False positive reaction in measurement of allergen-specific IgE--comparison of 3M IgE FAST-Plus Test using polystyrene well as adsorbent with Phadezym RAST]

Irrelevant IgE binding to cellulose discs is known to give false positive results in Phadezym RAST (Pharmacia) for the estimation of allergen-specific IgE in serum. We investigated FAST-Plus Test (3M Diagnostic Systems), an enzyme-linked sandwich type Fluoro-Allergo-Sorbent Test in which a particular allergen was coated to polystyrene well. Phadezym RAST and CAP RAST (Pharmacia) using cellulose-derivative discs as adsorbent were used as reference methods. Patients' sera which gave negative blank reactions to uncoated filter paper disc in the Phadezym RAST system were assayed for specific IgE to 6 allergens using FAST-Plus Test, CAP RAST and Phadezym RAST, and the results of the former two were compared with those of Phadezym RAST using a comparable class system. FAST-Plus Test showed variable correlations with Phadezym RAST, the correlation coefficients ranged from 0.41 to 0.97 (r = 0.462 in house dust 1, r = 0.713 in house dust 2, r = 0.412 in Candida albicans, r = 0.952 in Dermatophagoides peteronyssinus, r = 0.969 in Dermatophagoides farinae and r = 0.682 in Japanese cedar), although most of the results were within one class difference. Similar correlations were obtained between CAP RAST and Phadezym RAST. Of 3004 patients' sera tested in the past two years using Phadezym RAST, 132 (96 cases) displayed positive blank reactions to the uncoated filter paper disc. Of the 96 cases, 80 sera were assayed for binding of IgE to the uncoated cellulose-derivative disc in the CAP RAST system. 18 showed positive results up to 7 IU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Total serum IgE and specific IgE antibodies in children with bronchial asthma

Correlation between total serum IgE levels and RAST scores in a total of 342 asthmatic children were evaluated. The median and range of serum IgE were 1,050 IU/mL and 20 to 10,000 IU/mL respectively. Positive rates of RAST were highest for mites and house dust (87% to 91%) and lowest for milk, dogs, buckwheat, and eggs (2% to 8%). In general, patients with higher RAST scores had higher serum IgE (P less than .05, Mann-Whitney test). In individual cases, however, serum IgE levels did not significantly correlate with RAST scores and RAST was mandatory to estimate the levels of specific IgE antibodies.     

Comparison of cord blood immunoglobulin E concentrations and maternal allergy for the prediction of atopic diseases in infancy

Total serum IgE levels were determined in 136 newborns and their mothers and in 54 of their fathers, using the paper radioimmunosorbent test (PRIST) technique. IgE specific antibodies for house dust (Dermatophagoides pteronyssinus), orchard grass, timothy grass, and cow's milk were measured with the radioallergosorbent test (RAST). One hundred thirty-three RAST assays were negative in newborns, and in three cases RAST for cow's milk was positive. Cord blood IgE ranged from 0 to 5.5 IU/ml (mean 0.32 ± 0.54 IU/ml); levels were significantly (p < 0.05) higher when maternal IgE was over 100 IU/ml and when mothers had received progesterone therapy during the pregnancy. Salbutamol administration or tobacco smoking during pregnancy did not influence newborn IgE. A clinical follow-up study was conducted in 83 infants for 9 mo. Nine infants developed definite atopic disease, and possible allergic diseases were noted in eight other infants. The IgE level at birth appeared to be more predictive for the development of allergy in infancy than the family history.     

Comparative evaluation between two immunoenzymatic techniques (FAST and Phadezym) and the Phadebas RAST in food allergy

The diagnosis of food IgE-dependent hypersensitivity is based on the demonstration of specific IgE, completed by provocation tests. Two immunoenzymatic techniques, the Phadezym and the FAST, are compared with the Phadebas RAST, in 86 sera (23 controls, 28 from patients with a reported food allergy and 35 with a positive RAST to a food allergen). The within-run variation coefficient of class 0-2 sera was 9% for the Phadebas RAST, and higher than 20% for the Phadezym and the FAST. It was in order of 8.7%, 9.4% and of 17.6% respectively for Phadebas RAST, Phadezym and FAST when estimated with class 3-4 sera. The specificity was higher than 95% for the three techniques. The sensitivity was 75% for Phadebas and 43% for Phadezym and FAST. The FAST test is much less sensitive with allergens of vegetal origin than those of animal origin (P less than 0.01). This work indicates the high percentage of false negative results of immunoenzymatic techniques when food extracts are tested. This could be explained either by an enzyme-substrate reaction or by a non-specific inhibition of the enzyme linked to the anti-IgE IgG.     

纸片放射免疫吸附试验测定支气管哮喘病人血清总IgE

IgE是人血清中含量最少的一类免疫球蛋白,目前多用放射免疫或酶免疫技术作定量测定。Ceska等对纸片放射免疫吸附试验(PRIST)方法作了详述。此法简便、快速、敏感、特异。PRIST是一种因相双抗体夹心技术,先将抗IgE抗体偶联于溴化氰活化的滤纸片上,使与待测标本或IgE工作标准进行反应,洗净后加入~(125)I—抗IgE抗体再进行反应,洗净后测定滤纸片上复合物的放射能。后者随IgE量增多而增强。我们在建立PRIST方法之后,将其应用于检测支气管哮喘病人血清总IgE。现报告如下。材料与方法     

Relationship between IgE and specific aeroallergen sensitivity in Alaskan native children.

BACKGROUND: The relationship between atopic disease and serum IgE levels varies among populations and geographic regions. The close association of atopy with IgE may not occur in subarctic populations as it does in developed countries in temperate climates. OBJECTIVE: To evaluate the relationship between total and specific IgE concentrations and clinical atopy in 5- to 8-year-old Alaskan native children. METHODS: Medical record reviews, interviews, physical examinations, serum IgE measurements, and radioallergosorbent testing (RAST) were performed. RESULTS: The IgE geometric mean was 122.1 IU/mL. Fifty-eight percent of patients had IgE levels greater than 70 IU/mL, and 17% had levels greater than 1,000 IU/mL; 14% had RAST values greater than 0.35 kU/L. Both IgE levels greater than 70 IU/mL and greater than 1,000 IU/mL were associated with RAST values greater than 0.35 IU/L (P = .004) and early wheezing (P = .005) but not with current wheezing, asthma, eczema, or a history of allergies. A RAST value greater than 3.51 kU/L was associated with eczema (P = .04) but not with allergies or wheezing. Children with current wheezing were more likely to have allergies (P = .03) but not eczema, an IgE level greater than 70 IU/mL, or a positive RAST value. Children hospitalized with respiratory syncytial virus (RSV) were not more likely than controls to have current wheezing. CONCLUSIONS: Elevated serum IgE concentrations, including levels greater than 1,000 IU/mL, are common among Alaskan native children; positive RAST reactions to aeroallergens are not. The IgE levels do not relate to wheezing, eczema, a history of allergies, or past hospitalization for RSV infection but likely reflect infections other than RSV and environmental factors in subarctic indigenous populations.     

Comparison of Two Assays for the Determination of Specific IgE in Serum of Atopic and Nonatopic Subjects: the Allergenetics FAST™ and the Phadezym RAST®

The FAST assay (Allergenetics) for the determination of specific IgE has recently been introduced. The results of this test, graded in classes 0 (negative), 1 (equivocal) and 2 to 6 (positive) were compared with those of another commercially available test (Phadezym RAST, Pharmacia), graded from class 0 (negative) and classes 1 to 4 (positive). In 52 adults suffering from rhinitis/asthma, a total of 114 positive skin prick tests (SPT) were obtained for common respiratory allergens. In 67% of these tests the Phadezym RAST was positive and in 63% the FAST was positive (classes 2 to 6). In these patients there were 151 negative SPT: 6% corresponded to a positive Phadezym RAST (all class 1) and 34% to a positive FAST (classes 2 to 6). The serum of nine nonatopic volunteers who had negative SPT for 12 common allergens were tested. In none did the Phadezym RAST give any positive results; the FAST was positive in all nine sera, detecting between five and 11 allergens. When both assays were performed on 14 unselected cord blood samples, the Phadezym RAST was positive in three samples (with class 1 results to a maximum of two allergens), and the FAST was positive in 12 samples, detecting between one and seven allergens. Thus, in our hands, the FAST gave an abnormally high number of positive results in patients with negative SPT, in nonatopic volunteers, and in cord blood.     

Quantitative determination of total and specific human IgE with the use of monoclonal antibodies

We have used two monoclonal antibodies (BS17 and Le27) that recognize two different epitopes of the constant region of human IgE in order to determine the total and specific IgE contained in human serum. Both types of assay are based on classic PRIST and RAST procedures and involve one or two monoclonal antibodies labeled with 125I iodine. The performance of these two assays were compared systematically with those obtained with a commercially available polyclonal tracer. We first investigated the 125I-labeling conditions for monoclonal antibodies. In our hands the best results were obtained by use of a tracer of specific radioactivity close to 10 microCi/micrograms. We have been able to demonstrate that as a consequence of its limited affinity, the radioactive tracer is always incompletely bound to the solid-phase IgE. Nonetheless, the sensitivity of the assay is comparable to that obtained with the polyclonal antibody, since less than 0.5 IU/ml can be measured by the PRIST procedure. In contrast, the dilution curves obtained in the RAST with the monoclonal antibodies are very different from those observed with the polyclonal tracer. In fact, these curves are strictly parallel to the dilution curve and to the standard curve derived from the PRIST method, thus indicating that the monoclonal antibodies recognize all IgE equally well regardless of the way in which they are bound to the solid phase. On the basis of this observation, we propose a quantitative assay of specific IgE with the PRIST standard curve as reference. Our results demonstrate that this approach is indeed possible if high-capacity, solid-phase as well as long incubation times are used.     

Clinical evaluation of a new enzymo-assay for allergen-specific IgE

A new assay for the determination of specific IgE in serum, using a monoclonal anti-IgE (Stallerzym), was evaluated. Compared with the results of skin prick tests, the Stallerzym was found to be 93% specific and 73% sensitive. In sera from patients selected to be either clearly allergic or not allergic (as defined by the concordant results of case history, skin prick test, and provocation test), Stallerzym proved to be as accurate and reliable as the Phadezym IgE RAST currently available.     

Immunoglobulin E assayed after pepsin digestion by an automated and highly sensitive particle counting immunoassay: application to human cord blood

We describe in this article a fully automated, universal assay for serum IgE after pepsin digestion of the sample and subsequent assay of the released Fc" epsilon fragment by particle counting immunoassay (PACIA). The sensitivity and the range of the assay were easily modulated by changing the concentration of dextran in the reaction medium. In the application to cord serum, the sensitivity reached 0.1 IU/ml in 30 min incubation time and with a throughput of 50 analyses per hour. Within-day and between-day coefficients of variation did not exceed 7.6% for IgE levels covering a wide range of the standard curve. Dependable accuracy was demonstrated by linearity tests, analytical recoveries (89% to 112%), and correlation with PRIST on 48 samples from children ages 1 to 14 yr (y = 0.97x + 5.92; r = 0.987). Minor discrepancies between the two methods were attributed to a slight serum effect in PRIST. PACIA applied to 348 cord serum samples demonstrated a statistically significant influence of sex and race on the cord IgE level. In European neonates boys had significantly (p = 0.019) higher geometrical mean cord IgE levels (n = 142; 0.46 IU/ml; range less than 0.10 to 30 IU/ml) than girls (n = 146; 0.33 IU/ml; range less than 0.10 to 8.0 IU/ml), which was attributed to a predominance of boys (chi 2 = 4.29; p less than 0.05) having more often elevated cord IgE (less than 1.20 IU/ml) than girls. Neonates of African-Asian origin had significantly (p less than 0.00005) higher cord IgE levels (n = 60; 1.05 IU/ml; range less than 0.10 to 125.0 IU/ml) than European neonates (n = 288; 0.39 IU/ml; range less than 0.10 to 30.0 IU/ml).     

Application of microtitre plates and fluorescence reading to shorten handling of Phadezym RAST® and Phadezym IgE PRIST®

To enable shorter and more convenient testing, the Phadezym RAST® and Phadezym IgE PRIST® procedures for the determination of specific and total IgE were modified in three ways: (i) allergen-coupled paper discs were tested in microtitre wells; (ii) the incubation times were reduced to 1 hr with serum and 2 hr with the anti-IgE by shaking the plates at room temperature; and (iii) the fluorogenic substrate used reduced the development time to 15 min. Determination of IgE antibody specific for fifteen inhalation allergens by the modified fluorescence test (FEIA) and by the conventional Phadezym RAST® (EIA) was performed on the serum of thirty-two patients suffering from asthma/rhinitis: correlation studies for these sera showed that 96.1% of the results fell in the same class. In these patients, both FEIA and EIA detected the same proportion of skin-prick tests (SPT) positive results (67%). With the FEIA, 4/165 (2.4%) class 1 results were found in eleven non-atopic subjects (symptom free, fifteen negative SPT, total IgE lower than 80 kU/1), compared to 1/165 (0.6%) with the EIA. In twenty cord sera, both FEIA and EIA found 4/300 (1.3%) class 1 results. For the determination of total serum IgE, the microtitre FEIA showed a detection limit of 0.5 kU/1 and an excellent correlation with Phadezym IgE PRIST® (n= 66 serum, r= 0.99). These data indicate that the adaptation of Phadezym RAST® and Phadezym IgE PRIST® to microtitre plates and fluorescence technology has resulted in a time-saving and easy to perform within-day assay which provided results as reproducible, sensitive and specific as those of the conventional procedure.     

Usefulness of AlaSTAT, a new method for the measurement of IgE]

AlaSTAT is an enzyme-immunoassay method for the measurement of allergen specific IgE antibodies. This method has a special feature in that carbohydrate, nucleic acid and fat as well as protein allergens can be used as antigens. In this study, IgE antibodies were measured by the use of AlaSTAT kits which were able to detect IgE antibodies to protein or nucleic acid allergens, and the results were compared to those of skin tests and RAST. 1367 samples from 479 patients were examined with 20 allergens. The capacity in the AlaSTAT system was larger than that in the RAST system. The correlation coefficient between AlaSTAT and RAST was 0.90 (p less than 0.01) and the correspondence rate was 90.7%. When the rice allergen was used in RAST, a nonspecific reaction was observed at high concentrations of total IgE. However, no such reaction was observed in AlaSTAT, even with total IgE of 10,000 IU/ml. The sensitivity, specificity and correspondence rate to skin tests was 71%, 87% and 75% respectively. The specificity of AlaSTAT seemed to be slightly higher than that of RAST, but the sensitivity and the correspondence rate were similar to those of RAST. From these results, AlaSTAT appears to be a good or better method for the measurement of IgE antibodies in comparison with RAST.     

In vitro diagnosis of atopic allergy in children

A new multi RAST (Phadiatop) was compared with conventional RAST and total IgE determination (PRIST) for the diagnosis of IgE-mediated allergy in children. Serum specimens were tested from 100 children with a suspected IgE-mediated allergy, restricted to the eyes and/or the respiratory tract, to inhalant allergens. Compared with a RAST panel of 12 allergens representing seven groups, the new multi RAST showed a sensitivity of 95% and a specificity of 100% with a predictive value for positive test of 100% and for negative test of 91%. Corresponding figures for total IgE elevated greater than 2 SD above mean determined with PRIST were 47%, 88% 84% and 56% respectively. We conclude that multi RAST might be a good adjunct to the clinical diagnosis of IgE-mediated allergy to inhalant allergens.     

Isotopic and enzymatic IgE assays in non-allergic subjects

In order to establish normal values in an adult population for serum IgE concentration, sera were obtained from 446 ambulatory Canadian caucasian subjects with negative allergy histories. A standard isotopic procedure, the Phadebas paper radioimmunosorbent test (PRIST), was compared with the new enzymatic counterpart, the Phadezyme PRIST. By the isotopic method, serum IgE concentrations in women and men were comparable from one age group to another with no age-related trend in the seven age groups examined (15-20, 21-30, 31-40, 41-50, 51-60, 61-70, above 70). The median and 95th percentile units (U)/ml respectively were 17.5 and 145 for 224 women and 25.5 and 275 for 222 men. Mean values +/- 1 SD for women were 43 +/- 102 and for men, 58 +/- 137. Levels were significantly higher in men as a group. Sera with IgE concentrations above 100 U and a sampling of additional sera were tested for specific IgE antibodies to 13 common allergens by the radioallergosorbent test (RAST). After exclusion of RAST-positive sera, the mean U/ml values +/- 1 SD were 22 +/- 29 for 204 women and 37 +/- 54 for 196 men. Geometric mean U/ml values for these sera were 14.6 for women and 22.3 for men and the median and 95th percentile U/ml respectively for the women were 15 and 66, for the men, 24 and 135. These 95th percentile values are considered the upper limits of normal in this population. The RAST identification of antibodies to allergens to which sensitization was demonstrated provided a potential explanation for serum IgE concentrations above 100 U/ml in less than 30% of the sera in this population with negative allergy histories. The isotopic method and the counterpart enzymatic method (Phadezyme PRIST) were highly comparable; the correlation coefficient (r) for all the sera was +0.93 (P less than 0.001). IgE levels were significantly higher in male smokers than non-smokers by both methods but these differences were not significant in women.     

Application of the dot immunobinding assay to allergy diagnosis

The recently developed dot immunobinding assay is operationally simple and facilitates performance of multiple simultaneous assays. Here, its use as the basis for determination of total and allergen-specific IgE is established. For total IgE, the same commercially available polyclonal anti-human IgE was used on the solid phase and as a peroxidase conjugate in the liquid phase. After incubation with a chromogenic substrate, IgE was determined from the color intensity of the resulting dots with a scanning reflectance densitometer. The limit of sensitivity was 50 pg/ml of IgE. Standardized conditions gave the dynamic range 50 to 2500 IU/ml in serum. The IgE measured was not subject to interference by serum components, was labile at 56 °C, was soluble at 30% saturation (NH₄)₂SO₄, and was unaffected by anti-human immunoglobulins of other specificity. Coefficients of variation were 0.05 within run, and were 0.1 between run. Comparison with data on sera obtained with the PRIST method yielded a correlation coefficient of 0.96 and a linear regression of slope 1.15. Assays for allergen-specific IgE were established with bee venom and dust mite allergens in the solid phase. The same peroxidase-conjugated antibody was used as for total IgE. Comparisons with comparable RAST assays were performed.     

Sensitization to aeroallergens depends on environmental factors

Respiratory allergies and subclinical sensitization to aeroallergens were investigated in 129 rural and in 222 urban probands. The incidence of respiratory allergies was not significantly higher in the urban residents. Sensitization to aeroallergens was investigated with Phadezym RAST (house dust mite, Cladosporium, orchard grass, birch pollens) and the Phadiatop multi RAST and found to be significantly more frequent in polluted than in unpolluted areas. Allergen-specific IgE was detected n 37.8% of urban probands and in 25.6% of rural probands with Phadezym RAST (P less than 0.025) and a positive Phadiatop multi RAST was found in 43.7% versus 32.6 (P less than 0.05).     

Comparison of procedures for measurement of IgE protein in serum and secretions.

Because of conflicting reports in the literature concerning the value of various procedures for measurement of IgE in serum and secretions, we compared four different methods, the radioimmunosorbent test (RIST), the double-antibody radioimmunoassay (RIA), the paper disc immunosorbent test (PRIST), and radial immunodiffusion (RID). The standards used in the assays were tested initially in the double-antibody RIA with the use of the reference from the World Health Organization. The results showed that RID as expected was relatively insensitive and IgE was reliably measured only above approximately 1,000 international units (IU). Moreover, certain sera containing low levels of IgE by the other procedures gave distinct precipitin zones and presumably falsely high levels of IgE protein. Thus RID may yield apparently erroneous results when used as a screening procedure for measurement of IgE levels. Among the other procedures PRIST and the double-antibody RIA showed the best agreement. With serum samples RIST yielded values for IgE in the low level range higher than those given by the PRIST and double-antibody RIA. With breast milk and colostrum, values of IgE between 120 and 690 ng/ml were found by RIST, whereas IgE was not detected by double-antibody RIA and PRIST. No evidence of an inhibitor of IgE was found in breast milk, so that the apparent elevation of IgE in breast milk by the RIST is likely false. These findings confirm prior reports of spurious elevations of IgE with the RIST and indicate the usefulness of the PRIST and double-antibody RIA for the measurement of IgE in sera and secretions.     

Significance of IgE level in amniotic fluid and cord blood for the prediction of allergy

Intrauterine sensitization has been reported in animal and clinical studies. One study suggests that the amniotic fluid (AF) IgE level might be useful in predicting infant allergy. We followed for 1 year 83 newborns on whom we had 78 samples of AF, 82 of cord serum (CS), and 83 of maternal serum (MS). All infants were delivered by C-section at term. Amniotic fluid samples were aspirated through the exposed myometrium. Sanguineous specimens were excluded. Amniotic fluid, CS, and MS were tested for total IgE level and IgE RAST to three foods: cow's milk, egg white, and peanut. Data on family medical history, feeding history, and allergy symptoms were collected for 12 months. By 1 year 23% had probable allergy: recurrent wheezing = 8, food related G.I. symptoms = 7, and atopic dermatitis = 4. Allergy in formula-fed infants occurred more often than in those breast-fed for greater than 6 months. IgE in AF was greater than or equal to 0.5 IU/mL in 21/78 (27%); range = 0.5 to 5.9 and geometric mean = 0.76. No correlation was noted between AF total IgE and the appearance of allergy. RAST was equivocally positive in 1.2% AF. Cord serum total IgE was greater than or equal to 0.5 IU/mL in 6/82 (7%); range = 0.5 to 2.6 and geometric mean = 0.72. Allergy appeared in 67% of infants with CS total IgE greater than or equal to 0.5 IU/mL. RAST was negative in all CS samples. In this limited series, AF IgE did not seem to have predictive value of allergy in infancy.     

Evaluation of the Phadiatop® test in an epidemiological study

Sera from 204 adult patients with chronic airways obstruction were analysed with the Phadiatop, a new allergosorbent test with a paper disc containing the most relevant inhalant allergens, the PRIST for total IgE determinations, and a panel of seven RAST tests with the common inhalant allergens in The Netherlands as reference. The aim was to evaluate the Phadiatop as screening test in the in vitro diagnostic procedures in an epidemiological setting. The Phadiatop was classified positive or negative according to percentage binding, total IgE was considered elevated at values greater than 100 kU/l and the RAST panel positive when at least one RAST result was class greater than 1. The prevalence of inhalant atopy came to 27.9%. The predictive value of the Phadiatop for a positive RAST panel was 96.4%, and for a negative RAST panel 97.3%. For the PRIST these values were 51.9% and 87.2% respectively. The correlation between the log percentages binding of the Phadiatop and the RAST panel was 0.93. It is concluded that the Phadiatop is a valuable test for the screening of inhalant atopy, and as the percentage binding of the Phadiatop may reflect the degree of sensitization it could also be applied as a quantitative measure especially for epidemiological purposes.     

Improvement of the radioallergosorbent test (RAST) sensitivity by using an antibody specific for the determinant Dɛ2

A technical modification was introduced by the manufacturer (Pharmacia) of a commercially available RAST, with the use of an anti-IgE antibody specific for the domain ε2 of the IgE molecule. Results of the previous and of the new RAST were compared in 100 patients suffering from bronchial asthma or chronic rhinitis. When the same serum was assayed by both techniques, the new RAST technique disclosed a higher positivity in 26% of the comparisons. The new RAST improved the overall correlation between RAST and skin-tests by 6%: individually by 12% for house dust, 8% for Dermatophagoides pteronyssinus and 15% for animal danders. However, the level of correlation was decreased by 8% for the moulds. In patients with a total IgE concentration larger than 100 PRIST units, the new RAST allowed the demonstration of specific IgE in a number of cases twice as large as the previous technique. Thus, the modification of the RAST provides a more sensitive test, the results of which better correlate with those of other diagnostic methods.