857© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/B978-0-08-097037-0.0006...
858 The Immunoassay Handbook
3. Plant and animal food allergens (fruits, vegetables,
nuts, milk, eggs, shellfish, and fish):...
859CHAPTER 9.14 Allergy
DIAGNOSIS AND THERAPY
In diagnosis, atopic IgE-mediated allergy must be differ-
entiated from cond...
860 The Immunoassay Handbook
advantages and great limitations of these techniques have
fueled disagreement among clinician...
861CHAPTER 9.14 Allergy
maintained throughout adult life. The geometric mean of
truly healthy nonsmoking adults is about 1...
862 The Immunoassay Handbook
Law of Mass Action
(1)
(2)
(3)
When the assay is essentially affinity independent, i.e.,
when ...
863CHAPTER 9.14 Allergy
Preparation for human IgE (75/502). This allows true
quantitation of the specific antibodies provid...
864 The Immunoassay Handbook
REFERENCE VALUES
According to the rules of WHO for establishment of stan-
dards for biologica...
865CHAPTER 9.14 Allergy
UniCAP was also evaluated in six clinical studies including
894 consecutive patients. The clinical...
866 The Immunoassay Handbook
The characterization of the protein epitopes recognized
by the IgE is fundamental to the unde...
867CHAPTER 9.14 Allergy
Plebani, M. Clinical value and measurement of specific IgE. Clin. Biochem. 36,
453–469 (2003).
Pre...
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  1. 1. 857© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00068-3 Allergy Marcos Alcocer1 (Marcos.Alcocer@nottingham.ac.uk) Lars Yman2 1 This edition. 2 Previous editions. Allergic Disease In general, allergy is not an easy disease to define. In 1906, the Viennese physician Von Pirquet described it as an uncommon sensitivity against substances completely harmless to the majority. It belongs to a large group of inflammatory conditions involving several interacting sys- tems in the human body, sometimes described as hyper- sensitivity type I, II, III, or IV (Coombs and Gell, 1968). The social and economic impact of allergies is also not easy to describe. Although the statistics vary from different countries and within individual regions, it is now recog- nized that, regardless of the measuring indicator, hospital admissions for some systemic allergic diseases have risen sharply in the last decade (Gupta et al., 2007), which may indicate a rising incidence in these conditions. After extensive nomenclature revision (Johansson et al., 2004), allergy is currently defined as a set of reproducible symptoms (hypersensitivity) initiated by exposure to a defined harmless environmental stimulus at a dose tolerated by normal people that involves specific (cell and antibody related) immunological mechanisms. This hypersensitivity can cause a range of inflammatory symptoms such as rhinitis, asthma, urticaria, dermatitis, and potentially life-threatening (anaphylactic) immediate and late reactions. In critical cases, where the mucosal membranes of the airways or gastrointestinal tract are compromised, allergy is characterized by excessive activation of immune cells (mast cells and basophils) by antibodies of the IgE class in a TH2 process also known as hypersensitivity type I (Fig. 1). Mast cells and basophils play a central role in allergy, releasing mediators that stimulate glandular secretions and smooth muscle contraction, and increase the permeability of the small blood vessels. These typical features of the immediate phase of an allergic asthma attack or an out- break of allergic rhinitis are often followed by a late phase, initiated by the release of chemotactic factors from the mast cell. These activate cells, such as eosinophil leuko- cytes, to release substances like eosinophil cationic protein damaging the tissues (Svensson et al., 1990). A particular characteristic of mast cells and basophils is that they carry high-affinity receptors (Fcε) for immuno- globulin molecules of the IgE class on their surface. Although most people are able to produce IgE antibodies against parasitic antigens, allergic individuals tend to pro- duce excessive amounts of IgE antibodies to certain innocu- ous foreign macromolecules via inhalation, ingestion, or injection and hence develop allergic symptoms when reex- posed to the same material. The foreign materials or sensi- tizers that precipitate IgE production (sensitization) are referred to as allergens, and individuals with a tendency to become sensitized as atopic. In mucosal membranes, after the sensitization phase, mast cells and basophils are loaded with specific IgE through the Fcε receptors. Reexposed allergens will then cross-link the specific IgE antibodies on the surface of basophils/mast cells triggering, through a cas- cade of well-known events, the activation of these cells that will result in potentially life-threatening immediate and late allergic reactions (Fig. 1). ALLERGENS Substances capable of eliciting an allergic reaction are gen- erally referred to as allergens, regardless of the mechanism involved. This chapter is limited to allergens that can stim- ulate the synthesis of IgE antibodies, causing atopic allergy. Identification of the offending allergen can be complex and difficult. Although the timing of a seasonal allergy may give clues to the cause, most affected patients are sensi- tized by allergens from many sources with overlapping seasons of exposure and often with overlapping immuno- logical specificity. Many allergens have recently been identified and cloned with a number of databases set up to provide information on their molecular, biochemical, and clinical data. The current nomenclature utilizes the following format: Ggg(g) s(s) n.iivv, where g=genus (2–3 letters), s=species (1–2 let- ters), n=allergen number, i=isoallergen number (2 digits), v=variance number (2 digits). Hence, the main major pol- len allergen from Betula verrucosa (birch) is known as Bet v 1 and one of the isoallergens and variants by Bet v 1.0102. The official list of allergens issued by World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-committee (WHO/ IUIS) (www.allergen.org), the list available at the Aller- gome database (www.allergome.org) and others described elsewhere (Valenta et al., 2010) are good initial resources for current nomenclature and clinical data. Classification In generic terms, allergens can be classified as (Chapman, 2008): 1. Indoor allergens (mite, animal allergens, cockroach, and molds): proteolytic enzymes (serine and cyste- ine proteases), lipocalins (ligand-binding proteins), tropomyosins, albumins, calcium-binding proteins, protease inhibitors. 2. Outdoor allergens (grass, tree and weed pollens, and mold spores): plant pathogenesis-related (PR- 10) proteins, pectate lyases, b-expansins, calcium- binding proteins (polcalcins), defensin-like proteins, trypsin inhibitors. C H A P T E R 9.14
  2. 2. 858 The Immunoassay Handbook 3. Plant and animal food allergens (fruits, vegetables, nuts, milk, eggs, shellfish, and fish): lipid-transfer proteins, profilins, seed storage proteins, lactoglob- ulins, caseins, tropomyosins, parvalbumins. 4. Injected allergens (insect venoms and some thera- peutic proteins): phospholipases, hyaluronidases, pathogenesis-related proteins, asparaginase. Based on this classification and in order to illustrate the range and properties of allergens normally encountered in the environment, some examples of the source, name, and homologies of some common allergens are shown in Table 1. COMMON PROPERTIES OF ALLERGENS Despite advances in genomics, bioinformatics and evolu- tionary biology, and continuous effort by a large com- munity of experts worldwide, the attributes that define an allergen, i.e., the intrinsic characteristics that confer allergenicity to an otherwise harmless substance remain largely unknown. Only two mechanisms, both described in the Dermatophagoides pteronyssinus (house dust mite system), are currently known: a protease-dependent (Der p 1) and a protease-independent one (Der p 2). In the former Der p 1, protease activity has been shown to cleave CD23, CD25, and CD40, inhibiting IL-12 secre- tion (Ghaemmaghami et al., 2002) with the possible acti- vation of the PAR family of G-coupled proteins in the process (Kauffman et al., 2006). In the latter mechanism, it has been elegantly shown that Der p 2 structurally mimics MD2, a component of the TLR4 complex that can co-opt other signaling components to activate its tar- get cells (Trompette et al., 2009). Hence, the involve- ment of complex lipids and Toll receptors has been demonstrated. As most of the well-characterized aller- gens are not proteases, and it is unlikely that MD2 homology could be a general mechanism, it is possible that other mechanisms exist. The general observation that most of the families of allergens are found in a reduced number of families (only 2% of all sequence- based and 5% of all structural protein families) with a bias toward certain biochemical functions such as Ca2+, actin, and lipid binding, most of them involved in innate defense functions against pathogens, is interesting (Radauer et al., 2008). Allergens do not share common structural or functional properties, however, in general terms, most allergens are biological materials which, when inhaled or ingested, have to gain access to the body via either a lipophilic barrier (skin) or hydrophilic one by dissolving in mucosal secre- tions. Some allergens bypass these barriers either through stings, bites, or drug injections or by being produced endogenously by invading parasites (Aalberse et al., 2001). Allergens also tend to be comparatively easy to extract and dissolve in buffered solutions. Typically, the allergy source releases a complex mixture of potentially allergenic macro- molecules such as carbohydrates, complex lipids, polypep- tides, glycoproteins, and peptidoglycans. Atopic individuals may produce IgE antibodies to one or many of the aller- genic components, and there is strong evidence that indi- cates that the recognition of the allergen or its epitope profile remains constant during the natural course of the disease (Valenta et al., 2007). Thus, every allergic patient has a range of unique antibodies to a given allergen source. Because some allergenic components have a higher rela- tive concentration and greater immunogenic activity than others, it is possible to test allergic sensitivity qualitatively using selective combinations of a limited range of allergens. Ag Ag APC Lymphatic system Lymph node THo Cell B Cell IgE Mast Cells/ basophils FcE rec. Vasodilation Vascular permeability Adhesion molecules Bronchoconstriction IL5 IL4/IL13 TH2 Cell IL4 IL4 FIGURE 1 Main immunological cellular events taking place during sensitization and subsequent contact with the allergen (see text). Ag=antigen or allergen, APC=antigen presenting cells, TH =T helper Cell. Modified from Loewenstein and Mueller, 2009. (The color version of this figure may be viewed at www.immunoassayhandbook.com).
  3. 3. 859CHAPTER 9.14 Allergy DIAGNOSIS AND THERAPY In diagnosis, atopic IgE-mediated allergy must be differ- entiated from conditions initiated by other mechanisms, both immunological and non-immunological (Fig. 2). Normally, this involves testing and identifying specific IgE in serum. A raised level generally confirms a clinical diag- nosis and acts as a basis for treatment. Although in clinical practice, in vivo tests, such as prick and intradermal skin testing, are still widely used methods for demonstrating specific IgE antibody activity, the TABLE 1 Molecular Properties of Common Allergens Source Allergen MW (kDa) Homology Indoor House dust mite (Dermatophagoides pteronyssinus) Der p 1 25 Cysteine protease Der p 2 14 Lipid-binding protein Der p 3 30 Serine protease Der p 5 14 Unknown Cat (Felis domesticus) Fel d 1 38 Uteroglobin-like protein Dog (Canis familiaris) Can f 1 25 Lipocalin Mouse (Mus musculus) Mus m 1 21 Lipocalin Rat (Rattus norvegicus) Rat n 1 21 Pheromone-binding lipocalin Cockroach (Blattella germanica) Bla g 2 36 Inactive aspartic protease Outdoors Pollen/Grasses Rye (Lolium perenne) Lol p 1 28 Unknown Timothy (Phleum pratense) Phl p 5 32 Unknown Bermuda (Cynodon dactylon) Cyn d 1 32 Unknown Weeds Ragweed (Artemisia vulgaris) Art v 1 28 Defensin-like protein Trees Birch (Betula verrucosa) Bet v 1 17 Pathogenesis-related protein Bet v 2 15 Profilin (actin binding) Foods Milk Bos d 5 18 β-lactoglobulin Egg Gal d 1 28 Ovomucoid Codfish (Gadus callarias) Gad c 1 12 Ca-binding protein (parvalbumin) Shrimp (Penaeus aztecus) Pen a 1 36 Tropomyosin Peanut (Arachis hypogaea) Ara h l 63 Vicilin (seed storage protein) Celery (Apium graveolens) Api g 1 16 PR-10, Bet v 1 homologous Brazil nut (Bertolletia excelsa) Ber e 1 12 2S Albumin Venoms Bee (Apis mellifera) Api m 1 9.5 Phospholipase A2 Wasp (Polistes annularis) Pol a 5 23 Mammalian testis proteins Homet (Vespa crabro) Ves c 5 23 Mammalian testis proteins Fire ant (Solenopsis invicta) Sol i 2 13 Unknown Fungi Aspergillus fumigatus Asp f 1 18 Ribonuclease Asp f 2 37 Fibrinogen binding Alternaria altenata Alt a 1 29 Unknown Latex Hevea brasiliensis Hev b 1 15 Elongation factor Hev b 5 16 Unknown function Drugs Tetracycline Codeine Penicillin Sulfonamides N/steroid anti-inflammatory Modified from Chapman, 2004, 2008.
  4. 4. 860 The Immunoassay Handbook advantages and great limitations of these techniques have fueled disagreement among clinicians for decades and hence are out of the scope of this review. One of the major and irreplaceable advantages of in vivo methods, however, is the strong psychological patient–doctor component brought about by these techniques. Alternatively, current in vitro techniques for specific IgE detection present many advantages to clinical diagnostics such as reproducibility, sensitivity, specificity, and stability. Most of these greatly facilitate quality control and interlab comparisons. The major advantages of in vitro methods, however, are undoubtedly the avoidance of further potential sensitiza- tions and risks of systemic reactions. Once the eliciting allergen is known, allergy can gener- ally be treated by eliminating exposure to the allergen, injecting a natural or modified version of the allergen (specific immunotherapy), or administering antihista- mines, steroids, or other medications. The latter drugs inhibit the release of mast cell mediators or block their effects. As will be discussed later in the COMPONENT- RESOLVED DIAGNOSTICS section, the success and effectiveness of the modern and innovative immunother- apy are directly related to the high degree of specificity obtained by current in vitro methods. Analytes A unifying thread linking atopic conditions (e.g., dermati- tis, asthma, and rhinitis) is their occurrence in individuals with markedly elevated levels of IgE antibodies. Hence, due to their historic value, widespread use and importance, the principles of Phadebas radioallergosorbent test (RAST), still colloquially used to describe immunoassays for IgE will be initially described. The theoretical concepts of excess of antigen, developed successfully for earlier sys- tems, are still valid for new generations of profiling tools that will be discussed later. IgE is a very particular class of immunoglobulin that is highly catabolized with a short biological half-life of 1–5 days in peripheral blood. It does not cross the placenta or activate classical complement pathways. However, from a clinical perspective, the accurate assessment of sensitivity and specificity of IgE antibodies can identify causative allergens and aids the clinical history in the diagnosis of allergic diseases (Plebani, 2003). In regular clinical prac- tice, two measurements are normally carried out: total serum IgE and specific serum IgE. TOTAL SERUM IGE Although their clinical role has been increasingly con- tested (Kerkhof et al., 2003), historic habits, automation, and good reproducibility made the immunoassays for total serum IgE popular as an aid in the clinical diagnosis of atopic diseases. The tests generally use a wide range of assay principles: competitive double-antibody, solid-phase radioimmunoassays (RIA) coexist with immunometric (sand- wich) enzyme immunoassays. Solid phases in common use include microparticles, polystyrene beads and tubes, and cellulose foam. The signals measured can be radioactivity, color, fluorescence, and luminescence. Concentrations are expressed as kU/L defined by the WHO International Reference. Clinical Applications The serum total IgE level is generally raised in atopic dis- ease, although an IgE concentration within the normal range does not rule out IgE-mediated disease, especially if the dis- ease is due to a single allergen. Total IgE measurements have been used as an aid for clinical diagnosis to differentiate atopic from nonatopic disease and for prediction of allergy in children by measurement in cord blood or in newborns. However, great care must be exercised in their interpretation (see LIMITATIONS; Kerkhof et al., 2003). The serum IgE con- centration is increased in atopic allergy (asthma, rhinitis), especially in eczema patients who may exceed 20,000kU/L. The total serum IgE concentration is normally correlated with the number of allergens involved. IgE is also raised in parasitosis and some immunodeficiencies. Reference Intervals The serum total IgE level in healthy individuals increases during childhood from <1kU/L at birth to a peak at the age of about 10 years and then declines to levels that are FIGURE 2 Allergy-like symptoms. The majority of cases have perennial or irregular symptoms that may be IgE mediated or triggered by other conditions. Differentiation mostly requires extensive testing.
  5. 5. 861CHAPTER 9.14 Allergy maintained throughout adult life. The geometric mean of truly healthy nonsmoking adults is about 10kU/L and the mean+2SD is slightly above 100kU/L. It is recommended that allergy investigation is done on all children with levels higher than the mean for the age+1SD, and in adults if the level is higher than 100kU/L (Johansson and Yman, 1988). The validity of the original adult reference values, geomet- ric mean=13.2kU/L, mean+2SD=114kU/L (Zetter- ström and Johansson, 1981) was confirmed with UniCAP, testing 63 healthy blood donors without known allergy. Geometric mean=17.4kU/L, mean+2SD=112.9kU/L (Persson and Yman, 1997). Limitations Due to the low negative predictive value, total IgE levels are in general not efficient to rule out sensitization to com- mon inhalant allergens (Kerkhof et al., 2003). High total IgE might indicate a high probability of sensitization in younger subjects, but identification will still require fur- ther specific testing. Total IgE levels are less closely linked to allergic disease in areas with high incidence of parasitosis. Assay Technology A wide variety of immunoassay procedures are in routine use. The bulk of testing is performed in laboratory systems with varying degrees of automation. Desirable Assay Performance Characteristics An assay covering the range 0.5–5000kU/L would satisfy the practical clinical needs for IgE determinations from cord serum measurements on allergy risk babies to testing of subjects with atopic dermatitis or parasitosis. To keep imprecision (repeatability and reproducibility) as low as possible, this range is typically split into a routine range (preferably 2–2000kU/L) and a low range for primarily pediatric use. UniCAP low measuring range is equivalent to the range covered by the specific IgE assay, i.e., 0.35– 100kU/L. Although some other commercial assays include diluents as zero calibrators, the detection limits are always higher. An option to store calibration curves is desirable. Types of Sample CAP assays for total and specific IgE are generally vali- dated for serum and plasma. Measurements in tears (Kari et al., 1985; Sainte Laudy et al., 1994), nasal secretion (Deuschl and Johansson, 1977; Sensi et al., 1994), and feces (Sasai et al., 1992) have been reported. Applications out- side those described in the manufacturer’s instructions for use must be validated by the investigator. ALLERGEN-SPECIFIC IGE ANTIBODY Allergen-specific antibodies of a single immunoglobulin class have to be measured in the presence of other antibod- ies of the same class and antibodies of other classes specific for the same allergen. This requires specific recognition of the antigen-binding sites (Fab) and the class-specific epit- opes (Fc) in the same assay. In the case of IgE, by having the lowest concentration of any of the five immunoglobu- lin isotypes in the sera, the concentration of specific anti- body should be measurable down to picograms per milliliter in samples that may contain total IgE and com- peting antibodies of other classes well into the microgram per milliliter range. Early specific allergen detection tests lacked reproduc- ibility, mainly due to large batch-to-batch variation in sta- bility of the allergens following extensive extraction procedures. Hence, an important advance in allergy diag- nosis testing was the introduction of standardized allergen extracts achieved in the late 1970s and early 1980s. The standardization of allergens was responsible, not only for the improvement of the accuracy of in vivo tests, but also for the reliability of this test when coupled to the RAST test. During the 1990s, with the availability of protein sequences, large databases and advances in protein expres- sion, the production of large quantities of specific well- purified recombinant allergens became possible. Over the past decades, many platforms have been developed for specific IgE detection (Renault et al., 2007), however, as previously mentioned, Phadebas RAST remained collo- quially identified in the description of immunoassay for specific IgE test. Hence, the basic concepts involved in the design of the RAST test, the subsequent step with the introduction of a solid support with higher allergen capac- ity and automated systems, such as UniCAP, will be ini- tially discussed (Fig. 3). Later in the section, other platforms introduced to the market resulting from recent advances in genomics and automation will also be briefly mentioned. General Assay Characteristics IgE assays use a solid phase coated with allergen to bind the IgE antibody in the sample. There is a critical washing step after the initial allergen–IgE antibody reaction and before the incubation with labeled anti-IgE. The solid phase may alternatively be coated with ligands that catch allergen–IgE complexes before the washing step. A high- capacity solid phase, like the chemically activated foam of the Pharmacia CAP System and UniCAP, provides a large excess of allergen that maximizes the binding of the IgE antibody. This makes the assay less sensitive to competing antibodies of other classes. It also means that the bound IgE antibody can be detected with less amplification. The resulting lower concentration of anti-IgE, less intensive labeling, and lower substrate concentration minimizes sev- eral of the classic contributions to high and variable non- specific background. Allergen in Excess A high-capacity solid phase provides conditions for quan- titation of the sum of all antibodies regardless of specificity and affinity. The Law of Mass Action, applied to heteroge- neous, solid-phase immunoassays, predicts that when the available allergen concentration is increased to a level, where K (the affinity constant) multiplied by the allergen concentration Eqn (3) reaches ≥10, the proportion of IgE antibody bound to the solid phase will be ≥90% and essen- tially independent of the affinity between the antibody and the allergen (Peterman, 1991; Yman, 1994).
  6. 6. 862 The Immunoassay Handbook Law of Mass Action (1) (2) (3) When the assay is essentially affinity independent, i.e., when more than approximately 90% of the IgE antibody is bound, a further increase of the allergen concentration will have negligible effect on the measured value (Fig. 4). Anti-IgE Anti-IgE preparations must be IgE Fc specific and are pref- erentially combinations of, e.g., monoclonal antibodies with specificity against more than one epitope on the Fc fragment and with complementary dose–response characteristics. Labeled anti-IgE has been prepared using several signal gen- eration systems, in each case aiming at a high sensitivity. Calibration Calibrators for both specific and total IgE measurements should be traceable to the WHO International Reference FIGURE 3 Principle of Phadebas RAST®, Pharmacia CAP System™ RAST®, and UniCAP® -specific IgE. FIGURE 4 Diversity of IgE antibody patterns against soy bean allergen in sera from 21 individuals. The allergenic components were separated by SDS-g-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Allergen-IgE complexes were detected by radiola- beled anti-IgE and autoradiography (Perborn, 1990, unpublished data).
  7. 7. 863CHAPTER 9.14 Allergy Preparation for human IgE (75/502). This allows true quantitation of the specific antibodies provided that the allergen is in excess. The calibrator range of UniCAP-spe- cific IgE is 0.35–100kU/L. The design of the assay should permit storage of calibration curves up to 1 month using curve controls in each assay to verify the validity of the stored calibration curve. Test Selection, Use, and Interpretation Allergy tests should be used selectively, taking into account the local distribution of the allergens and the clinical history of the individual patient. Unless there is a clear link between the appearance of symptoms and occasions of exposure to one allergen, multisensitivity should always be suspected. Multisensitive Patients – Phadiatop® Before trying to identify offending allergens, patients with allergy symptoms should first be tested to confirm that the disease is atopic. If not, further searches for offending allergens are rarely successful. Testing with Phadiatop detects at least 90% of patients with atopic inhalant allergy (Eriksson, 1990). Neither Phadiatop nor any other com- mercial test with this type of indication gives quantitative results. Phadiatop was designed to be a test for atopic allergy with its main use for patients with respiratory symptoms. This means that common inhaled allergens form the base. Furthermore, it means that cases primarily sensitized by food or insect sting, by occupational exposure, or by keep- ing more or less exotic pets, like birds, guinea pigs, etc., cannot be detected unless the patients are also sensitive to common inhalants. The allergens selected for Phadiatop, and the way they are combined, are aimed at achieving an approximately 90% probability of correct classification of atopics and nonatopics. The combined effect of exposure, multisensi- tization, and cross-reactivities between allergens gives a high cumulative efficiency already for a relatively limited combination of species. The order of the allergens in terms of contribution to the cumulative efficiency varies between populations depending on relative intensity of exposure, but it has been possible to select three combinations that cover the needs of Europe, North America, and Japan. Standardization and Evaluation Allergy tests are used to help in the diagnosis and monitor- ing of allergic patients. Assays need to be sensitive and spe- cific and permit quantitative measurements over a wide range (Bernstein, 1988). As with all immunoassays, there is a need for common standards and definitions of perfor- mance parameters. In the case of total serum IgE, the WHO standard serves its purpose but for the measure- ment of allergen-specific antibodies, there is no official standard collection of units and there are no uniform definitions. In reality, however, Phadebas RAST, first available in 1974, became a working standard against which all subsequent commercial tests were compared. Phadebas RAST includes a reference consisting of several dilutions of a serum containing IgE antibodies specific for birch pol- len allergen. The dilutions were selected to permit the construction of a calibration curve and evaluation of test results in arbitrary units (PRU/mL) or semiquantitative classes. This standard was internally calibrated against the WHO IgE standard (Lundkvist, 1975). In the other gen- erations of RAST (Pharmacia CAP System and UniCAP), the birch reference was replaced by a direct substandard to the WHO standard 75/502 for IgE (Yman, 1990) covering the range 0.35–100kU/L. Concentrations lower than the 0.35kU/L calibrator are considered to be undetectable. Tests for all allergens are evaluated against this IgE standard. All other commercial test kits largely adhere to this principle. The apparent agreement in terms of classes or units is, however, usually not supported by data defining the true concentrations of the calibrators. Direct compari- sons are therefore difficult. Modified scoring systems are used routinely by labora- tories and physicians. Recent instrument and micropro- cessor technology development permit the storage of calibration curves for each reagent batch, although con- trol sera should always be run with the patient samples under test. Multiallergen reagents, such as group-specific combinations or Phadiatop, are not designed to be quan- titative and test results should be interpreted as detect- able or undetectable by means of a clearly defined cutoff. QUANTITATIVE MEASUREMENT OF ALLERGEN-SPECIFIC IGE ANTIBODIES The IgE molecule has a large number of epitopes on the Fc part. Combinations of capture–anti-IgE and conjugate– anti-IgE can be found that make the calibrator IgE and allergen-bound IgE appear identical. To what extent this applies to a certain assay can only be judged on the basis of experimental data and not on general assumptions. In the case of the Pharmacia CAP System, it has been shown in model experiments with several allergens that the mea- surement of allergen-bound IgE is equivalent to IgE pro- tein (Fig. 5). This is possible because practically all the IgE antibod- ies of the sample are bound to the solid phase (Borgå et al., 1992). For instance, in the case of the timothy ImmunoCAP, the capacity to bind IgE antibodies was shown to be 25 times greater than is required to bind all IgE antibodies at a concentration corresponding to the high end of the measuring range (100kUA/L). This is a large excess and referring to the Law of Mass Action, it is clear that formation of allergen–IgE complex is favored. As mentioned above, the excess is large enough to make the antibody binding independent of the association con- stant of the interaction (the affinity), regardless of the IgE antibody concentration. Furthermore, if all the relevant allergenic components of the source are present on the solid phase and shown to be capable of binding IgE anti- bodies (Fig. 6), the system is capable of measuring the sum of allergen-specific antibodies and satisfies the requirements of a quantitative assay (Perborn et al., 1995).
  8. 8. 864 The Immunoassay Handbook REFERENCE VALUES According to the rules of WHO for establishment of stan- dards for biological materials, an International Reference Preparation for IgE was established in 1968. The prepara- tion was assigned arbitrary mass units (International Units) and submitted to collaborative trials. Later the Interna- tional Unit was shown to correspond to 2.42ng pure IgE by parallel chemical and immunological measurement (Bazaral and Hamburger, 1972). The International Unit is well established for total IgE measurements, and it is evi- dent that measurements of specific IgE antibodies also should move toward increasing standardization and that the antibody concentrations ultimately should be expressed in the same mass unit of IgE protein. QUALITY CONTROL Adequate controls should be included in every assay. Uni- versally available IgE antibody control sera with defined concentration and allergen specificity are preferred over in-house pools. National and international quality control programs provide regular assessment of reagent and labo- ratory performance (Fifield et al., 1987). The most common causes of error in RAST-type assays are inadequate washing and failure to remove washing solution effectively and evenly from all the reaction ves- sels. Measurement in fluids other than those specified in the directions for use should be avoided if possible. How- ever, if this is essential, then proper negative and positive controls should be formulated and run, and dilution and recovery should be confirmed. The quality documentation that should be produced for an IgE antibody assay by the manufacturer includes: G quality and reproducibility of allergen source materials; G specificity and dose–response characteristics of labeled anti-IgE; G capacity and reproducibility of solid-phase binding; G calibration; G linearity and parallelism of calibrator and sample dilu- tions regardless of allergen; G recovery; G signal-to-noise ratio for the lowest calibrator; G freedom of interference from nonspecific IgE and com- peting IgG antibodies; G high-dose hook data; G stability of reagents and reagent combinations; G reproducibility; G correlation to reference method; G clinical sensitivity and specificity calculated on relevant patient populations. Clinical Applications DIAGNOSIS OF ATOPIC DISEASE AND IDENTIFICATION OF OFFENDING ALLERGEN The clinical sensitivity and specificity of IgE antibody measurements should be judged in relation to the allergy specialist’s diagnosis of groups of consecutive patients. The groups must be representative of the populations for which the test is intended in terms of prevalence of disease and degree of sensitization. Extensive clinical trials in accordance with this principle on more than 2000 indi- viduals showed the average diagnostic efficiency of the Pharmacia CAP System to be about 90% (Yman, 1990). FIGURE 5 Measurement of the change of concentration of IgE antibody and IgE protein after stepwise depletion of specific IgE antibody from serum. The allergen-specific antibody unit (UA) is equivalent to the international IgE unit (U). Lindqvist et al., 1995. FIGURE 6 Immunoblotting analysis of four sera with wheat-specific IgE antibodies before (1) and after (2) contact with a wheat ImmunoCAP. Antibodies of all subspecificities were bound to the solid-phase allergens.
  9. 9. 865CHAPTER 9.14 Allergy UniCAP was also evaluated in six clinical studies including 894 consecutive patients. The clinical sensitivity and speci- ficity of UniCAP-specific IgE derived from 5170 compari- sons to clinical diagnosis were 89% and 91%, respectively (Paganelli et al., 1998). COMPONENT-RESOLVED DIAGNOSTICS (CRD) The term component-resolved diagnostic (CRD) has been coined to designate a more precise diagnostic test based on pure allergen molecules that are either produced by recombinant (r) expression of allergen-encoding cDNAs or by purification from natural (n) allergen sources (Valenta et al., 2007). CRD is recommended for identify- ing specifically the allergenic reactivity profile of a patient along with their potential cross-reactivity interactions. This is particularly important for the development of strategies for allergen immunotherapy. Although some of the immunotherapies have been known and in use since 1911, the need for identification of the disease-eliciting molecules was a major obstacle for prescription of allergy vaccines. The selection of patients for immunotherapy must take into consideration, among other factors, the presence of an IgE-mediated allergic sensitization by the demonstration of allergen-specific IgE antibodies. The current success of these therapies depends on fac- tors such as accurate diagnostics, vaccine quality, and degree of immunoresponse to treatment. CRD as a con- cept was greatly aided by the availability of pure proteins in the CAP system but evolved to a higher degree of sophistication with the development of profiling systems such as protein microarrays. Interestingly, the monitoring of success of treatment by some of the current immuno- therapies is also associated with the development of aller- gen-specific tests for other classes of immunoglobulins (IgG4, IgG1, IgG2) (Valenta et al., 2007). Another impor- tant application of CRD is related to the establishment of geographical patterns of IgE sensitization, i.e., demo- graphic sensitization studies where the prevalence of spe- cific IgE responses within a particular population is able to give important clues regarding the sensitizing allergen. More recently, the observation that some classical specific allergen markers do correlate with the degree of severity of allergic diseases is also an important new development (Nicolaou et al., 2010 – Manchester study). Altogether, the acquired ability to carry out CRD, and the current results in different platforms suggest that potential uses of this technique are just beginning to be unraveled. ALLERGEN MICROARRAYS The move toward miniaturization of diagnostic testing is a popular one, not just for economic but also functional rea- sons. In the past 10 years, the ability to produce microarrays has provided the technology for monitoring the expression of thousands of genes, under many different conditions, on a solid-based medium the size of a microscope slide. Ini- tially used for DNA, the technique has evolved to other more sophisticated applications using different attachment methods in order to determine protein functionality, pro- tein interactions, and enzymatic activity among others (Renault et al., 2007). The mechanics of protein array production are now very well established, and a number of comprehensive reviews are available describing this area (Harwanegg et al., 2003; Predki, 2004; Bertone and Snyder, 2005; Merkel et al., 2005; Molloy et al., 2005). Essentially protein microarray represents a multi-analyte, solid-phase immunoassay, where proteins are immobilized onto the solid surface and micro-quantities of the serum samples are then incubated under standard condi- tions. The high concentration of proteins in the spot assures the selection of high-affinity antibodies. Hence, antibodies to the particular protein present in the patient’s serum are captured by the immobilized protein, and after the slides are washed, the bound antibodies are detected with a high- sensitivity fluorescently labeled anti-isotype antibody and a laser-based scanner (Fig. 7). The same assay format has been further developed using different and improved solid surfaces. Many aspects of the array technology such as the solid support for immobilization, “direct” vs. “sandwich” format, and methods used for signal amplification multi-analyte detection are reviewed elsewhere. With the availability of a large number of well-charac- terized recombinant proteins, arrays of different complex- ity have been produced employing purified recombinant allergens (Harwanegg et al., 2003), and commercial prod- ucts such as ImmunoCAP ISAC (Phadia) are in place. The results obtained from these arrays are very encouraging, and a number of published examples have now shown that the sensitivity of these tests is similar to those from RAST, ELISA, and immunoblot tests. Pure proteins vs. extracts: Despite the benefits of the use of pure proteins, the sheer numbers, complexity, and logistics of producing a comprehensive assay that covers all known protein families are sometimes overlooked. In reality, a very small number of pure proteins are avail- able. Hence, several groups have proposed the use of comprehensive protein extracts. One advantage of using heterogeneous protein mixtures from allergen extracts on arrays instead of purified proteins should be the detection of underrepresented proteins or protein com- plexes in allergenic products (Kim et al., 2002). Some promising results have been reported in studies con- ducted with over 150 different crude extracts (Bacarese- Hamilton et al., 2005), and complex platforms covering a large number of extracts with simultaneous detection of four classes of immunoglobulins have been introduced (Renault et al., 2011). BB B BIgE Fluorescence FIGURE 7 Diagram showing the basic components of a standard protein array. Antibodies from the patient’s serum recognize the immobilized protein molecules on the surface of the chip. Secondary antibodies and fluorescent markers allow the target molecule to be detected and quantified. Renault et al., 2007. (The color version of this figure may be viewed at www.immunoassayhandbook.com).
  10. 10. 866 The Immunoassay Handbook The characterization of the protein epitopes recognized by the IgE is fundamental to the understanding of the mech- anisms leading to allergy and for the designing of safe immu- notherapeutics. In its simple format, overlapping peptides rather than proteins can be immobilized in slides with an average of 10–20 amino acids in length. Cluster analysis has shown a high degree of correlation between patient sera and their reactivity to specific peptides (Sampson, 2004, 2005; Shreffler et al., 2005). While mapping a large number of peptide epitopes can provide prognostic information about the patient and some information on protein antigenicity, it has the drawback of missing the important nonlinear epit- opes. In a more elaborate approach, the proof-of-concept of using engineered, properly folded, chimeric proteins con- taining swapped, structural domains has been established in the mapping of the three-dimensional IgE epitope of a nut protein (Alcocer et al., 2004). With advances in the domain swapping technologies by commercial in vitro recombina- tion systems, it is expected that the knowledge accumulated will further our understanding of how the body’s immune system sees and reacts to new proteins. Regarding the array format, whether the reaction in future successful platforms takes place on flat, solid surface slides or on encoded beads or suspension arrays will depend more on commercial considerations than technical constraints. One important development in the area involves coupling the diversity of the protein microarray with the biological output of basophilic cell degranulation (Lin et al., 2007). High- throughput diagnostic tests are now possible, and multiple allergen detection marks the newest tool available to pro- duce rapid, accurate, and immunological functional allergy profiling. The progression from laboratory-based tests for specific Ig binding to clinical practice is a long way from being realized; however, the available molecular and cellular technologies are closing fast the gap to a fully in vitro system. Potentially, these in vitro biological assays might provide unique insight into the relationship between immunoglobu- lins and effector systems in type I hypersensitivity. This is a significant improvement to the reality faced by conventional allergy clinics worldwide. References and Further Reading Aalberse, R.C., Akkerdaas, J.H. and van Ree, R. Cross-reactivity of IgE antibodies to allergens. Allergy 56, 478–490 (2001). Alcocer, M.J.C., Murtagh, G.J., Wilson, P.B., et al. The major human structural IgE epitope of the Brazil nut allergen Ber e 1: a chimaeric and protein microar- ray approach. J. Mol. Biol. 343, 759–769 (2004). Bacarese-Hamilton, T.G.J., Ardizzoni, A. and Crisanti, A. Allergen microarrays. Methods Mol. Med. 144, 195–207 (2005). Bazaral, M. and Hamburger, R.N. Standardization and stability of immunoglobulin E (IgE). J. Allergy Clin. Immunol. 49, 189–191 (1972). Bertone, P. and Snyder, M. Advances in functional protein microarray technology. FEBS J. 272, 5400–5411 (2005). Bernstein, I.L. Proceedings of the task force on guidelines for standardizing old and new technologies used for diagnosis and treatment of allergic diseases. J. Allergy Clin. Immunol. 82, 487–526 (1988). Borgå, Å, Karlsson, T., Perborn, H., Lindqvist, A. and Yman, L. Standardization of timothy, peanut and Aspergillus fumigatus allergens for immunoassay of specific IgE (Pharmacia CAP System). J. Allergy Clin. Immunol. 89(1, Part 2), 148 (1992). Chapman, M.D. Allergen nomenclature. In: Allergens and Allergen Immunotherapy, (eds Lockey, R.F., Bukantz, S.C. and Bousquet, J.), 51–64 (Marcel Decker, New York, 2004). Chapman, M.D. Allergen nomenclature. In: Allergens and Allergen Immunotherapy, 4th edn (eds Lockey, R.F. and Ledford, D.K.), 47–58 (Informa Healthcare, New York, 2008). Chapman, M.D., Pomes, A., Breiteneder, H. and Ferreira, F. Nomenclature and structural biology of allergens. J. Allergy Clin. Immunol. 119, 414–420 (2007). Coombs, R.R.A. and Gell, P.G.H. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In: Clinical Aspects of Immunology, (eds Gell, P.G.H., Coombs, R.R.A. and Lachman, P.J.), 317 (Blackwell, Oxford, 1968). Deuschl, H. and Johansson, S.G.O. Specific IgE antibodies in nasal secretions from patients with allergic rhinitis and with negative or weakly positive RAST in the serum. Clin. Allergy 7, 195–202 (1977). Eriksson, N.E. Allergy screening with Phadiatop® and CAP Phadiatop® in combi- nation with a questionnaire in adults with asthma and rhinitis. Allergy 45, 285–292 (1990). Fifield, R., Bird, A.G., Carter, R.H., Ward, A.M. and Whicher, J.T. Total IgE and allergen-specific IgE assays: guidelines for the provision of a laboratory service. Ann. Clin. Biochem. 24, 232–245 (1987). Ghaemmaghami, A.M., Gough, L., Sewell, H.F. and Shakib, F. The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level. Clin. Exp. Allergy 32, 1468–1475 (2002). Gupta, R., Sheikh, A., Strachan, D.P. and Anderson, H.R. Time trends in allergic disorders in the UK. Thorax 62, 91–96 (2007). Harwanegg, C., Laffer, S., Hiller, R., et al. Microarrayed recombinant allergens for diagnosis of allergy. Clin. Exp. Allergy 33, 7–13 (2003). Johansson, S.G.O. and Yman, L. In vitro assays for immunoglobulin E. Clin. Rev. Allergy 6, 93–139 (1988). Johansson, S.G.O., Bieber, T., Dahl, R., Friedman, P.S., Lanier, B.Q., Lockey, R.F., Motala, C., Martell, J.A.O., Platts-Mills, T.A.E., Ring, J., Thien, F., Cauwenberger, P.V. and Williams, H.C. Revised nomenclature for allergy for global use: report of the nomenclature review committee of the World Allergy Organisation, october 2003. J. Allergy Clin. Immunol. 113, 832–836 (2004). Kari, O., Salo, O.P., Björksten, F. and Backman, A. Allergic conjunctivitis, total and specific IgE in the tear fluid. Acta Ophthalmol. 63, 97–99 (1985). Kauffman, H.F., Tamm, M., Timmerman, J.A.B. and Borger, P. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease independent mechanisms. Clin. Mol. Allergy 4, 5 (2006). Kerkhof, M., Dubois, A.E., Postma, D.S., Schouten, J.P. and de Monchy, J.G. Role and interpretation of total serum IgE measurements in the diagnosis of allergic airway disease in adults. Allergy 58, 905–911 (2003). Kim, T.E., Park, S.W., Cho, N.Y., et al. Quantitative measurement of serum allergen-specific IgE on protein chip. Exp. Mol. Med. 34, 152–158 (2002). Kristjánsson, S., Strannegård, I.L. and Wennergren, G. Inflammatory markers in childhood asthma. Ann. Med. 28, 395–399 (1996). Lin, J., Renault, N., Haas, H., Schramm, G., Vieths, S., Vogel, L., Falcone, F.H. and Alcocer, M.J.C. A novel diagnostic tool for the detection of food allergy combining protein microarrays with human basophils. Clin. Exp. Allergy 37, 1854–1862 (2007). Lindqvist, A., Ikezawa, Z., Tanaka, A. and Yman, L. Seafood specific IgE in atopic dermatitis. Ann. Allergy 70, 58 (1993). Lindqvist, A., Maaninen, E., Zimmerman, K., Rimland, A., Andersson, O., Holmquist, I., Karlsson, T. and Yman, L. Quantitative measurement of aller- gen specific IgE antibodies applied in a new immunoassay system, UniCAP. In: XVI European Congress of Allergology and Clinical Immunology ECACIÌ95, (eds Basomba, A., Hernandez, F. and de Rojas, M.D.), 195–200 (Monduzzi Editore, Bologna, 1995). Loewenstein, C. and Mueller, R.S. A review of allergen-specific immunotheraphy in human and veterinary medicine. Vet. Dermatol. 20, 84–98 (2009). Lundkvist, U. Research and development of the RAST technology. In: Advances in Diagnosis of Allergy: RAST, (ed, Evans, R.), 85–99 (Symposia Specialists, Miami, 1975). Merkel, J.S., Michaud, G.A., Salcius, M., et al. Functional protein microarrays: just how functional are they? Curr. Opin. Biotechnol. 16, 447–452 (2005). Molloy, R.M., McConnell, R.I., Lamont, J.V., et al. Automation of biochip array technology for quality results. Clin. Chem. Lab. Med. 43, 1303–1313 (2005). Nicolaou, N., Poorafshar, M., Murray, C., Simpson, A., Winell, H., Kerry, G., Woodcock, A., Ahlstadt, S. and Custovic, A. Allergy or tolerance in children sensitised to peanut: prevalence and differentiation using component-resolved diagnostics. J. Allergy Clin. Immunol. 125, 191–197 (2010). Paganelli, R., Ansotegui, I.J., Sastre, J., Lange, C.-E., Roovers, M.H.W.M., de Groot, H., Lindholm, N.B. and Ewan, P.W. Specific IgE antibodies in the diagnosis of atopic disease. Clinical evaluation of a new in vitro test system, UniCAP, in six European allergy clinics. Allergy 53, 763–768 (1998). Perborn, H., et al. Standardization of allergen reagents for immunoassay of aller- gen-specific IgE antibodies (UniCAP). Allergen excess and component speci- ficity. In: XVI European Congress of Allergology and Clinical Immunology ECACIÌ95, (eds Basomba, A., Hernandez, F. and de Rojas, M.D.), 191–194 (Monduzzi Editore, Bologna, 1995). Persson, E., Yman, L., 1997. Unpublished data. Peterman, J.H. 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