Measurement of IgE antibodies by the radioallergosorbent test: I. Technical considerations in the performance of the test

doi:10.1016/0091-6749(75)90005-6

Journal of Allergy and Clinical Immunology

Volume 55, Issue 5, May 1975, Pages 334-345

https://doi.org/10.1016/0091-6749(75)90005-6 Get rights and content

Abstract

Since its introduction by Wide, Bennich, and Johansson in 1967, the radioallergosorbent test (RAST) has been increasingly used for measurement of specific IgE antibodies. In this study we have varied the conditions during the performance of the RAST and have investigated the effect of altering: (1) the speed of rotation of tubes, (2) the number of washes in both steps of the test, (3) the length of rotation of tubes, (4) the temperature, (5) the conditions during centrifugation, (6) the quantity of antibody to IgE in the second step of the test, (7) the composition of buffers in the reactions, (8) the type of tube employed in the test, (9) the quantity of antigen employed during the coupling procedure, and, finally, (10) the iodine isotope, ¹²⁵I or ¹³¹I, used to radiolabel anti-IgE. Of these variables the most significant were the quantity of anti-IgE used in the second step of the test and the quantity of antigen used in the coupling procedure. Other variables of importance were the choice of radioiodine, whether the tubes were rotated or not, the type of tube used, and finally the diluent employed.

References (26)

L. Wide et al.
Diagnosis of allergy by an in-vitro test for allergen antibodies
Lancet
(1967)
R. Evans et al.
An immunologic evaluation of ragweed-sensitive patients by newer techniques
J. Allergy Clin. Immunol
(1972)
K. Aas et al.
The radioallergosorbent test in the in-vitro diagnosis of multiple reaginic allergy
J. Allergy Clin. Immunol
(1971)
B. Stenius et al.
Reaginic antibody (IgE), skin and provocation tests to Dermatophagoides culinae and house dust in respiratory allergy
Lancet
(1969)
T. Foucard et al.
Concentration of IgE antibodies, PK titers, and chopped lung titers in sera from children with hypersensitivity to cod
J. Allergy Clin. Immunol
(1973)
G.J. Gleich et al.
Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test
J. Allergy Clin. Immunol
(1974)
J.W. Yunginger et al.
Measurement of ragweed antigen-E by double antibody radioimmunoassay
J. Allergy Clin. Immunol
(1972)
M. Ceska et al.
Radioimmunosorbent assay of allergens
J Allergy Clin. Immunol
(1972)
S.G.O. Johansson et al.
The clinical significance of IgE
G.J. Gleich et al.
Immunoglobulin E and atopic diseases
South Dakota J. Med
(1973)

T. Berg et al.

In-vitro diagnosis of atopic allergy. I. A comparison between provocation tests and the radiollergosorbent test

Int. Arch. Allergy Appl. Immunol

(1971)

T. Berg et al.

In-vitro diagnosis of atopic allergy. IV. Seasonal variations of IgE antibodies in children allergic to pollens

Int. Arch. Allergy Appl. Immunol

(1971)

L.M. Lichtenstein et al.

IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy

J. Clin. Invest

(1973)

Cited by (48)

An electrochemiluminescence (ECL)-based assay for the specific detection of anti-drug antibodies of the IgE isotype
2013, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
In vitro test methods for drug-specific IgE antibodies have been documented. A radioimmunoassay was employed for the detection of specific IgE antibodies to erythromycin [16,17]. The ImmunoCap platform has been adapted for the detection of IgE antibodies specific to several biologics [14,18,19].
To address a possible linkage between the occurrence of the hypersensitivity reactions and the induction of IgE anti-drug-antibodies (ADA), a drug specific IgE ADA assay was developed using electrochemiluminescence (ECL) technology. In the assay a drug-specific IgE isotype chimeric antibody was generated and used as an ADA positive control. The biotinylated drug X (an antibody) and ruthenium-labeled omalizumab (an anti-human IgE antibody) were used as capture and detection reagents, respectively.
The binding affinities of the chimeric IgE isotype positive control have been shown to be highly comparable to drug X and drug Y (drug X is the 2nd generation of drug Y), indicating that it could serve as a highly useful control to compare and contrast the relative ability of the two generations of drug to elicit IgE ADA responses. The assay cut point factor (CPF) was estimated to be 1.13. The cut point factor derived from normal human serum samples was statistically equivalent to the cut point factor determined from targeted population samples. The assay could detect less than 250 ng/mL of IgE antibodies in the presence of 300 μg/mL drug X. The assay sensitivity was <0.2 ng/mL. A minimal prozone was observed at 100 μg/mL IgE ADA, but the sample remained highly detectable. The inter-assay precision was within 12%. The assay was not susceptible to non-specific matrix effects. The performance specifications ensured that the assay was suitable for validation. The combination of the chimeric IgE positive control and the detection antibody (ruthenium-labeled omalizumab) used for the assay could potentially provide a general bioanalytical approach for other biopharmaceuticals for the detection of IgE ADA responses.
Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase
2005, Journal of Allergy and Clinical Immunology
Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens.
To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent.
Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis.
The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r² = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001).
The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.
In vitro assays for the diagnosis of IgE-mediated disorders
2004, Journal of Allergy and Clinical Immunology
Citation Excerpt :
It is the most complex and highly variable reagent in IgE antibody assays, in part because of the heterogeneity of most allergen extracts and the different chemistries used to insolubilize or label the allergenic proteins. In an attempt to improve on the antibody-binding capacity of the paper disc, a variety of carbohydrate-based allergosorbents (other than Sephadex and paper), such as microcrystalline cellulose and agarose, were historically used in research (Table II).4,5 The most significant advance for clinical assays, however, was the development of an encapsulated hydrophilic carrier polymer to which allergen was covalently coupled.6
Advances in technology have provided new laboratory tools for the quantitation of allergen-specific IgE antibodies in serum and on the surface of basophils. This review examines the evolution from qualitative IgE antibody assays of the late 1960s to the present-day, third-generation, automated and quantitative allergen-specific IgE assays. The latest technology trend is toward microarrays in which crude or purified native and recombinant allergens can be spotted in microdot arrays on silica chips to permit extensive panels of specific IgE measurements to be performed with small quantities of serum. Although these technologies hold promise, their diagnostic performance requires further assessment once their technical details have been optimized. Potential abuses of this newer IgE antibody technology include the use of allergosorbent specificities (eg, especially food and drugs) that lack validation, application of IgE antibody measurements in the diagnosis of non-IgE–dependent disorders (eg, aspirin sensitivity), and modification of IgE antibody assays to measure food-specific IgG antibody for which there is no clinical indication. Basophil mediator release assays have evolved to include flow cytometric methods that can quantitatively detect the presence of cell surface–bound allergen-specific IgE antibodies. Assays for histamine and leukotriene C₄ released after in vitro basophil activation are now more accurate and standardized. Current analytic methods for IgE antibodies provide more quantitative and reproducible measurements of IgE than ever before, although still with less sensitivity that traditional skin testing. The current challenge is to translate the quantitative IgE antibody results into a more accurate diagnosis of allergic disease.
Clinical value and measurement of specific IgE
2003, Clinical Biochemistry
The diagnosis of human allergic diseases involves the combined use of a careful clinical history, physical examination, and in vitro and in vivo assay methods for the detection of IgE antibodies of defined allergen specificities. In vivo (skin testing) and in vitro (measurement of specific IgE in serum) techniques cannot be considered interchangeable, the former reflecting not only the presence of IgE but also mast cell integrity, vascular and neural responsiveness. Both techniques have similarities and differences, advantages and disadvantages. Recently introduced “second generation” immunoassays have continued to improve the analytical sensitivity and reproducibility thanks to automation and improved reagent quality. Quantitative assays may allow the use of specific clinical thresholds able to differentiate symptomatic from asymptomatic patients. False-negative and false-positive results should derive from lability of some major extracts, and from possible cross-reactivities, respectively. Characterization of allergens at a molecular and submolecular level and, where necessary, the use of recombinant allergens can reduce cross-reactions and further improve the quality of immunoassays.
Role of clinical laboratory in allergy testing
1994, Clinical Biochemistry
The clinical laboratory has a well defined role to play in the diagnosis and management of patients with allergy. Elevated serum levels of total IgE and/or allergen-specific IgE indicate that an IgE mediated event has occurred. Methods such as basophil degranulation and basophil or leukocyte histamine release can provide similar information. Sensitive and precise methods suitable for automation are available for quantitation of histamine in whole blood or plasma. Methyl histamine can be assayed in urine. Eosinophil cationic protein levels in serum can be used as an indicator of eosinophil activation in disorders such as asthma and atopic dermatitis. Similarly, serum mast cell tryptase levels can confirm or exclude an anaphylactic reaction both in life and as a cause of death. This review documents and compares commercially available methods for these assays and discusses their application to screening, diagnosis, and management of patients with allergy.
Effect of immunotherapy on the serum concentrations of allergen-specific IgG antibodies in dog sera
1989, Veterinary Immunology and Immunopathology
An ELISA assay which uses horseradish peroxidase conjugated anti-canine IgG and polystyrene microtiter wells for detection of allergen-specific IgG in the serum of dogs is described. Individual allergen blanks were used to account for the variable nonspecific binding among various allergens, and the results observed in milliunits of absorbance were normalized using four reference sera. The coefficients of variation for the intraassay and interassay variability ranged from 1.34 to 12.50% and 4.62 to 9.77%, respectively. The relationship between ELISA results and serum concentrations of allergen-specific IgG was quantified. IgG antibodies with specificity for various allergens were found in the majority of non-atopic individuals and in all atopic subjects. Specific immunotherapy resulted in a rise in the serum concentration of allergen-specific IgG.

View all citing articles on Scopus

^☆: Supported by grants from the National Institutes of Health, Division of Biologic Standards, NIH-DBS-72-2087, and the National Institute of Allergy and Infectious Diseases, AI-11483.

View full text

Article preview

Journal of Allergy and Clinical Immunology

Abstract

References (26)

Diagnosis of allergy by an in-vitro test for allergen antibodies

Lancet

An immunologic evaluation of ragweed-sensitive patients by newer techniques

J. Allergy Clin. Immunol

The radioallergosorbent test in the in-vitro diagnosis of multiple reaginic allergy

J. Allergy Clin. Immunol

Reaginic antibody (IgE), skin and provocation tests to Dermatophagoides culinae and house dust in respiratory allergy

Lancet

Concentration of IgE antibodies, PK titers, and chopped lung titers in sera from children with hypersensitivity to cod

J. Allergy Clin. Immunol

Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test

J. Allergy Clin. Immunol

Measurement of ragweed antigen-E by double antibody radioimmunoassay

J. Allergy Clin. Immunol

Radioimmunosorbent assay of allergens

J Allergy Clin. Immunol

The clinical significance of IgE

Immunoglobulin E and atopic diseases

South Dakota J. Med

In-vitro diagnosis of atopic allergy. I. A comparison between provocation tests and the radiollergosorbent test

Int. Arch. Allergy Appl. Immunol

In-vitro diagnosis of atopic allergy. IV. Seasonal variations of IgE antibodies in children allergic to pollens

Int. Arch. Allergy Appl. Immunol

IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy

J. Clin. Invest

Cited by (48)

An electrochemiluminescence (ECL)-based assay for the specific detection of anti-drug antibodies of the IgE isotype

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

In vitro assays for the diagnosis of IgE-mediated disorders

Clinical value and measurement of specific IgE

Role of clinical laboratory in allergy testing

Effect of immunotherapy on the serum concentrations of allergen-specific IgG antibodies in dog sera

Measurement of IgE antibodies by the radioallergosorbent test: I. Technical considerations in the performance of the test☆

Abstract

Lancet

J. Allergy Clin. Immunol

J. Allergy Clin. Immunol

Lancet

J. Allergy Clin. Immunol

J. Allergy Clin. Immunol

J. Allergy Clin. Immunol

J Allergy Clin. Immunol

The clinical significance of IgE

Immunoglobulin E and atopic diseases

South Dakota J. Med

In-vitro diagnosis of atopic allergy. I. A comparison between provocation tests and the radiollergosorbent test

Int. Arch. Allergy Appl. Immunol

In-vitro diagnosis of atopic allergy. IV. Seasonal variations of IgE antibodies in children allergic to pollens

Int. Arch. Allergy Appl. Immunol

IgE antibody measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy

J. Clin. Invest