M. Michael Glovsky Summary
About 40 yr ago, two groups of investigators identified a new class of immunoglobulins, IgE. By exchanging their results and reagents, they proved that the immunoglobulin responsible for immediate hypersensitivity was IgE. From that day forward the science of allergy was greatly advanced. Within a few years of the IgE discovery, an assay for IgE was developed. This test was named the radio allergosorbent test. The specific IgE testing methodology has matured in the last four decades. Different means of detecting IgE bound to allergen is the subject of this review. We have included methods for measuring specific IgE using the ImmunoCAP 1000 instrument. The methodology for measuring basophile histamine release is also detailed in this chapter.
Key Words: Allergy; RAST; immunoassay; allergen-specific IgE; ImmunoCAP
IgE, the immunoglobulin isotype related to immediate hypersensitivity (allergy), was identified by two groups of investigators in 1966 and 1967. The Ishizakas, in Denver, extracted a new class of immunoglobulins (not IgG, IgA, IgM, or IgD) from reaginic sera of individuals highly allergic to ragweed pollen (1). When the isolated immunoglobulin was passively injected into human skin and the skin was challenged with ragweed extract, a wheal and flare reaction occurred. Within the same years Bennisch and Johanson in Sweden had identified a patient, N. D., with an unusual myeloma protein not identified by antisera to the known immunoglobulin isotypes (2). They called this myeloma protein Ig N. D. The two groups compared the myeloma protein with the isolated immunoglobu-lin and proved that the reagenic antibody and IgE N.D. were similar in structure, antibody reactivity, and function, and named the allergy antibody "IgE" (2).
Diagnostic testing for allergy has progressed since the discovery of IgE. In 1967, Wide and colleagues introduced an in vitro test for specific allergens (3). They produced antibody to human IgE in rabbits and coupled specific allergen protein (such as ragweed or ryegrass extract) to cellulose discs. Next, they added sera of individuals allergic to ragweed, ryegrass, or other allergens to the specific allergen-coated cellulose discs. After washing the discs to remove unbound antibody, they added I125 labeled anti-IgE to the paper discs. The I125 that bound to the discs was analyzed to determine the quantity of specific IgE. This test was named the Radio Allergo Sorbent Test, or RAST (Fig. 1). Since its initial development, the RAST has been modified and automated. Other versions of the specific IgE test have been formulated, such as the MAST, AlaSTAT®, and Hycor-EIA tests. All are based on similar methodologies. This review will focus on the ImmunoCAP-FEIA methodology, thought by some investigators to be the most accurate and best-validated in vitro specific IgE test available.
ImmunoCAP is a third-generation quantitative test developed by Pharmacia Diagnostics from the original RAST technology (4). Also included in this chapter is an overview of newer methods, including flow cytometry, upregulation of basophile receptors CD63 and CD203, and silica chip evaluation of specific IgE using recombinant allergens.
The third-generation Pharmacia ImmunoCAP-specific IgE technology uses an activated sponge-like matrix to absorb specific allergens. The tests are sandwich immunoassays using a flexible, hydrophilic carrier polymer encloses in a capsule as the solid phase (the ImmunoCAP). Serum is added to the Immuno-CAP and incubated, and the cap tube is then washed. Enzyme-labeled anti-IgE is added to the cap, and after further washing the bound IgE is quantified using a fluorescent substrate. Results are reported quantitatively using a kU/L scale. The calibrator is IgE bound to anti-IgE caps using a six-point quantitative curve. Calibration ranges from <0.35 kU/L to >100 kU/L. Results can be reported by a second system using six classes (0, 1, 2, 3, 4, 5, and 6) (Table 1).
CAP testing has been highly automated on several instruments such as the AutoCAP 100, ImmunoCAP 250, and ImmunoCAP 1000; the numeric suffixes indicate the numbers of tubes that can be analyzed.
2.1. Method for Measurement of Specific IgE Using the Unicap 1000 Instrument
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The human body And Todays chemical infested world. Here is a news flash You are not allergic to pollen, pet dander, or whatever it is that makes your body revolt Rather, your body just can not handle that one thing, what ever it is, anymore, due to the massive barrage of toxic chemicals you and everyone else are ingesting every single day.