Levels of IgE in serum from normal children and allergic children as measured by an enzyme immunoassay

Roger E. Lindberg, Ph.D., and Carlos Arroyave, M.D., Ph.D. North Chicago

and Chicago, Ill.

Serum IgE levels of 346 nonallergic and 301 allergic children were measured by an enzyme immunoassay. The data are presented as the geometric mean t 95% confidence interval for each age from less than I to 12 years of age with those 13 to 16 years of age, pooled. The geometric mean for nonallergic patients increases from 0.9 IUiml at less than I year of age to 45.4 IlJlml at 13 to 16 years of age. By use of an IgE value between confidence intervals of allergic and nonallergic subjects, a cutoff value for each age was established to differentiate allergic from nonallergic children. This value increases from 10 Wlml at less than 1 year to 100 IUlml at 13 to 16 years of age. The diagnostic sensrtivity and speciJicity of the assay, based on the cutoff values, average 83% and 91%, respectively. (J ALLERGY CLIN IMMUNOL 748:614-t?, 1986.)

Elevated levels of IgE are observed in serum of most patients with atopic disorders, such as aller- gic rhinitis. extrinsic asthma, urticaria, and atopic eczema,‘-s as well as some disorders not related to allergy, including parasitic diseases,6 some cases of bronchiolitis and bronchopulmonary aspergillosis,’ and some immunodeficiency diseases such as Wiskott- Aldrich syndrome ,* DiGeorge’s syndrome,’ and hy- per-IgE syndrome.‘” Elevated levels of IgE are also helpful in predicting the subsequent development of allergies in childre:n. “-I3 The definition of what con- stitutes an elevated level of IgE has been the subject of several articles,‘4-24 but most of these articles were studies of European populations or included subjects who were combined in age groups of various sizes. Since age.2.3, II_ 12 genetic background,16, 25-27 and en- vironmental factors2’ have all been demonstrated to be important determinants of IgE levels, the reported normal values of IgE may not be directly applicable to children m the United States.

This study reports the use of a new enzyme im- munoassay (the Abbott IgE EIA) to compare IgE levels in allergic and nonallergic children to determine levels that may be useful in differentiating allergic from nonallergic individuals.

From the Clinical Immunology Group, Abbott Laboratories, and Children’s Memorial Hospital, Chicago, Ill.

Received for publication Sept. 25, 1985. Accepted for publication March 18, 1986. Reprint requests: Roger E. Lindberg, Ph.D., Dept. 908, AP 9,

Abbott Laboratories. Abbott Park, IL 60064.

614

Abbreviation used CVs: Coefficients of variation

1.6 r

I 40 80 120 160 200

IgE (lU/ml)

FIG. 1. A typical standard curve of the enzyme immuno- assay for total serum IgE. The five standards used to es- tablish the standard curve include samples containing 0, 10, 40, 7E. and 200 IUiml of IgE from a diluted myeloma specimen.

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IgE measured by enzyme immunoassay 615

TABLE I. Number of subjects studied, geometric mean, and range of IgE values for allergic and nonallergic children

--

(nonaiergic) Range (NJ/ml) Geometric mean Range W/ml) Geometric mean

Age WI (nonallergic) (nonallergic) (alle?gicl (allergic) (allergic)

<I 31 0 to 6.6 0.9 27 IO to 750 41.3 1 60 0 to 20 2.3 29 7 to 195 38.9 2 23 0.1 to 15.8 3.4 24 0.2 to 700 24.1 3 22 0 to 29.2 4.2 20 2 to 540 78.3 4 25 0.3 to 25 3.7 2s 0.4 to 246 47.9 5 24 0.2 to 17.6 5.5 29 1.7 to 680 59.5 6 16 0.2 to 13.1 5.1 31 2.6 to 1000 101.9 7 20 0.3 to 46.1 8.9 21 0.5 to 2491 166.3 8 23 1.8 to 60.1 11.9 25 3.8 to 630 84.3 9 16 3.6 to 81 24.4 4 169 to 900 432.3

10 12 8 to 95 33.1 14 10 to 895 168.2 11 12 1.5 to 99.7 33.0 19 3.6 to 1273 134.8 12 17 3.9 to 83.5 32.0 14 4.7 to 1046 94.5 13 to 16 45 3.3 to 188 45.4 16 70 to 793 338.7

MATERIAL AND METHODS IgE measurements

Serum IgE levels were determined by the Abbott IgE EIA (Abbott Laboratories, North Chicago, Ill.) with fresh or recently frozen sera. This test is a sandwich enzyme im- munoassay in which samples are incubated with polystyrene beads coated with rabbit antihuman IgE. After a 30-minute incubation, unbound material is removed by washing, and the beads are incubated for another 30 minutes with goat antihuman IgE conju,gated with horseradish peroxidase. Af- ter a second wash, beads are incubated for 30 minutes with o-phenylenediamine containing hydrogen peroxide, and the reaction is stopped by the addition of 1 N H,SO,. IgE levels are calculated from absorbance values at a wavelength of 492 nm automatically by the Abbott Quantum TM spectro- photometer.

Populations studied

The population studied included 647 children who were referred to Children’s Memorial Hospital in Chicago, Ill. The 346 nonatopic children selected for study (192 male and 154 female children of which 74% were white, 16% were Hispanic, and 9% were black) were admitted for plastic or orthopedic surgery, had no evidence of rheumatic dis- eases, were without tumors or parasitic infections, and had no family history of allergic disease but were not skin tested. The 30 I atopic subjects included in the study (185 male and I16 female subjects of which 68% were white, 19% His- panic, 12% black, and 1% Oriental) were classified as al- lergic on the basis of family history, personal history, and at least one positive skin test.

Statistics

Measurements of serum IgE were tabulated for age groups less than 1 year and each age 1 to 12 years with those 13 to 16 years, pooled. Since the data are skewed, a

log tran&rmation was done, and geometric means 4 95% confidence intervals were calculated for each age group according to the method previously described.”

RESULlrS

A typical standard curve of the Abbott IgE EIA is presented in Fig. 1. The reproducibility of the assay was evaluated at four clinical sites by use of a panel of 15 serum samples. Results from these sites yielded intrarun percent CVs that ranged from 5.3% to 17.8%, and interrun percent CVs that ranged from 7.3% to 20.2%. The between laboratory percent CVs ranged from 7.8% to 17.4%.“’ Linear regression analysis of 397 sample results obtained by use of the Abbott IgE EIA and the Pharmacia IgE EIA or RIA (PRIST) demonstrated a correlation coefficient of 0.995, a slope of 0.850, and a y-intercept of -4.3. The an- alytical sensitivity of the assay is defined as the mean absorbance of duplicate values of the zero standard plus 2 SD. The mean absorbance of the zero standard was 0.063, and plus 2 SD yields a value of 0.079 or a theoretical sensitivity of 0.46 IU/ml. Results from three experiments done in replicates of six indicate that accurate IgE values down to 0.5 IU/ml can be reliably achieved in dilution studies.“’

The number of subjects studied, the geometric mean, and the range of IgE values for both allergic and nonallergic subjects at each age from less than 1 to 12 years of age with those 13 to 16 years, pooled, is illustrated in Table I. There was no significant dif- ference in IgE levels between male and female subjects or between the races studied (p > 0.05 by analysis of variance in comparing Hispanics to whites at each age, but there was too small a number of blacks to

616 Lindberg and Arroyave J. ALLERGY CLIN. IMMUNOL. OCTOBER 1986

TABLE II.

l Allergic Patients

n Nonallergic Patients

100 L b I I I I I I I I I I I I I < 1 1 2 3 4 5 6 7 8 9 10 11 12 13-16

PATIENT AGE (YRS 1

FIG. 2. The geometric mean k 95% confidence interval of IgE levels from allergic and nonallergic children of various ages is compared. Significant difference:: (p < 0.05) are observed at all age groups except at 11 and 12 years of age.

The diagnostic sensitivity and specificity of the assay by use of cutoff values that increase with age

Age (yr) Cutoff (IUlml) Sensitivity* Specificityt

<I 10 100 1 15 90 2 15 63 3 1.5 90 4 15 88 5 15 72 6 15 90 I 15 91 8 25 88 9 50 100

10 75 79 II 15 79 12 15 46 13 to 16 loo 88

100 97 96 91 84 92

100 80 85 81 83 92 94 98

*Percent of allergic patients correctly identified. TPercent of nonallergic subjects correctly identified.

do meaningful comparisons), therefore, data from male and female subjects of all races have been pooled in establishing statistical values. The geometric mean for nonallergic children increases from 0.9 IU/ml at less than 1 year of age to 45.4 IU/ml at 13 to 16 years of age. Although the geometric mean for allergic in- dividuals is higher at all ages, there is considerable

overlap in IgE values at all ages except less than 1 year of age.

The standard deviations from the means are wide- spread for all age groups and do not provide a useful guide to differentiate the allergic and nonallergic group; however, when the geometric mean -+ 95% confidence interval is compared, significant differ- ences between allergic and nonallergic subjects can be observed (Fig. 2). With an IgE value midway between the upper limit of the 95% confidence interval for nonallergic and the lower limit of the 95% con- fidence interval for allergic subjects, a cutoff value was established, and the diagnostic sensitivity (i.e., percent of allergic individuals correctly identified) and specificity (i .e . , the percent of nonallergic individuals correctly identified) of the assay were determined (Table II).

Table III presents a comparison of the geometric means of IgE values of children from this study with those reported for children from other countries. Al- though I[gE levels in children less than 1 year of age are sliglntly higher in Mexican than in Swedish or American children, large differences do not appear until children reach 4 years of age, at which time Mexican children have somewhat higher IgE levels (13.6 ILJlml) than either Swedish (8.6 IU/ml) or American (3.7 NJ/ml) children. At 10 years of age, differences between American and Mexican children disappear (33.1 versus 32.4 IU/ml), whereas Swedish

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IgE measured by enzyme immunoassay 617

TABLE III. Comparison of normal IgE levels among nonallergic children of different nationalities

Age (W United States Mexicoz4 Swedenlz Venezuela=

<l 0.9 2.0 CO.82 1 2.3 4.3 3.5 2 3.4 5.0 3.0 3 4.2 6.2 1.8 4 3.7 13.6 8.6 5 5.5 14.8 6 5.1 12.4 7 8.9 24.2 12.9 51 8 11.9 26.3 53 9 24.4 74

10 33.1 32.4 23.7 52 II 33.0 38 12 32.0 28 13 to 16 45.4 20.1

children have lower IgE levels than either American or Mexican children (23.7 IU/ml).

Venezuelan children have much higher IgE levels than Mexican, American, or Swedish children be- tween 7 and 10 years of age, but their IgE levels fall to a point that, when the children reach 12 years of age, they are very similar to American children (28 versus 32 IU/ml, respectively).

DISCUSSION

Since total IgE has been demonstrated to be im- portant in predicting the subsequent development of allergies among children,‘*, ** we have limited our study to individuals younger than 17 years of age. No extensive studies of serum IgE in American children have been reported by use of an enzyme immunoassay. We used the Abbott IgE EIA test and found it to be easy to use and reliable for the estimation of serum IgE levels. The assay is reproducible and sensitive down to 0.5 IU/ml, making it particularly useful for the analysis of pediatric specimens in which low levels of IgE are expected.

There was no significant difference in IgE levels between male and female subjects, which agrees with previous studies.‘. .“. 32

Although comparisons between geometric means are helpful in comparing IgE levels among individuals from different ethnic backgrounds and geographical locations, a more useful statistic for predicting the likelihood of an allergy based on a single IgE deter- mination is the diagnostic sensitivity and specificity of a particular assay with regard to the population studied. We used a value midway between the upper limit of the 95% confidence limit of nonallergic sub- jects and the lower limit for allergic subjects to es- tablish a cutoff value that could be used to determine

the diagnostic sensitivity and specificity of the assay (Table II). With 100 IU/ml as the cutoff between allergic and nonallergic subjects, the diagnostic sen- sitivity and specificity of the assay for subjects 13 to 16 years, of age (pooled) were 88% and 98%, respec- tively. Wittig et a1.3 also found that a cutoff value of 100 is a.cceptable for all age groups except infants, but no mention was made of cutoff values for younger age groups. Our study indicates that for younger age groups, a lower cutoff provides a better combination of diagnostic sensitivity and specificity. For example, a cutoff of 10 IU/ml for children less than 1 year of age provided 100% specificity and sensitivity in the 27 allergic and 3 1 nonallergic children that we studied. A cutoff of 15 IU/ml for children 1 to 7 years of age provided 83% sensitivity and 92% specificity (179 allergic and 190 nonallergic children). The cutoff in- creases fivefold between 7 and 10 years of age, in- creasing from 15 IU/ml to 75 IU/ml. The cutoff values suggested include patient material that has not been selected on the basis of a follow-up and may include “nonatopic” children with pathologic IgE levels that may be present before the appearance of symptoms.33

A comparison of normal IgE values from this study and those reported by other investigators is presented in Table III. Other studies have also been done but have been excluded from this comparison because they have pooled data from allergic and nonallergic individua;ls’* or have pooled data from children of different ages.3 Variations in the assays used and dif- ferences in the number of individuals studied at each age make direct comparisons difficult; however, it ap- pears that children of different nationalities reach peak IgE levels at different ages, i.e., Japanese children at age 2 years,” Venezuelan children at age 9 years,” Swedish children at age 10 years,‘* Mexican children

618 Lindberg and Arroyave

at age 10 year,s or later,24 and American children at age 13 to 16 years. Whether this difference is due primarily to environmental factors or genetic factors is unknown.

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