Underestimation of house dust mite–specific IgE with extract-based ImmunoCAPs compared with molecular ImmunoCAPs

House dust mites (HDMs) are one of the most important allergen sources causing severe forms of respiratory and skin allergy.1Sanchez-Borges M. Fernandez-Caldas E. Thomas W.R. Chapman M.D. Lee B.W. Caraballo L. et al.International consensus (ICON) on: clinical consequences of mite hypersensitivity, a global problem.World Allergy Organ J. 2017; 10: 14Crossref PubMed Scopus (37) Google Scholar A major problem associated with the use of natural allergen extracts for diagnostic testing is that they show large variability regarding the contents of the major HDM allergens groups 1 and 2 and might lack clinically important allergens, such as Der p 5, Der p 7, Der p 21, and Der p 23.2Casset A. Mari A. Purohit A. Resch Y. Weghofer M. Ferrara R. et al.Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.Int Arch Allergy Immunol. 2012; 159: 253-262Crossref PubMed Scopus (108) Google Scholar In addition to skin testing, intradermal and provocation testing, measurement of allergen-specific IgE levels in serum is a valid method to detect HDM-specific IgE sensitization. However, there are also problems with HDM allergen extract–based serologic tests. HDM allergen extracts can contain bacterial antigens, which can cause false-positive IgE test results in subjects.3Dzoro S. Mittermann I. Resch-Marat Y. Vrtala S. Nehr M. Hirschl A.M. et al.House dust mites as potential carriers for IgE sensitization to bacterial antigens.Allergy. 2018; 73: 115-124Crossref PubMed Scopus (29) Google Scholar With the introduction of component-resolved diagnosis or molecular allergy diagnosis, purified HDM allergen molecules are available for identification of HDM-sensitized subjects.4Valenta R. Lidholm J. Niederberger V. Hayek B. Kraft D. Gronlund H. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT).Clin Exp Allergy. 1999; 29: 896-904Crossref PubMed Scopus (449) Google Scholar However, the panel of HDM allergen molecules comprising the majority of HDM-specific IgE epitopes needs to be defined. To identify the most important HDM allergens, we have established IgE reactivity profiles to a panel of 13 HDM allergen molecules using ImmunoCAP ISAC technology5Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (100) Google Scholar in 2 cohorts of patients with HDM allergy. Table E1 in this article's Online Repository at www.jacionline.org describes the characteristics and demographics of the patient population. One cohort (ie, cohort 1) comprising 27 patients with a doctor's diagnosis of HDM sensitization (see Table E1) and a second cohort (ie, cohort 2) of 26 patients who also had symptoms of rhinitis on controlled HDM allergen exposure were obtained from the Vienna Challenge Chamber (see the Methods section and Table E1 in this article's Online Repository at www.jacionline.org). Six (ie, Der p 1, 2, 5, 7, 21, and 23) of the 13 tested HDM allergen molecules were identified as relevant allergens by using ImmunoCAP ISAC, according to prevalence of IgE recognition and allergen-specific IgE levels (Table I), and testing with these allergens confirmed IgE sensitization to HDM in each of the tested patients (see Fig E1 in this article's Online Repository at www.jacionline.org). Results obtained for different methods of IgE detection (ie, RAST-based dot blot, ImmunoCAP ISAC, and ELISA) were highly correlated, yielding best results for ImmunoCAP ISAC and the highly sensitive RAST-based assay (see Table E2 in this article's Online Repository at www.jacionline.org).Table ISerologic characteristics of patients with HDM allergy from cohorts 1 and 2Cohort 1 (n = 27)Minimum-maximumMeanTotal IgE (kUA/L)42.9-3425.0576.5CAP extract (kUA/L)0.7-117.029.0CAP allergen (kUA/L)1.1-201.643.6ISAC (ISU)Prevalence (n)PercentageMinimum-maximum of positiveMean of positiveClinically important Dermatophagoides pteronyssinus allergens nDer p 12385.20.3-11.32.4 rDer p 22281.50.3-52.416.7 rDer p 51451.90.4-30.05.0 rDer p 7725.90.3-39.16.0 rDer p 211348.10.3-8.92.8 rDer p 231970.40.3-24.84.7Other D pteronyssinus allergens rDer p 4414.80.3-1.50.7 rDer p 10414.80.3-0.30.3 rDer p 1100.0—— rDer p 1400.0—— rDer p 1500.0—— rDer p 18518.50.8-6.42.2 clone 16311.11.1-2.31.7Cohort 2 (n = 26)Minimum-maximumMeanTotal IgE (kUA/L)18.0-2104.0261.2CAP extract (kUA/L)1.5-42.114.1CAP allergen (kUA/L)0.2-117.427.8ISAC (ISU)Prevalence (n)PercentageMinimum-maximum of positiveMean of positiveClinically important D pteronyssinus allergens nDer p 11765.40.4-9.93.7 rDer p 22492.30.4-81.619.3 rDer p 5934.60.3-38.511.8 rDer p 7934.60.3-26.67.6 rDer p 21830.80.4-27.76.9 rDer p 231973.10.4-27.78.4Other D pteronyssinus allergens rDer p 400.0—— rDer p 1027.70.3-2.72.0 rDer p 1100.0—— rDer p 1400.0—— rDer p 1500.0—— rDer p 1800.0—— clone 16415.40.3-3.91.8ISU, ISAC standardized units. Open table in a new tab ISU, ISAC standardized units. We then prepared molecular ImmunoCAPs containing the 6 important HDM allergen molecules (ie, Der p 1, 2, 5, 7, 21, and 23) by using Streptavidin ImmunoCAP technology (o212 ImmunoCAP; Thermo Fisher Scientific/Phadia, Uppsala, Sweden) and compared them with the commercial allergen extract–based ImmunoCAP (d1) and the ImmunoCAP ISAC for quantification of HDM-specific IgE levels (see Fig E2 in this article's Online Repository at www.jacionline.org). The best correlation was found between the extract-based ImmunoCAP and molecular ImmunoCAP containing a mix of Der p 1, 2, 5, 7, 21, and 23 (CAP allergen) (r = 0.95 and P < .0001 in cohort 1 and r = 0.82 and P < .0001 in cohort 2) (see Figs E2 and E3 in this article's Online Repository at www.jacionline.org). Interestingly, HDM-specific IgE levels determined with the molecular ImmunoCAP were considerably greater than those measured with the allergen extract–based ImmunoCAP as follows: cohort 1, 43.6 kUA/L (1.1-201.6 kUA/L) versus 29 kUA/L (0.7-117.0 kUA/L); cohort 2, 27.8 kUA/L (0.2-117.4 kUA/L) versus 14.1 kUA/L (1.5-42.1 kUA/L; Table I; Fig 1: cohort 1, P = .0136; cohort 2, P = .0009; and cohort 1 plus cohort 2, P < .0001). All patients were given a diagnosis with the molecular ImmunoCAP (see Table E3 in this article's Online Repository at www.jacionline.org). IgE levels measured with the molecular ImmunoCAP were considerably greater (mean, 97%) in 39 (74%) of the 53 patients than those measured with the allergen extract (see Table E3). Only in a few patients (14/53 [26%]) were slightly greater HDM-specific IgE levels (mean, 29%) measured with the extract–based ImmunoCAP (see Table E3). There are several possible explanations for this finding. First, it is possible that certain allergens are underrepresented in natural allergen extracts, and as a consequence, allergen-specific IgE levels measured for patients with IgE sensitization to the underrepresented components will be low. Second, natural allergen extracts contain large quantities of nonallergenic proteins in addition to the allergens, which can then preferentially couple to the solid phase used for immobilization of the allergens, resulting in underestimation of allergen-specific IgE levels. Third, both of the first 2 factors might play a role. Therefore we performed a more detailed analysis. We grouped sera according to HDM allergen-specific IgE levels in sera containing levels of greater than 20 kUA/L and sera containing levels of less than 20 kUA/L and found that significantly greater HDM allergen-specific IgE levels were measured with the molecular ImmunoCAP versus the extract-based ImmunoCAP in patients with HDM allergen-specific IgE levels of greater than 20 kUA/L compared with those with HDM-specific IgE levels of less than 20 kUA/L, indicating that important allergens can be coupled in quantities that are too low in the allergen extract–based ImmunoCAPs (see Fig E4, A, in this article's Online Repository at www.jacionline.org). We then analyzed patients with IgE reactivity to 1 to 3 HDM allergens of the panel Der p 1, 2, 5, 7, 21, and 23 and those with IgE reactivity to 4 to 6 of the allergens (see Fig E4, B). In both groups HDM-specific IgE levels were greater when measured with the molecular ImmunoCAP, especially in the group with IgE reactivity to 4 to 6 allergens, but there was no significant difference in the group recognizing only 1 to 3 allergens (see Fig E4, B). Finally, we grouped the patients according to the presence or absence of IgE specific for the 6 important allergens (see Fig E4, C-H). We found that Der p 2, Der p 5, Der p 21, and Der p 23 seemed to be underrepresented in natural allergen extracts because specific IgE levels measured with the molecular ImmunoCAPs were greater for patients with specific IgE to these allergens compared with those in patients without specific IgE to these allergens (see Fig E4). For Der p 5, this difference was even statistically significant (see Fig E4, E). Therefore it seems that the factors mentioned above, underrepresentation of certain allergens and/or competition by nonallergenic proteins, are responsible for IgE levels measured with HDM allergen extract–based ImmunoCAPs being lower than those measured with molecular ImmunoCAPs. Our finding might be of great clinical relevance because the accurate measurement of allergen-specific IgE levels is important. Allergen-specific IgE levels inform about allergen exposure and whether measures, such as allergen avoidance, have been successful; they are important as thresholds for certain symptoms such as rhinitis versus asthma; and they might help predict severe allergic reactions.6Maloney J.M. Rudengren M. Ahlstedt S. Bock S.A. Sampson H.A. The use of serum-specific IgE measurements for the diagnosis of peanut, tree nut, and seed allergy.J Allergy Clin Immunol. 2008; 122: 145-151Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar Among the limited number of patients tested in our study, we did not obtain evidence that greater HDM-specific IgE levels are associated with certain manifestations of HDM allergy, but we have evidence that threshold IgE levels can be established for symptomatic and silent IgE sensitization to HDM (S. Vrtala, unpublished). Therefore the question is how the measurement of HDM-specific IgE levels could be improved. One possibility would be to add recombinant allergen molecules to natural HDM allergen extracts, as has been done previously for other complex allergen sources.7Andersson K. Ballmer-Weber B.K. Cistero-Bahima A. Ostling J. Lauer I. Vieths S. et al.Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking.Allergy. 2007; 62: 897-904Crossref PubMed Scopus (30) Google Scholar The other possibility would be to prepare molecular ImmunoCAPs that contain the most important allergens, as shown in our study. The advantage of such molecular ImmunoCAPs would be that cross-reactive allergens, which are also present in other unrelated allergen sources, can be omitted so that it is possible to identify patients with a genuine sensitization to the given allergen source with molecular tests. Such molecular tests are very useful not only because they have high specificity and sensitivity for detection of IgE sensitization but also because they facilitate the accurate prescription of allergen-specific immunotherapy.8Stringari G. Tripodi S. Caffarelli C. Dondi A. Asero R. Di Rienzo Businco A. et al.The effect of component-resolved diagnosis on specific immunotherapy prescription in children with hay fever.J Allergy Clin Immunol. 2014; 134: 75-81Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Therefore we suggest replacing allergen extract–based tests for the detection of HDM sensitization with molecular tests that are highly reproducible and not dependent on varying qualities of allergen extracts. Sera from patients with HDM allergy (cohort 1, n = 27; cohort 2, n = 26) were obtained in 2 centers in Vienna (Department of Pathophysiology and Allergy Research, Medical University of Vienna, and Vienna Challenge Chamber), Austria. Patients had a positive case history and perennial airway symptoms indicative of HDM allergy (ie, rhinitis, asthma, or both). IgE sensitization to HDM was confirmed by means of detection of specific IgE antibodies to Dermatophagoides pteronyssinus by using ImmunoCAP (d1 ImmunoCAP; Thermo Fisher Scientific/Phadia). Sera from both cohorts were also tested for IgE reactivity to a panel of 176 microarrayed allergen molecules by using ImmunoCAP ISAC technology (Thermo Fisher Scientific/Phadia).E1Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (165) Google Scholar Patients from cohort 2 underwent controlled HDM allergen exposure in the Vienna Challenge Chamber and had a Total Nasal Symptom Score of greater than 6.E2Zieglmayer P. Lemell P. Chen K.W. Schmutz R. Zieglmayer R. Pfaar O. et al.Clinical validation of a house dust mite environmental challenge chamber model.J Allergy Clin Immunol. 2017; 140: 266-268.e5Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar For control purposes, serum from at least 1 nonallergic subject was included in all analyses. Table E1 provides a demographic and clinical characterization of the patients from cohorts 1 and 2. Serologic characteristics are summarized in Table I. Patients' sera were anonymously analyzed for allergen-specific IgE reactivity with approval from the Ethics Committee of the Medical University of Vienna (EK 1641/2014) after written informed consent was obtained. Natural purified Der p 1 was obtained from Professor W. R. Thomas.E3Chua K.Y. Dilworth R.J. Thomas W.R. Expression of Dermatophagoides pteronyssinus allergen, Der p II, in Escherichia coli and the binding studies with human IgE.Int Arch Allergy Appl Immunol. 1990; 91: 124-129Crossref PubMed Scopus (56) Google Scholar rDer p 2, rDer p 5, rDer p 21, and rDer p 23 were expressed in Escherichia coli strain BL21-Gold (DE3; Thermo Fisher Scientific/Agilent Technologies, Santa Clara, Calif) and purified, as previously described.E4Weghofer M. Grote M. Dall'Antonia Y. Fernandez-Caldas E. Krauth M.T. van Hage M. et al.Characterization of folded recombinant Der p 5, a potential diagnostic marker allergen for house dust mite allergy.Int Arch Allergy Immunol. 2008; 147: 101-109Crossref PubMed Scopus (41) Google Scholar, E5Weghofer M. Dall'Antonia Y. Grote M. Stocklinger A. Kneidinger M. Balic N. et al.Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.Allergy. 2008; 63: 758-767Crossref PubMed Scopus (67) Google Scholar, E6Weghofer M. Grote M. Resch Y. Casset A. Kneidinger M. Kopec J. et al.Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.J Immunol. 2013; 190: 3059-3067Crossref PubMed Scopus (115) Google Scholar rDer p 7 was expressed in E coli BL21-Gold (DE3) and purified by means of hydrophobic interaction chromatography and hydroxyapatite chromatography. Endotoxins were removed from purified rDer p 5, 7, 21, and 23 by using immobilized polymyxin resins (Affi-Prep Polymyxin Matrix; Bio-Rad, Austin, Tex), and endotoxin from rDer p 2 was removed by using the Endotoxin Removal Kit (ProteoSpin Endotoxin Removal Maxi Kit; Norgen Biotek, Ontario, Canada). Endotoxin levels were measured with the Limulus-Amebocyte-Lysate assay (Lonza, Basel, Switzerland). Endotoxin contents of Der p 2, 5, 7, 21, and 23 were less than 250 endotoxin units/mL, and the endotoxin content of Der p 1 was 1300 endotoxin units/mL. The other HDM allergens on the chip were purified and characterized, as previously described.E1Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (165) Google Scholar IgE ELISA and the RAST-based IgE dot blot assay were performed, as previously described.E7Swoboda I. Bugajska-Schretter A. Linhart B. Verdino P. Keller W. Schulmeister U. et al.A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.J Immunol. 2007; 178: 6290-6296Crossref PubMed Scopus (141) Google Scholar, E8Banerjee S. Weber M. Blatt K. Swoboda I. Focke-Tejkl M. Valent P. et al.Conversion of Der p 23, a new major house dust mite allergen, into a hypoallergenic vaccine.J Immunol. 2014; 192: 4867-4875Crossref PubMed Scopus (48) Google Scholar A customized microarray based on ImmunoCAP ISAC chip technology was produced by Phadia Austria GmbH (Vienna, Austria).E1Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (165) Google Scholar Detection of IgE binding was performed as described, and levels of IgE antibodies were shown in ISAC standardized units, with a cutoff of 0.3 ISAC standardized units.E1Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (165) Google Scholar The quantification of allergen-specific IgE antibodies to D pteronyssinus allergen extract (Allergen d1) and of total IgE was performed by using ImmunoCAP technology, according to the manufacturer's instructions, on an ImmunoCAP 100 instrument (Thermo Fisher Scientific/Phadia). Streptavidin ImmunoCAPs (o212 ImmunoCAP, Thermo Fisher Scientific/Phadia) were used to prepare ImmunoCAPs containing a mix of Der p 1, 2, 5, 7, 21, and 23, as previously described.E9Erwin E.A. Custis N.J. Satinover S.M. Perzanowski M.S. Woodfolk J.A. Crane J. et al.Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase.J Allergy Clin Immunol. 2005; 115: 1029-1035Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar For this purpose, the allergens were dialyzed against a buffer containing 0.1 mol/L sodium bicarbonate and 1 mol/L sodium chloride and biotinylated with a 5 times molar surplus of biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester (B3295; Sigma-Aldrich, St Louis, Mo) at pH 8.0. After 2 hours of incubation at room temperature, allergens were dialyzed against PBS to remove unbound biotin. Different amounts of biotinylated allergens were tested (1, 0.5, and 0.1 μg), showing that 0.1 μg of biotinylated allergen was sufficient for IgE detection (ie, reaching the plateau for IgE binding). Accordingly, 0.1 μg of each of the biotinylated allergens (Der p 1, 2, 5, 7, 21, and 23) were mixed (in 50 μL of PBS) and coupled to streptavidin CAPs for 30 minutes at room temperature. Measurement of specific IgE levels in sera from patients with HDM allergy was performed according to the manufacturer's instructions on an ImmunoCAP 100 (Thermo Fisher Scientific/Phadia). Allergen-specific IgE levels of greater than 0.1 kUA/L were considered positive. Correlation between different IgE-binding assays was determined by calculating the Spearman correlation coefficient (r) for patients from the 2 cohorts. The comparison of allergen-specific IgE levels measured with the commercial allergen extract–based D pteronyssinus ImmunoCAPs and molecular ImmunoCAPs containing a mix of the 6 allergens (Der p 1, 2, 5, 7, 21, and 23) was analyzed by using the Student t test for patients from both cohorts and patients who had been stratified according to (1) IgE levels (ie, >20 kUA/L or <20 kUA/L) measured with the molecular ImmunoCAP and (2) IgE reactivity to 1 to 3 or 4 to 6 of the relevant allergens and (3) for patients with and without IgE reactivity to each of the 6 allergens. A P value of less than .05 was considered statistically significant. Cohort 1 (n = 27) was used for comparison of different methods for measuring allergen-specific IgE levels and for quantification of HDM-specific IgE levels, and cohort 2 (n = 26) was used for quantification of HDM-specific IgE levels.