothy grass pollens, respectively (unpublished).

HSee ref. 28. ( Standard deviations for group I and group II determinations for timothy tend to be higher because of the faintness of the RID circles. ” Values determined by Dr. John Ohman using his “old” standard.‘” The weight estimates are only approximate micrograms in the case of

the determinations of Cat-l. The cat, W, preparation was also found to contain 3.06 ? 0.27 FDA U/mg in assays performed in our laboratory based on the FDA El-Ct reference

wheal 15 min after puncturing the skin. The tracing was transferred to a 0.1 by 0.1 inch graph paper with transparent surgical tape (Transpore; 3M Co., Minneapolis, Minn.). Skin tests that were difficult to interpret or poorly repro- duced in duplicate tests with the same antigen were repeated on a different occasion.

A series of pilot studies, including comparison with mea- surements obtained by counting 0.1 mm squares within the wheal. were used to determine the most accurate way to evaluate skin tests. Any wheal response was considered negative if its diameter was less than 2 mm (not distinguish- able from the needle puncture) after subtraction of any wheal response to the diluent control. For positive wheal reactions with diameters 4 mm or less, the diameter of the wheal was recorded as the average of the maximum length plus the length of the perpendicular bisector of that maximum length. For the generally irregular wheal reactions

with diameters greater than 4 mm, the numbers of whole and partial 0.1 inch squares within the wheal tracing were counted. Equating a partial square with 50% of a whole square, we converted the count to area (mm2) and computed the diameter (mm) of a circle having the equivalent area.

Computation of allergic sensitivity

As assessed by skin testing, a patient’s allergic sensitivity toward a particular allergen may be expressed by the allergen concentration that gives rise to a wheal of a particular diameter on the linear portion of the wheal diameter vs. log[antigen] titration curve. 3o For intradermal titration, we have assessed this end point as the concentration giving rise to a “2-plus” reaction (8 to 10 mm wheal with erythema) after injection of 0.05 ml of allergen solution.31 In the pres- ent study, we wished to compute a similar sensitivity end point from a puncture test performed at a single allergen

278 Freidhoff et al. J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1983

concentration and to relate the computed end point to that used for the intradermal method.

Therefore seven rye grass-sensitive clinic patients were tested, in duplicate, with a partially purified sample of Rye II antigen* at concentrations from 3 X 10-l to 10:’ pgiml (puncture method) and from lO-fi to 10m2 pgiml (intrader- ma1 titration) in half-log,, steps until unequivocally positive reactions were obtained for at least four concentrations with each procedure. Wheal diameters (calculated as described above) were averaged for the duplicate determinations and the slope of the regression line for wheal diameter vs. log[antigen] was computed for each subject. From these data. we determined the relationship between the puncture and intradermal methods and computed sensitivity end points for puncture tests at single concentrations in all study patients (see Results).

Computation of the allergy index (Allergy Index II) and other indices

In developing an allergy index based on a person’s sensitivity toward the entire panel of allergens, we took two approaches: (I) making few assumptions about the nature of the population from which our data were drawn (a nonparametric approach) and (2) making those assumptions generally applied to a population of interest (a parametric approach), including normality of the distribution of the responses in that population. By showing a strong correlation between the two resulting distributions, we hoped to be able to use not only the generally greater power and efficiency of the parametric techniques but also to substan- tiate any primary conclusions with appropriate nonparametric tests. We also developed indices for atopic sensitivity toward each antigen in the panel. Evidence of site-to-site differences in reactivity along the forearm (see footnote to Table I and Results), led us to consider each skin-test site separately.

The computation of the nonparametric index (Allergy Index I) proceeded as follows:

I. All individuals reacfing positively to an antigen at a particular test site were arranged sequentially from the least to the most sensitive, on the basis of the computed puncture-test end points.

2. An integer from 1 to N was assigned to each subject within the sequence, and an individual index for each allergen was computed as follows:

Sequence Nb, x loo N

Two individuals with the same sensitivity to an allergen had the same index for that allergen and all negative reactions were assigned a score of zero.

3. The values for the replicated sites of (I) short ragweed

*This antigen fraction was chosen because patients were available who exhibited a wide range of sensitivities toward this particular material. Complete purification of Rye II (sallergen y) has been described previously.25

(24 hr) and (2) rye grass were each averaged for all individuals who reacted positively at one or both sites. The final Allergy Index I assigned to each subject was the average of the individual scores to the following six allergens: short ragweed (24 hr), rye grass, cat, dog, house dust, and Alter- nariu. Timothy grass. short ragweed (16 min), and histamine were not included (see Results).

The computation of the parametric index (Allergy Index II) proceeded as follows:

1. For each antigen site, the mean and standard deviation of the sensitivities of subjects reacting positively were calculated. In order to adjust for quantitative differences in the subjects’ responses toward each extract (dependent on allergen quality, etc.; see Results and Discussion), the mean of each distribution was (arbitrarily) adjusted to the mean of the distribution for rye grass at site 2.

2. The resulting set of distributions was placed on a log,, scale of 0 to 8 corresponding to the adjusted sensitivity end points of IO” to lo-” pgiml.

3. Since the minimum value for each distribution was slightly different when the means were adjusted, a value of 0.1 unit less than the minimum value for that site was assigned to all negative reactions at that site. Therefore a skin test-negative subject was assigned an average “negative” score instead of zero (cf. computation of Allergy Index I).

4. The final Allergy Index II was generated, first by averaging the separate indices for six allergens (cf. computation of Allergy Index I) and, second. by subtracting the average “negative” score for the six selected allergens. The latter adjustment was necessary in order to assign a value of zero to subjects who were skin test-negative to all the allergens.

The individual indices for ragweed [ragweed (24 hr) indices I and II] were also used in certain computations.

Total serum IgE

A variation of the direct RIST assay was used to determine total serum IgE levels. :j2 Sepharose CL-4B-anti-IgE beads were incubated with each subject’s serum (or a standard serum or negative control) at an appropriate dilution (or set of dilutions), and the beads were washed and incubated with 12”1-anti-IgE (e-specific purified antibody). The beads were then rewashed and counted, and the quantity of IgE was computed by reference to a titration curve for the standard serum. This standard serum had initially been care- fully standardized against the WHO primary standard in three assays using individually prepared dilutions of both standards. All analyses were performed in triplicate in at least two assays and discrepant values (coefficient of variation > 10%) within or between assays were repeated in order to ensure a high degree of accuracy in the final data set. The final results were expressed in nanograms per milliliter tak- ing 1 IU s 2.42 ng.

Measurement of specific IgE antibody to ragweed

Analysis of IgE antibody levels to short ragweed allergen were performed by a modified RAST technique, essentially

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FIG. 1. CIE analyses of (A) short ragweed (24 hr. Westinghouse study); (B) IUIS short ragweed standard; (C)short ragweed (16 min, Westinghouse study). Antiserum to 24 hr short ragweed at 22 ~l/cm* was placed in all anodic, and antiserum to 16 min short ragweed at 16 yllcmz in all cathodic second-dimension gels. The experiments were performed at 15” C with a potential gradient of 10 V/cm for 30 min in the first dimension and 1 V/cm for 18 hr in the second dimension. Three hundred micrograms of antigen were used in A and C; in B, the amount of antigen applied was adjusted to the same AgE equivalence as in A. In these and subsequent CIE plates the anode is to the right in the first dimension and to the top in the second dimension.

as described by Schellenberg and Adkinson.a* Ragweed- coupled Sepharose CL4B beads were used at a concentration of 1% (v/v), which was found to be optimal after careful titration against several serum pools having high IgE and/or IgG antibody titers. Each serum was tested in triplicate and those with aberrant values (coefficient of variation >lO%) were repeated. All IgE antibody results are expressed in terms of nanograms per milliliter by reference to a titration curve for a standard serum from an individual having a known high IgE antibody content previously determined by RAST adsorption.“”

RESULTS Antigens

Table II summarizes the antigenic analyses of the two short ragweed extracts, the two grass pollen extracts, and the cat extract used in the Westinghouse study in comparison with other well-standardized reference extracts. In comparing the national (FDA) and international (IUIS) standards with our skin test materials, we based the ‘ ‘pg/mg ” determinations on yields of nondialyzable solids we have previously obtained by extracting the corresponding pollens in our laboratory (see footnote to Table II). The values for the short ragweed extract (24 hr) used in the Westing- house study compared favorably with corresponding determinations of four FDA standards, the new IUIS standard, and a reference ragweed preparation used by Lowenstein and Marsh in extensive CIE and CRIE

analyses of ragweed.%, 34, 35 The short ragweed extract (16 min) contained relatively little AgE but was rich in the basic antigens Ra5 and Ra6 (cf. ref. 19). The rye and timothy grass pollen extracts used for the Westinghouse study had higher group I and group II contents than the corresponding FDA and IUIS standards. There were no national or international standards available for the cat, dog, Alternaria, or house dust extracts. However, the Westinghouse cat extract contained levels of Cat-l and cat albumin similar to Dr. Ohman’s standard. No antigen quantitations were performed on the dog, Alternaria, or house dust extracts since appropriate standards were not available. However, some qualitative CIE analyses were kindly performed on the Alternaria and house dust extracts by Dr. Henning Lowenstein and Lise Nyholm in the Protein Laboratory, University of Copenhagen.

Figs. 1 to 3 show CIE analyses of most of the allergenic extracts used for our study. Comparisons with reference materials from the FDA or IUIS are included where available. As noted in the legend to the figures, these reference reagents were analyzed at the same AgE or group I equivalence as the Westing- house ragweed or grass extracts, respectively. The 24 hr short ragweed extract used for the Westinghouse study (Fig. 1, A) was similar qualitatively and quanti- tatively in antigen content to the IUIS reference (Fig. 1, B), in keeping with the high degree of similarity in

280 Freidhoff et al. J. ALLERGY CLIN. IMMUNOL.

SEPTEMBER 1983

FIG. 2. CIE analyses of (A) rye grass, Westinghouse study, vs. anti-rye grass; (B) rye grass (FDA ref. E8Ry) vs. anti-rye grass; (C) timothy grass, Westinghouse study, vs. anti-timothy grass; (D) timothy grass (IUIS standard) vs. anti-timothy grass. All experiments were performed at 15” C with a potential gradient of 10 V/cm for 45 min in the first dimension and 10 V/cm for 17 hr in the second dimension at 2 V/cm. Three hundred micrograms of antigen were used in A and C; the amounts of antigen used in Band D were adjusted to the same group I equivalence as in A and C, respectively. The antibody concentrations in the second-dimension gels were 29 ~l/cm* in A and B and 9 ~I/cm2 in C and D.

the contents of AgE, Ra3, Ra5, and Ra6 in the two preparations. On the other hand, the 16 min extract was relatively rich in basic antigens (Fig. 1, C), as noted above (see also ref. 19). The rye and timothy extracts used for the Westinghouse study (Fig. 2, A and C) showed an overall qualitative similarity but some quantitative differences as compared with the respective reference materials obtained from the FDA and the IUIS (Fig. 2, B and D, respectively). No reference materials were available with which to compare the cat, dog and Alternaria extracts shown in Fig. 3.

Qualitative CIE analysis of the Alternaria extract revealed the presence of amounts of Agl (=Alt-1) comparable to those that the Copenhagen group have found in their best Alternaria materials (unpublished data). Qualitative CIE analyses of the house dust ex-

tract revealed substantial amounts of Derrnatophag- oides, Mucor, cow, pig, cat, and dog antigens, together with lesser amounts of Afternaria, Clado- sporium, Penicilliutn, Aspergillus, grass pollen, ragweed pollen, horse dander, and guinea pig dander antigens.

Calibration of the puncture method and its relationship to the intradermal method

Our study of the seven clinic patients, skin-tested by the puncture and intradermal methods using serial dilutions of antigen Rye II, allowed us to determine puncture-sensitivity end points and compare the sensitivities of the puncture and intradermal methods. Fig. 4, A, illustrates the results of skin test titration experiments using the puncture method. The seven regression lines were parallel, except for the line of

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FIG. 3. CIE analyses of (A) cat extract, Westinghouse study vs. anti-cat; (B) dog extract, Westing- house study vs. anti-dog; C, Alterrtaria extract, Westinghouse study vs. antidlternaria. All experiments were performed at 12” C with a potential gradient of 10 V/cm for 30 min in the first dimension and 1 V/cm for 24 hr in the second dimension. One hundred fifty micrograms of antigen were used in each experiment. The antibody concentrations in the second-dimension gels were 15 ~l/cm2 in A and B and 10 ~I/cmz in C.

least slope (1.99; closed circles), which was significantly different from only one of the other six slopes (p < 0.05). In order to compute skin-test sensitivity end points for our Westinghouse study subjects, who were tested at single antigen concentrations, we determined the mean slope and mean y-intercept for the seven clinic patients, which gave the regression line shown on the right hand side of Fig. 4, B. Assuming that this dose-response relationship held for all tests, we could then calculate the log[antigen] that would produce a wheal diameter of any specified size. The size selected for the sensitivity end point determina- tion was 6 mm, which was roughly halfway along the dose-response curve and was 4 mm greater than the minimum detectable wheal (2 mm). (Note that the 2-plus end point for the intradermal test was 9 mm, which is also halfway along the linear portion of the dose-response curve and about 4 mm larger than the minimum detectable reaction in this procedure.)

We compared the sensitivities of the puncture and intradermal tests, using the means of the slopes and y-intercepts of the regression lines obtained in the seven clinic patients by the two methods (Fig. 4, B). Using sensitivity end points of 6 mm for the puncture method and 9 mm for the intradermal method, we obtained a sensitivity difference of 5.7 logs (-500,000-fold).

Skin-test results for the Westinghouse study subjects

One hundred ninety-two of the 485 subjects (320 randomly selected and 165 self-reported allergic in-

dividuals) were skin test positive to one or more of our panel of crude allergenic extracts (Table III). Skin test-positive responses to the pollens and house dust were the most prevalent, and positivity to dog dander and Alternaria were the least prevalent (Table III). Having included in our skin test panel duplicate tests of several materials, we looked at the reproducibility of wheal reactions (within each subject). For short ragweed (24 hr) and rye, the correlation coefficients were r = 0.76 (p < 0.01) and r = 0.79 (p < O.Ol), respectively. For all materials that were tested in duplicate (short ragweed, rye, timothy, and histamine), a few individuals were weakly positive at one of the duplicate sites but not the other. Five of the 192 skin test-positive subjects (2.6%) were rated as apparent negative reactors to histamine. Four of these atypical individuals gave small reactions of similar size to both the diluent, which contained HSA, and to histamine, which did not. Thus these four subjects appeared to be reacting weakly to both HSA and histamine, and there appeared to be only one out of the 192 skin test- positive subjects who was definitely negative to histamine at 500 pg/ml.

The cumulative distributions of the skin-test sensitivity end points for the subpopulations of individuals responding positively at each site are shown in Fig. 5. Statistical analyses of these data are presented in Table III. By the Kolmogorov-Smimov one-sample test,“6 none of the distributions for responses to the allergens was significantly different from a normal distribution, which supports the use of a parametric approach in computing Allergy Index II. On the other

282 Freidhoff et al. J. ALLERGY CLIN. IMMUNOL SEPTEMBER 1983

TABLE III. Statistics for the distributions of allergen concentrations log&g/ml], required to elicit a 6 mm diameter wheal reaction for each site tested

Percent skin test positive to individual allergens

Antigens

Group skin test Antigen concentration No. of positive positive (log[~g/ml]) eliciting 6 mm wheal

Site reactions Total group to 21 allergen tested* (~2 mm dia.) N=485 N = 192 Mean S.D. Max. value Min. value

Pollens Short ragweed

24 hr

16 min Rye grass

Tirnothy grass

Animal danders Cat Dog

Other: House dust Altrrnaria

Histamine

I 119 25 62 2.46 1.04 4.18 -0. I7 15 128 26 67 2.38 1.00 4.24 -0.29 II 106 22 55 2.57 1.23 4.51 -0.98

2 120 25 63 1.57 1.20 3.91 -2.1 I I4 119 25 62 1.46 I.18 4.27 .- 1.24 3 117 24 61 1.64 1.25 3.78 -2.50

13 I14 32 68 1.74 1.17 4.00 -1.72

4 104 21 54 3.62 0.98 5.07 0.23 5 60 12 31 4.23 0.66 5.30 2.64

6 II7 24 61 3.96 0.83 5.30 1.26 7 70 14 36 2.86 1.36 4.84 -1.72 9 456 94 97 3.55 0.54 4.75 1.86

12 333 92 95 3.24 0.53 4.66 1.50

*Other sites were used for diluent controls or for antigens not tested uniformly throughout the population.

hand, the distributions for both histamine tests (sites 9 and 12) were significantly different from normal.

Allergy indices

In order to determine the two allergy indices, we excluded skin sensitivity data for ragweed (16 min), timothy grass, and histamine. The distributions for rye and timothy grass extracts were not significantly different. Only two subjects who were weakly skin test positive to rye were skin test negative to timothy, whereas three subjects who were weakly skin test positive to timothy were skin test negative to rye. All subjects who were skin test positive to 16 min short ragweed were positive to 24 hr short ragweed, but not vice versa. The correlation between ragweed index II for the 24 hr and the 16 min extracts was r = 0.71 (p < 0.001) for 106 subjects who were sensitive to 16 min short ragweed.

Allergy indices I and II were computed from the sensitivity end points for each individual toward each allergen as described in Materials and Methods. The corresponding cumulative distributions (Fig. 6) were both different from normal distributions (p = 0.05) by the Kolmogorov-Smimov one-sample test. The means for allergy indices I and II were 26.5 (range 0.32 to 85.2) and 0.96 (range 0.02 to 3.4), respec-

tively. Note that the maximum possible theoretical value for the nonparametric Allergy Index I is 100, which would be the case for a person who had the highest sensitivity toward each of the six allergens used in compiling the index. The corresponding maximum possible value for Index II cannot be defined because the highest possible sensitivity level toward each allergen used in compiling this index is un- known.

In order to generate Allergy Index II, the means of the distributions shown in Fig. 5, A, were adjusted to the mean of the distribution for rye grass at site 2 (1.57 log,, pg/ml), which is shown by the solid line farthest to the left on the figure. The rationale for adjusting the means is based on the following con- siderations: (1) the variability in the allergen content of different types of extracts; (2) site-to-site differences in the sensitivity of the skin; and (3) differences in the mean exposure of the population toward the different allergens (see Discussion). The differences in allergen content are obvious from the cumulative distributions presented in Fig. 5 and are well known throughout the practice of allergy. Evidence for site- to-site differences along the forearm is illustrated by the slopes of the regression lines for individuals who were positive at one or both sites: for example, short

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-1 0 I 2 3

ANTIGEN CONCENTRATION. Log, [PQh]

2 d : 4 I . .

FIG. 4. A, Linear regression analysis of antigen concentration, log[~g/ml] vs. wheal diameter for the puncture tests in each of the seven subjects who were tested with serial dilutions of antigen Rye II. B, Relationships between the mean of the seven linear regression lines for antigen concentration, log[cLg/ml] vs. wheal diameter for both the intradermal (left) and puncture (right) titrations. The mean slopes of the linear regression lines for the intradermal and puncture methods were the same (p > 0.5). The regression lines for each subject by either test were also parallel except for one subject (open squares in both figures) whose regression slopes (b = 1.59 for intradermal method and b = 2.83 for puncture method) were significantly different at p < 0.05.

ragweed (sites 1 vs. 15), b = 0.71, and rye grass (sites 2 vs. 14), b = 0.78. Both of these slopes were significantly different from 1 .OO @ < 0.01). Thus we have confirmed the finding of Santilli et a1.15 that the lower part of the forearm is less sensitive than the upper part.

Since our intention was to use primarily the more powerful approach of parametric statistics in future analyses employing the concept of an “allergy index, ” we examined the relationship between the indices obtained by nonparametric and parametric approaches (allergy indices I and II, respectively). In the 192 skin test-positive subjects, the two indices were significantly correlated by the Spearman Rank Order Test (rs = 0.98, p < 0.001). Furthermore, both Al- lergy Index I and Allergy Index II were significantly

100

c 80 5

B a 60 I

-2 -I 0 I 2 3 4 5

ANTIGEN CONCENTRATION,lqC [&mlj

5 BO Y 6 n. 60

Y

s _I 40

:

2 20

I , 1 I 1 I , , -2 -I 0 I 2 3 4 5

ANTIGEN CONCENTRATION, log,,[yg/ml]

FIG. 5. Cumulative distributions of the puncture-test end points (concentrations of allergen required to elicit a 6 mm wheal) in subjects who were skin test positive at each particular site. A, Allergens used to generate the allergy indices. B, Remaining substances used in skin testing. Dashed line, Data for sites 14, 1, 3, and 12 for rye, 24 hr short ragweed, timothy, and histamine, respectively; so/id lines, other sites for these allergens.

correlated with the number of positive skin test reactions (rs = 0.89, p < 0.001, and r, = 0.88, p < 0.001, respectively).

The cumulative distribution of log[total serum IgE] in the 192 skin test-positive subjects was not different from a normal distribution (Fig. 7). The correlation between log[total serum IgE] and Allergy Index II was r = 0.39 (p < 0.01) for the 192 skin test- positive subjects (Fig. 8). The correlation between log[total serum IgE] and Allergy Index I was essentially the same (rs = 0.41, p < 0.01). None of the subjects who had low total serum IgE levels was highly sensitive in the skin test (i.e., had a high Al- lergy Index II). However, there were subjects with high total serum IgE levels who appeared to be only moderately or slightly allergic according to our skin test data (i.e., had a low or moderately high Allergy Index II).

We also investigated the relationship between the indices for a specific allergen vs. specific serum anti-

284 Freidhoff et al. J ALLERGY CLIN. IMMUNOL.

SEPTEMBER 1983

v , I I / I I 20 40 60 00

ALLERGY INDEX I

; eo- Y F5 a 60-

Y F 3 40-

z

ci 20-

I h i ALLERGY INDEX II

FIG. 6. Cumulative distributions of (A) Allergy Index I and (B) Allergy Index II in 192 skin test-positive subjects (solid lines). Dashed lines, Normal cumulative distributions generated from the means and standard deviations of the distributions for allergy indices I and II, respectively. In- sets, Frequency distributions of Allergy Index I and Al- lergy Index II.

body level. Specific IgE to 24 hr ragweed was mea- sured by RAST in 102 of the ragweed-positive subjects for whom sufficient serum was available. Log[specific IgE to ragweed] was significantly correlated with both Ragweed Index I and Ragweed Index II (r, = 0.64, p < 0.01, andr = 0.63, p < 0.01, respectively).

We next tested whether the allergy indices were related to overall allergic symptomatology as per- ceived by skin test-positive subjects in longitudinal reports. Using the weekly symptom diaries (see Ma- terials and Methods), we examined the correlation between average reported symptoms during the first 12 mo of the study and several measures of allergic sensitivity including the average number of days per week the subject had symptoms of “sneezing . . . ” and/or “wheezing” and the average number of anti-

TOTAL SERUM IgE (rig/ml)

FIG. 7. Cumulative distribution of total serum IgE level in the 192 skin test-positive subjects (solid line). Dashed fine, Normal cumulative distribution generated from the mean and standard deviation of the distribution of log[lgE]. Inset, Frequency distribution.

histamine tablets taken per week. The average weekly symptom, medication, and total (symptom-plus-medication) scores in the remaining 22 individuals were not significantly correlated with either of the indices or the number of positive skin tests by Spearman Rank Order Test (rs = 0.2 to 0.4). Surprisingly, log[total IgE] was actually negatively correlated with weekly symptom and medication scores (rs = -0.03 to - 0.3) and showed that there is clearly no relation between this parameter and overall allergic symptomatology reported by the study group.

Fourteen subjects skin test positive to ragweed kept daily diaries during the ragweed pollination season (see Materials and Methods). Significant correlations were found between the average seasonal symptom scores and total (symptom-plus-medication) scores and Ragweed Index I, Ragweed Index II, and log[specific IgE to ragweed] (p < 0.05; Table IV).

DISCUSSION This paper presents an innovative technique for

quantitating a person’s overall level of IgE-mediated skin sensitivity to a panel of well-standardized, high quality extracts of common environmental allergens. In each of 192 skin test-positive individuals (out of 485 tested), sensitivity end points for eight allergens were computed from puncture test results performed at a single concentration of each allergen. The end point results @g/ml required to induce a 6 mm wheal) for six of the allergens formed the basis for two allergy indices, which assessed each individual’s aver-

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age degree of skin sensitivity toward these allergens. Emphasis was placed on the careful measurement of the whole continuum of sensitivity rather than the discrete categorization of negative and positive reactions, which has been used in previous studies of the prevalence of skin test response.‘-‘” Although wer4 and others’” have previously found that skin-test positivity decreases significantly over the age range of 20 to 60 yr, evaluation of this response as a continuum seems to us to be an appropriate model due to the relative stability of the actual wheal size induced by tests performed within shorter periods of time.‘“. 37

A most important feature of our study was the use of high-quality, immunochemically standardized extracts of ragweed, grass, and cat allergens and partially standardized dog and Alternaria extracts. This should enable future investigators, using similarly well-characterized allergens, to examine the validity of our results in other populations. It is well known that similarly labeled allergens from different manufacturers (or from different lots from the same manu- facturer) and of the same apparent concentration (w/v or PNU) often vary widely in their allergenic reactivity.20. 38 In their population study, Haahtela et a1.12 found that two extracts of the same allergen obtained from two manufacturers gave “widely different prev- alences of positive reactions” when used, in parallel, in the same subjects. We have avoided the problem of lot-to-lot consistency by using the same lots of well- standardized extracts throughout our study. Whereas we could standardize the ragweed, grass, and cat extracts in terms of their contents of certain important allergenic components, there is still a residual problem of further standardization with the dog and Alter- naria extracts for which appropriate purified antigen standards were not available. Also, our decision to include house dust in order to broaden the number of allergenic specificities covered by our allergen panel was at the expense of using material that could not be rigorously standardized immunochemically.

It may also be argued that we did not properly define an “allergic” subject because we failed to use a sufficiently large panel of skin test reagents. In order to make our study more manageable, we limited our initial panel of skin test reagents to eight common inhalant allergens (Table I), of which two were excluded from the computation of the allergy indices on the grounds of their cross-reactivity with other allergens that were included (see Results). Addition- ally, 124 of our random Westinghouse subjects were tested with materials such as common weed and tree pollens and fungal extracts. Of these, 10 (8%) were positive or equivocally positive to one or two (one subject) of the allergens in the new panel. Three of the

I . . . . . : . l

. .

. . . l . .

. .

.=. . . . . . 2 -* .

7 .--

:*.*; . . . l . . .

. l . . . : l

. . - - . l . . ..*.* . . . . M . . .*: . l -.:j l - ,

. . . . .

-=:t. ..: . . . . m .

l .* .* - ‘i 2. .

. . . . . m - .*‘r\ “. ..a”

lb 100 L&m lo,ooo TOTAL SERUM IqE (q/ml )

FIG. 8. Correlation between total serum IgE level and Al- lergy Index II (r = 0.39, p < 0.01). For comparison, log- [IgE] vs. Allergy Index I, r = 0.41, p i 0.01.

124 subjects tested (2%) were positive to the new allergens but not to any of the allergens in the original panel. We conclude therefore that the allergens chosen for skin testing are probably among the most relevant in defining the allergy indices of an individual residing in the Baltimore area. Probably, one would have to increase the size of the allergen panel several- fold in order to achieve a significant improvement.

In terms of their relationship with other parameters associated with atopic disease, there is little to choose between the nonparametric Allergy Index I and the parametric Allergy Index II. The two indices correlated well with one another (rS = 0.98) and both showed similarly strong correlations (rS - 0.9) with the number of positive skin reactions. The correlations between the indices and log[totaI serum IgE] were weak (r or rS -0.4), but significant and positive. The low correlations are perhaps not surprising in view of the numerous environmental factors influenc- ing total IgE levels,3g as well as undefined influences of ingested, injected, and, possibly, parasitic allergens .

Both of the allergy indices were positively, although not significantly, correlated with the allergic symptom and medication scores reported by a small group of 22 allergic subjects over a period of 1 yr. The lack of significant correlations is probably a result of the subjectivity in symptom reporting and the “in- terference” caused by person-to-person differences in allergen exposure, respiratory infections, etc., during the year. On the other hand, quantitation of speci$c ragweed sensitivity by our method leads to quite good correlations (r or r,-- 0.6, p < 0.01) between both ragweed indices I and II and log[IgE to ragweed].

286 Friedhoff et al. J ALLERGY CLIN. IMMUNOL.

SEPTEMBER 1983

TABLE IV. Correlation of average daily symptom scores with Ragweed Index I, Ragweed Index II and log[lgE to ragweed]

Average daily symptoms during the ragweed season (N =14)

Ragweed Index I Ragweed Index II Log[IgE to ragweed]

rs P* rs Pi rs PX

Symptom scores 0.60 <0.0.5 0.60 <0.05 0.68 <O.Ol Medication scores 0.54 0.55 0.75 <O.Ol Total scores 0.59 co.05 0.59 co.05 0.69 <O.Ol

*Only p values SO.05 are given.

Also, weaker correlations, but significant ones (p < 0.05), were found between these indices and reported symptoms during the ragweed pollination season. The data of Norman et al.“” show similar correlations using ragweed skin-test sensitivity data determined by the more time-consuming intradermal titration method.

In future studies, it is our intention to use Allergy Index II because of the generally greater power of parametric methods of statistical analysis that can be employed. As noted previously, the computation of this index involves adjustment of the mean sensitivity levels of each allergen to the same value in order to correct for differences in allergen quality and site- to-site variability in skin reactivity. Such an adjustment would also correct for differences in the mean exposure of the population toward different allergenic materials and possible differences in the immunizing potency of different allergens. It is also assumed that each individual in the population receives a similar degree of exposure to each allergen, which is probably reasonably true for the pollens but not for animal danders, for example. However, assessment of such differences is likely to be extremely difficult or im- possible.

Finally, we should mention the potential theoretical and practical usefulness of Allergy Index II (and other indices). First, one can use this continuous measure of allergic sensitivity in epidemiologic studies where the relationship between allergic status and age, sex, disease susceptibility, etc., are examined. Second, one can use Allergy Index II in genetic studies investigat- ing the relationship between allergic phenotypes, and HLA type, IgE level, etc. Such analyses are currently in progress in random samples and in allergic subjects and will be reported in subsequent publications. Also, we envisage that the specific indices for individual allergens, which are adjusted for allergen quality, could be used clinically as aids for diagnosis and in decisions for immunotherapeutic intervention.

We are particularly pleased to thank Mr. Phil Weber and the employees of the Westinghouse Electric Corp., Hunt

L. m Tuft L, Heck VM, Gregory DC: Studies in sensitization as

3.

4.

applied to skin test reactions. III. Influence of age upon skin reactivity. J ALLERGY 26:359, 1955. Lindblad JH, Fan RS: The incidence of positive intradermal reactions and the demonstration of skin sensitizing antibody to extracts of ragweed and dust in humans without history of rhinitis or asthma. J ALLERGY 32:392, 1961. Curran WS, Goldman G: The incidence of immediately reacting allergy skin tests in a “normal” adult population. Ann Intern Med 55~777, 1961.

5.

6.

7.

Hagy GW, Settipane GA: Bronchial asthma, allergic rhinitis, and allergy skin tests among college students. J ALLERGY 44~323, 1969. Williams H, McNicol KN: Prevalence, natural history and relationship of wheezy bronchitis and asthma in children. An epidemiological study. Br Med J 4~321, 1969. Hannaway PJ, Hyde JS: Scratch and intradermal skin testing: a comparative study in 250 atopic children. Ann Allergy 28:413, 1970.

8.

9.

10.

11.

12.

13.

14.

Hagy GW, Settipane GA: Prognosis of positive allergy skin tests in an asymptomatic population. A three year follow-up of college students. J ALLERGY CLIN IMMIJNOL 48~200, 1971. Smith JM: Skin tests and atopic allergy in children. Clin Al- lergy 3~269, 1973. Barbee RA, Lebowitz MD, Thompson HC, Burrows B: Im- mediate skin-test reactivity in a general population sample. Ann Intern Med 84~129, 1976. Davis JB: Asthma and wheezy bronchitis in children. Clin Allergy 6:329, 1976. Haahtela T, Bjorksten F, Heiskala M, Suoniemi I: Skin prick test reactivity to common allergens in Finnish adolescents. Allergy 35:425, 1980. Barbee RA, Brown WG, Kaltenbom W, Halonen M: Allergen skin-test reactivity in a community population sample: correlation with age, histamine skin reactions, and total serum immunoglobulin E. J ALLERGY CLIN IMMUNOL 68: 15, 1981. Freidhoff LR, Meyers DA, Bias WB, Chase GA, Hussain R, Marsh DG: A genetic-epidemiologic study of human immune

Valley, Md., for their generous cooperation and participa- tion in this study. We also thank Bonnie Middleman, Mary Conrad, Eva Ehrlich-Kautzky, Edward Siekierski, and Cheryl Carouge for technical assistance and Drs. Wilma Bias, Philip Norman, Marianne Roebber, and John Santilli for helpful suggestions in preparing this paper.

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15. Santilli J, Potsus RL, Goodfriend L, Marsh DG: Skin reactivity to purified pollen allergens in highly ragweed sensitive individuals. J ALLERGY CLIN IMMUNOL 65:406, 1980.

16. Norman PS, Rhyne MB, Mellits ED: Evaluation of agents for treatment of seasonal respiratory allergies. In Lasagna L, editor: Clinical pharmacology. Oxford, 1966, Pergamon Press, vol. 2, p. 639.

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as a tool for the analysis and isolation of allergens. Prog Al- lergy 25: 1, 1978.

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29. Garvey JS. Cremer NE, Sussdorf DH: Isolation of immuno- globulins, antibodies, and their subunits. In Methods in immunology. ed. 3. Reading, Mass.. 1977, W. A. Benjamin. Inc.

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35. Lowenstein H, Marsh DG: Antigens ofAmbrosia elurior (short ragweed) pollen. III. Crossed radio immunoelectrophoresis of ragweed-allergic patients’ sera with special attention to quantification of IgE responses. J Immunol 130:727, 1983.

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Documents

The structuring of an allergy index based on IgE-mediated skin sensitivity to common environmental allergens