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Progress in molecular diagnostics of allergy
Res. Immunol.
© INSTITUT PASTEUR/ELSEVIER
Pads 1998
1998, 149, 253-254
Progress in molecular diagnostics of allergy H. LCwenstein and J.N. Larsen ALK Research, Boge All~ 10-12, 2970 Horsholm (Denmark)
The first approach to an understanding of the mechanism behind allergic reactions was taken in 1921 when Prausnitz and Ktistner showed that the ability to elicit an allergic reaction could be transferred from one individual to the other by unknown serum components. They proposed the existence of a compound, "serum reagin" in the serum of allergic individuals. Some 45 years later, Ishizaka and colleagues showed that serum reagin was a new class of immunoglobulins, lgE. Ever since then, the detection of IgE has played a central role in molecular diagnosis of type 1 immediate hypersensitivity or allergy. Clinical history and serum samples from allergic individuals have been and continue to be important tools for the identification of allergen sources. The most important allergen sources have long been known and are constituted by the quantitatively dominating particles present in the air breathed by humans. Exceptions to this rule are the venoms of stinging insects. Allergen sources of world-wide importance are, above all, house dust mites and grass pollens, but weed pollens, cat and dog dander, and fungi such as Altemaria and Cladosporium are also frequently causing allergic reactions. On a regional basis pollens from ragweed, birch, olive or parietaria are dominant allergen sources. From all these sources genes encoding the major allergens have been cloned and sequenced, and current research is aimed at the determination of three-dimensional structures for the most important allergens. Thus far, detailed studies of molecular aspects of allergens have failed to detect a common struc-
Received April 1, 1998.
tural motif or a common activity associated with the ability to elicit allergic reactions. A common aspect of major allergens, however, is the presence in quantitatively dominating amounts on airborne particles of a well-defined size. While investigating other approaches to molecular allergy diagnosis, the detection of specific IgE is also likely to play an important role in the foreseeable future. Having identified the allergenic source material, the next step is to convert it into material suitable for laboratory testing. This is done by aqueous extraction. E x t r a c t i o n is an e s t a b l i s h e d pharmaceutical t e c h n i q u e ; however, allergen extracts are complex mixtures of several proteins and other components. The producers of allergen extracts need to apply standardization measures in order to control and ensure consistency in composition and potency. Since all antigens, i.e. proteins, are potential allergens, the allergen extract should ideally comprise all proteins present in the source material. Quantitative measurement of selected major allergens or marker proteins will indirectly control the relatiw~ composition of the extract, and finally, quantitation of total IgE binding capacity will determine the allergen extract potency. A standardization procedure in keeping with the times will comprise these three steps. The radioallergosorbent test, RAST, was originally described in 1967 and has been the in vitro diagnostic test principle ever since. The quality of a RAST test is dependent on the quality of the allergen extract. In its original version, the allergen extract was covalently coupled to paper discs.
254
11. L O W E N S T E I N A N D J.N. L A R S E N
The paper discs were incubated with patient serum, and bound IgE was detected using a radiolabelled anti-IgE. Today, the RAST principle is still used despite the further development of the solid phase, the detection system and the degree of automation. Total IgE can be quantified in a similar setup if solid phase-coupled anti-IgE is substituted for allergen extract. The molecular diagnosis of serum IgE reactivity can be further refined to identify individual allergens. A number of techniques are used which may be divided into two groups on the basis of principle of operation. According to one principle, the allergen extract is separated into its individual components by electrophoretic migration in a gel, resulting in a reproducible pattem from which individual allergens can be identified. The resulting sample is incubated with patient serum and developed for the identification of IgE-reactive components. Crossed radioimmunoelectrophoresis (CRIE) and SDSpolyacrylamide gel electrophoresis are in the most widespread use in this category. The other principle is the detection of IgE directed against one individual component. The assay may be based on the purified allergen or on monospecific reagents, most often rabbit or murine antibodies raised by immunization of the animals with the purified allergen in question. RAST or ELISA (enzyme-linked immunosorbent assay) can be used for this purpose. Analyses of allergens by two-dimensional gel e l e c t r o p h o r e s i s and m o l e c u l a r c l o n i n g and sequencing have revealed that allergens are not homogenous entities. Several isoallergens and variants exist, displaying from one to several amino acid substitutions. Up to 25 % of the amino acids may be substituted, a number corresponding to the number of substitutions comparing homologous sequences of major allergens from different species. This level of variability is expected to result in differences between patients' reactivity with isoallergens, both with respect to IgE and T cells. 2-D gel electrophoresis and immunoblotting can be used to evaluate the reactivity of serum IgE to various subsets of spots, however, with some uncertainties when it comes to quantitation of IgE to individual isoallergens. Quantitative assays based on homogenous recombinant allergens can precisely determine the "average" affinity of IgE from serum samples towards individual isoforms.
The continuous refinement of the techniques available for allergen characterization will enable a deeper understanding of the interaction between the immune system and the allergen. Three-dimensional structures of the important major inhalant allergens are now becoming available, facilitating the direct visualization of antibody-allergen interactions. Alignment of multiple sequences between isoallergens or between homologous allergens from different species will allow the identification of conserved residues forming patches on the molecular surface of the structure. These patches are candidates for epitopes shared by isoallergens or allergens from different species, and they form a comprehensible basis for the phenomenon of cross-reactivity. Crystallized allergen-Fab complexes based on murine model antibodies will enable the study of the precise effect on affinity constants of naturally occurring or artificial amino acid substitutions. Allergen-specific IgE is still the prevailing diagnostic marker for allergic disease in spite of the intensified search for more general markers. These include markers of allergic inflammation and genetic atopy markers. Genetic markers encoding factors involved in the regulation of the allergic immune response at a more general level have been identified, but so far many genes seem to be involved, and the evolving picture is far too complicated to be used for routine diagnostic testing. A n o t h e r possibility is HLA typing exploiting the association of certain HLA-D types and dominant T-cell epitopes of major allergens. HLA and allergy studies were encouraged by the finding of a tight association between HLA-D types and IgE to Amb a 5, a minor ragweed allergen. A large multicenter study was initiated in search for associations between HLA-D types and IgE towards the major inhalant allergens. Unfortunately, no associations were found and at the same time reports of multiple T-cell epitopes scattered over the entire sequences of several of the most important major allergens appeared. However, recent reports indicate that the diversity in T-cell epitopes may have been exaggerated and that the term "dominant T-cell epitope" may be meaningful, indicating an association between not a single HLA-D allele but perhaps two or three HLA-D alleles and the presence of IgE directed towards individual major allergens.
© INSTITUT PASTEUR/ELSEVIER
Pads 1998
1998, 149, 253-254
Progress in molecular diagnostics of allergy H. LCwenstein and J.N. Larsen ALK Research, Boge All~ 10-12, 2970 Horsholm (Denmark)
The first approach to an understanding of the mechanism behind allergic reactions was taken in 1921 when Prausnitz and Ktistner showed that the ability to elicit an allergic reaction could be transferred from one individual to the other by unknown serum components. They proposed the existence of a compound, "serum reagin" in the serum of allergic individuals. Some 45 years later, Ishizaka and colleagues showed that serum reagin was a new class of immunoglobulins, lgE. Ever since then, the detection of IgE has played a central role in molecular diagnosis of type 1 immediate hypersensitivity or allergy. Clinical history and serum samples from allergic individuals have been and continue to be important tools for the identification of allergen sources. The most important allergen sources have long been known and are constituted by the quantitatively dominating particles present in the air breathed by humans. Exceptions to this rule are the venoms of stinging insects. Allergen sources of world-wide importance are, above all, house dust mites and grass pollens, but weed pollens, cat and dog dander, and fungi such as Altemaria and Cladosporium are also frequently causing allergic reactions. On a regional basis pollens from ragweed, birch, olive or parietaria are dominant allergen sources. From all these sources genes encoding the major allergens have been cloned and sequenced, and current research is aimed at the determination of three-dimensional structures for the most important allergens. Thus far, detailed studies of molecular aspects of allergens have failed to detect a common struc-
Received April 1, 1998.
tural motif or a common activity associated with the ability to elicit allergic reactions. A common aspect of major allergens, however, is the presence in quantitatively dominating amounts on airborne particles of a well-defined size. While investigating other approaches to molecular allergy diagnosis, the detection of specific IgE is also likely to play an important role in the foreseeable future. Having identified the allergenic source material, the next step is to convert it into material suitable for laboratory testing. This is done by aqueous extraction. E x t r a c t i o n is an e s t a b l i s h e d pharmaceutical t e c h n i q u e ; however, allergen extracts are complex mixtures of several proteins and other components. The producers of allergen extracts need to apply standardization measures in order to control and ensure consistency in composition and potency. Since all antigens, i.e. proteins, are potential allergens, the allergen extract should ideally comprise all proteins present in the source material. Quantitative measurement of selected major allergens or marker proteins will indirectly control the relatiw~ composition of the extract, and finally, quantitation of total IgE binding capacity will determine the allergen extract potency. A standardization procedure in keeping with the times will comprise these three steps. The radioallergosorbent test, RAST, was originally described in 1967 and has been the in vitro diagnostic test principle ever since. The quality of a RAST test is dependent on the quality of the allergen extract. In its original version, the allergen extract was covalently coupled to paper discs.
254
11. L O W E N S T E I N A N D J.N. L A R S E N
The paper discs were incubated with patient serum, and bound IgE was detected using a radiolabelled anti-IgE. Today, the RAST principle is still used despite the further development of the solid phase, the detection system and the degree of automation. Total IgE can be quantified in a similar setup if solid phase-coupled anti-IgE is substituted for allergen extract. The molecular diagnosis of serum IgE reactivity can be further refined to identify individual allergens. A number of techniques are used which may be divided into two groups on the basis of principle of operation. According to one principle, the allergen extract is separated into its individual components by electrophoretic migration in a gel, resulting in a reproducible pattem from which individual allergens can be identified. The resulting sample is incubated with patient serum and developed for the identification of IgE-reactive components. Crossed radioimmunoelectrophoresis (CRIE) and SDSpolyacrylamide gel electrophoresis are in the most widespread use in this category. The other principle is the detection of IgE directed against one individual component. The assay may be based on the purified allergen or on monospecific reagents, most often rabbit or murine antibodies raised by immunization of the animals with the purified allergen in question. RAST or ELISA (enzyme-linked immunosorbent assay) can be used for this purpose. Analyses of allergens by two-dimensional gel e l e c t r o p h o r e s i s and m o l e c u l a r c l o n i n g and sequencing have revealed that allergens are not homogenous entities. Several isoallergens and variants exist, displaying from one to several amino acid substitutions. Up to 25 % of the amino acids may be substituted, a number corresponding to the number of substitutions comparing homologous sequences of major allergens from different species. This level of variability is expected to result in differences between patients' reactivity with isoallergens, both with respect to IgE and T cells. 2-D gel electrophoresis and immunoblotting can be used to evaluate the reactivity of serum IgE to various subsets of spots, however, with some uncertainties when it comes to quantitation of IgE to individual isoallergens. Quantitative assays based on homogenous recombinant allergens can precisely determine the "average" affinity of IgE from serum samples towards individual isoforms.
The continuous refinement of the techniques available for allergen characterization will enable a deeper understanding of the interaction between the immune system and the allergen. Three-dimensional structures of the important major inhalant allergens are now becoming available, facilitating the direct visualization of antibody-allergen interactions. Alignment of multiple sequences between isoallergens or between homologous allergens from different species will allow the identification of conserved residues forming patches on the molecular surface of the structure. These patches are candidates for epitopes shared by isoallergens or allergens from different species, and they form a comprehensible basis for the phenomenon of cross-reactivity. Crystallized allergen-Fab complexes based on murine model antibodies will enable the study of the precise effect on affinity constants of naturally occurring or artificial amino acid substitutions. Allergen-specific IgE is still the prevailing diagnostic marker for allergic disease in spite of the intensified search for more general markers. These include markers of allergic inflammation and genetic atopy markers. Genetic markers encoding factors involved in the regulation of the allergic immune response at a more general level have been identified, but so far many genes seem to be involved, and the evolving picture is far too complicated to be used for routine diagnostic testing. A n o t h e r possibility is HLA typing exploiting the association of certain HLA-D types and dominant T-cell epitopes of major allergens. HLA and allergy studies were encouraged by the finding of a tight association between HLA-D types and IgE to Amb a 5, a minor ragweed allergen. A large multicenter study was initiated in search for associations between HLA-D types and IgE towards the major inhalant allergens. Unfortunately, no associations were found and at the same time reports of multiple T-cell epitopes scattered over the entire sequences of several of the most important major allergens appeared. However, recent reports indicate that the diversity in T-cell epitopes may have been exaggerated and that the term "dominant T-cell epitope" may be meaningful, indicating an association between not a single HLA-D allele but perhaps two or three HLA-D alleles and the presence of IgE directed towards individual major allergens.