Detection Of Allergen-specific IgE Research Articles

Microfluidic Immunosensing Platform Based on a Rolling Circle Amplification-Assisted DNA Dendrimer Probe for Portable and Sensitive Detection of Allergen-Specific IgE.

The robust point-of-care platform for sensitive, multiplexed, and affordable detection of allergen-specific IgE (sIgE) is an urgent demand in component-resolved diagnostics. Here, we developed a microfluidic immunosensing platform based on a rolling circle amplification-assisted DNA dendrimer probe for sensitive detection of multiple sIgEs. The versatile multichannel microfluidic whole blood analytical device integrates cell filtration, recombinant antigen-modified magnetic enrichment, and DNA dendrimer probe-amplified signal transduction for portable on-chip analysis. Three sIgEs against common oyster allergens were simultaneously detected in blood samples by simple smartphone-based imaging without any pretreatment. The quantitative detection of multiple allergen-specific antibodies on the platform was achieved with limits of detection of less than 50 pg/mL, exhibiting superior sensitivity compared to most point-of-care testing. The detection results of 55 serum samples and 4 whole blood samples were 100% consistent with the ELISA results, confirming the accuracy and stability of our platform. Additionally, the reversible combination of hexahistidine6-tag and Ni-IMAC magbead was elegantly utilized on the immunosensing platform for desired reversibility. With the advantages of general applicability, high sensitivity, and reversibility, the DNA dendrimer-based microfluidic immunosensing platform provides great potential for the portable detection of immune proteins as a point-of-care platform in disease diagnostics and biological analysis.

Gastrointestinal manifestations, spectrum and frequency of sensitization to food allergens in Kyiv’s adult citizens with allergic rhinitis: a cross-sectional study

Background. Food allergy affects 1–3 % of adults worldwide. More than 160 foods can cause allergic reactions. Food allergy may precede and stimulate allergic rhinitis. Aim of the study was to assess the spectrum and frequency of sensitization to food allergens in patients with allergic rhinitis from Kyiv, Ukraine. Materials and me­thods. The investigation was conducted as a cross-sectional study. One hundred and seventy-five Kyiv adult patients with allergic rhinitis were enrolled in the trial. Sensitization to allergens and their components was determined by skin prick tests and in vitro detection of allergen specific IgE by ELISA in blood serum — a multicomponent Allergy Explorer2 ALEX2 test. Results. It was shown that sensitization to following inhalant allergens was predominant: to timothy grass — in 50.3 %, to ragweed — in 48.6 %, to birch — in 44 %, to wormwood — in 24 %, to Alternaria alternata mold — in 15.4 % of patients. One hundred and thirty-one (74.9 %) participants have co-sensitization to at least one food allergen, among them 77 (58.8 %) had gastrointestinal symptoms. Sensitization to following food allergens was predominant: to hazelnut — 27.5 %, apple — 26.3 %, peanut — 21.7 %, celery — 14.3 %, soy — 13.1 %, carp — 11.4 %, kiwi — 9.1 %, crab — 9.1 %, codfish — 8.6 %, oyster — 8.6 %, peach — 8 %, lobster — 8 %, carrot — 7.4 %, anisakis — 6.3 %, prawn — 6.3 %, tiger prawn — 5.7 %, to beef — 5.1 % of cases. It was found that the frequency of sensitization to cross-reactive proteins PR-10 was 20.5 % (95% confidence interval (CI); 9.7–33.9 %) in patients with allergic rhinitis without food co-sensitization comparing to 51.1 % (95% CI; 42.5–59.7 %) in participants with both allergic rhinitis and food co-sensitization, p < 0.001; to nsLTP — 2.3 % (95% CI; 0–8.9 %) and 19.8 % (95% CI; 13.4–27.2 %), respectively, p = 0.011. Conclusions. Among Kyiv adult patients with allergic rhinitis, approximately three quarters have food co-sensitization that may be the cause of gastrointestinal symptoms and exacerbations of allergic rhinitis after consuming vegetables, fruits and nuts due to cross-reactivity with inhalant allergens. This should be considered when manage such patients.

Influence of Pore Size in Protein G'-Grafted Mesoporous Silica Nanoparticles as a Serum Pretreatment System for In Vitro Allergy Diagnosis.

Particles with the capacity to bind to immunoglobulin G (IgG) can be used for the purification of IgG or to process clinical samples for diagnostic purposes. For in vitro allergy diagnosis, the high IgG levels in serum can interfere with the detection of allergen-specific IgE, the main diagnostic biomarker. Although commercially available, current materials present a low IgG capture capacity at large IgG concentrations or require complex protocols, preventing their use in the clinic. In this work, mesoporous silica nanoparticles are prepared with different pore sizes, to which IgG-binding protein G' is grafted. It is found that for one particular optimal pore size, the IgG capture capacity of the material is greatly enhanced. The capacity of this material to efficiently capture human IgG in a selective way (compared to IgE) is demonstrated in both solutions of known IgG concentrations as well as in complex samples, like serum, from healthy controls and allergic patients using a simple and fast incubation protocol. Interestingly, IgG removal using the best-performing material enhances in vitro IgE detection in sera from patients allergic to amoxicillin. These results highlight the great translation potential of this strategy to the clinic in the context of in vitro allergy diagnosis.

Issues of specific <i>in vitro</i> allergological diagnosis of atopic conditions

There is a steady increase in the prevalence of allergic diseases of atopic origin worldwide, e.g., atopic bronchial asthma (ABA) and atopic dermatitis (AD). Identification of a causally significant allergen in allergic patients is crucial for the diagnosis, therapy and prevention of allergic diseases. Korea has developed the Allergy-Q multiplex test to detect specific IgE. Allergy-Q is based on an immunoblotting method using a nitrocellulose membrane as a solid phase for allergen immobilization and can detect allergen-specific IgE simultaneously to 107 allergens. Our aim was to conduct a comparative analysis for detectable allergen-specific IgE antibodies to food, fungal, pollen, household, epidermal allergens in blood serum by immunoblotting method using the Allergy-Q test system in patients with atopic dermatitis, atopic bronchial asthma and psoriasis.
 The study included patients with atopic dermatitis (AD, group 1, n = 9), atopic bronchial asthma (ABA, group 2, n = 14) and psoriasis (PS, group 3, n = 17). The concentration of total immunoglobulin E and allergen-specific immunoglobulins of class E in blood serum to 32 most common food, fungal, pollen, household, epidermal allergens was determined by the immunoblotting method using the Allergy-Q test system (Korea).
 We have found that sensitization of atopic origin was observed in all patients with AD (n = 9), in 85.7% (n = 12) of patients with atopic bronchial asthma, and in 47.1% (n = 8) of patients with psoriasis. Polyvalent sensitization was shown to prevail in all groups of the examined persons. When studying the spectrum of sensitization to food allergens, a significantly increased frequency of positive reactions to cows milk protein was found in the group of patients with AAA as compared with AD and PS groups. Among all studied groups, sensitization to the Alternaria fungi was found at the highest frequency in the group of patients with ABA. Sensitization to ragweed pollen was very common in all groups of patients. Sensitization to household and epidermal allergens in the groups with AD and AAA was noted for all studied allergens with the highest positivity rates for the feline epithelium and dog dander.
 In the present study, the Allergy-Q system showed an agreement with preliminary data from a specific allergological examinations. This relationship suggests a potential for usage of the Allergy-Q immunoblotting method as a highly effective alternative to other in vitro tests for diagnosing atopy. An advantage of the Allergy-Q Multiplex Serum Allergen-Specific IgE Detection Kit is a short processing time, small amount of blood sample, and broader clinical information on the causative allergens.

A light-initiated chemiluminescent assay for the detection of children's milk protein-specific IgE with excellent ability to avoid interference of specific IgG

The detection of allergen-specific IgE is of value for the diagnosis of children’'s milk allergy. However, its accuracy will interfere with the presence of high levels of specific IgG in the serum of children with milk allergy. To solve this problem, we established a light-initiated chemiluminescent assay (LICA) based on nanomicrospheres, which neutralized the interference of specific IgG by increasing the amount of antigen coated on the microspheres. The ability of this method to resist IgG interference was confirmed by adding extra specific IgG to the serum of allergic patients. Finally, the positive rate of allergen-specific IgE was increased to 85%, which was better than the indirect ELISA (70%), indicating that this method has certain advantages for the detection of specific IgE in children with milk allergy.

Isotype-Specific Detection of Serum Immunoglobulins Against Allergens.

Type-I hypersensitivity is commonly characterized by increased levels of antigen-specific immunoglobulin (Ig) E. Therefore, it is important for clinical and research investigators to reliably measure serum levels of IgE in allergic patients and animal models. While current ELISA-based methods are simple and commonly performed for the detection of allergen-specific IgE using serum or plasma, they may produce misleading results. This is in part due to decreased sensitivity for IgE in the presence of other Ig isotypes in the same sample, such as IgG, that are typically more abundant than IgE. When assessment of multiple Ig isotypes is necessary, performing optimized assays for individual isotypes requires high sample volumes. Here, we describe an approach to increase the sensitivity for IgE detection while conserving the sample volume needed. This method not only improves the accuracy of serum IgE measurements but also allows simultaneous analysis of other allergen-specific immunoglobulins.

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF–SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

Specifically immobilizing His-tagged allergens to magnetic nanoparticles for fast and quantitative detection of allergen-specific IgE in serum samples

Fast and accurate detection of specific IgE (sIgE) is essential for diagnosis of allergic diseases. In this study, we developed a nano-capturer Fe3O4@SiO2-NTA based on magnetic nanoparticles with surface modification of Ni-NTA moieties. Results showed that our immunosensor based on the specifically immobilization of recombinant allergen protein on the Fe3O4@SiO2-NTA could realize fast, high efficiency and low-cost detection of sIgEs in serum samples. In vitro experiments demonstrated that the chemiluminescence (CL) response of the immunosensor to sIgEs showed an obviously discrimination between positive and negative serum samples. The CL intensity values were proportional to the sIgE concentrations in the range of 2.52–14.02 ng/mL with detection limit of 0.35 ng/mL, which offered a promising platform for fast and quantitative determination of sIgEs. Furthermore, our method was successfully utilized for allergic disease diagnosis in 46 serum samples and satisfied results were achieved in comparison with the commercial ELISA kit. In particular, by simply changing various recombinant allergen proteins immobilized on the surface of Fe3O4@SiO2-NTA, our strategy showed great potential for high-throughput analysis of allergen screening.

Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo

Introduction and objectivesThis study aimed to compare the effects of different biotinylation methods on the performance characteristics of allergen-specific IgE detection. Materials and methodsThe Gal d 6 gene was cloned into the pAN6/pAC6 vector, resulting in rGal d 6-Bio/Bio-rGal d 6 vector. The fusion protein was expressed in Escherichia coli AVB101 and simultaneously biotinylated in a site-specific manner. The Gal d 6 gene was amplified via PCR and cloned into the pET-28a vector and transformed into E. coli BL21 and purified via Ni-NTA, followed by chemical biotinylation using Sulfo-NHS-LC-Biotin. Twenty-eight patients allergic to hen's egg white were examined for sensitization against egg yolk. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed to detect allergen-specific IgE. ResultsrGal d 6, Bio-rGal d 6, and rGal d 6 were prepared using different biotin binding modes to detect allergen-specific IgE. rGal d 6-Bio (Kd=0.6154) and Bio-rGal d 6 (Kd=0.6698) had a markedly better detection performance than rGal d 6 (Kd=28.93), and the rGal d 6-Bio had a better detection performance in small-volume serum samples. ConclusionsrGal d 6-Bio improved the sensitivity for the detection of allergen-specific IgE.

NPY-mRFP Rat Basophilic Leukemia (RBL) Reporter: A Novel, Fast Reporter of Basophil/Mast Cell Degranulation.

Humanized rat basophilic leukemia (RBL) reporter cell lines are increasingly used for the detection of allergen-specific IgE and other purposes, such as the detection of allergens and standardization of allergen preparations. Existing reporter systems have many strengths and advantages but can be expensive or require longer incubation times. The new NPY-mRFP reporter cell line addresses such problems, as it requires neither expensive substrates nor overnight incubation for detection of activation. The fusion of Neuropeptide Y (NPY) with monomeric Red Fluorescent Protein (mRFP) results in localization of the fluorescent protein in granules.AsNPY-mRFP is preformed in granules, the reporter systemactivation can be assessed using fluorescence measurements after as soon as 45-60min, as described in this chapter, without the need to add any substrates.

A paper-based chemiluminescence immunoassay device for rapid and high-throughput detection of allergen-specific IgE.

The fast and precise detection of potential allergen-specific immunoglobulin E (sIgE) is imperative for the diagnosis and appropriate treatment of allergic diseases. In this study, we have successfully fabricated a novel paper-based immunoassay device for the detection of sIgE in allergic diseases. We used Can f 1, one of the main dog allergens, as a model allergen to detect sIgE in human sera. To achieve excellent performance, the experimental parameters were optimized. Further, we extended this device for potential applications in the clinical diagnosis of allergic diseases: worthwhile clinical performance in the detection of allergens was achieved as compared to that achieved by commercial enzyme-linked immunosorbent assay (ELISA) kit. Therefore, it was proven that this strategy has the advantages of high-throughput, rapid, sensitive, and highly accurate detection of trace amounts of sIgEs. Furthermore, by simply changing the antigen and antibody, this device could be used for the high-throughput detection of other allergens, so as to achieve multiallergen detection and appropriate desensitization therapy, thereby making it promising in the determination of allergic diseases in clinics.

Current status of allergy diagnostic testing in vitro of type I allergy

Type Ⅰ allergy is one type of allergic reaction mediated by IgE antibody. Simple, rapid and accurate allergen detection method can be used to identify the allergen of patients, which is of great significance for the prevention, diagnosis and treatment of allergic diseases. In this article, different techniques for in vitro diagnosis of allergen specific IgE antibodies in type Ⅰ allergic reactions were described, important factors affecting in vitro diagnosis of allergens were analyzed, and the future development trend of in vitro diagnosis of allergen specific IgE antibodies were prospected. In order to help clinicians to select more suitable in vitro detection methods in the process of in vitro diagnosis of type Ⅰ allergy and to better understand the internal relationship between the allergen specific IgE detection report and clinical symptoms.(Chin J Lab Med, 2018, 41: 889-892) Key words: Type I allergy; Allergens; Immunoglobulin E; in vitro diagnosis

Immunotheraphy in Allergic Diseases.

The prevalence of allergic diseases is increasing worldwide. It is estimated that more than 30% of the world population is now affected by one or more allergic conditions and a high proportion of this increase is in young people. The diagnosis of allergy is dependent on a history of symptoms on exposure to an allergen together with the detection of allergen-specific IgE. Accurate diagnosis of allergies opens up therapeutic options. Allergen specific immunotherapy is the only successful disease-modifying therapy for IgE-mediated allergic diseases. New therapeutic strategies have been developed or are currently under clinical trials. Besides new routes of administration, new types of allergens are being developed. The use of adjuvants may amplify the immune response towards tolerance to the antigens. In this review, we analyze different antigen-specific immunotherapies according to administration route, type of antigens and adjuvants, and we address the special case of food allergy.

Prevalence and clinical characteristics of local allergic rhinitis to house dust mites.

Local allergic rhinitis (LAR) is a recently classified subtype of rhinitis defined by a nasal allergic response in patients without systemic evidence of atopy. Recent studies have reported the prevalence, clinical course, culprit allergens, diagnostic methods and treatment outcomes of LAR. The purpose of this review is to summarize the most relevant and updated scientific evidence for LAR, especially focusing on its prevalence and clinical characteristics. LAR is found in a significant proportion (3.7-61.9%) of patients previously diagnosed with nonallergic rhinitis, but the prevalence may differ among ethnic groups and countries. Common allergens of LAR are similar to those of allergic rhinitis, in which house dust mites are the most common cause, followed by grass pollen, tree pollen, weed pollen and animal dander confirmed by provocation tests. Although the nasal provocation test to a single allergen is considered the gold standard method, the detection of allergen-specific IgE and other inflammatory mediators from nasal secretions and the basophil activation test can assist in the diagnosis of LAR. Conjunctivitis and asthma are the most common comorbid conditions, and the occurrence rate of asthma increases over period. However, the conversion rate to allergic rhinitis was not significantly different between LAR and healthy controls. LAR is a well-differentiated entity of rhinitis, which should be considered in patients with persistent and severe symptoms without any systemic evidence of atopy. Further research is needed to investigate the long-term outcome, and geographic and ethnic differences of LAR.

Allergen detection and etiological study of adenoidal hypertrophy in children

Objective:To investigate the etiology of adenoidal hypertrophy by detection of allergen and total serum IgE in children.Method:Using the German Allergy Screen Test Kit testing system, total serum IgE was detected and quentitative detection of allergen specific IgE in 19 groups in 782 children with adenoidal hypertrophy, the proportion of total IgE in serum and the positive rate of allergens were counted, the proportion of atopy and the characteristics of the allergens were analyzed. Result:The proportion of total IgE in serum of 782 children with adenoidal hypertrophy was 66.8%. The top ten allergens (by positive rate) were: milk（32.0%），house dust mite（27.7%），moulds（25.3%），dog hair（22.4%），egg whites（16.5%），cashew nuts（10.5%），house dust（10.4%），cat hair（9.5%），crab（5.1%），beef（3.7%）. Conclusion:In present study, 66.8% adenoidal hypertrophy children with atopy. The following inhalation allergens positive rate of top three in children with adenoidal hypertrophy are: house dust mite, moulds, dog hair. The positive rate of food intake of the first three are: milk,egg whites, cashew nuts.

Involvement of cross-reactive carbohydrate determinants-specific IgE in pollen allergy testing.

BackgroundSpecific IgE antibodies against the low-molecular-weight carbohydrate antigen that does not bridge IgE molecules on mast cells are not associated with clinical symptoms. Cross reactivity can be determined in allergen-specific IgE detection assays when the carbohydrate structures between pollen allergens and plant derived food allergens are similar; in such cases, false positive results for grain or legume allergens can be reported for pollen allergic patients who are not sensitized to those allergens. This phenomenon arises owing to the presence of cross-reactive carbohydrate determinants (CCDs).ObjectiveThis study aimed to assess the impact of CCD interference on the results for pollen allergen-specific IgE antibodies in the general adult population and to perform CCD inhibition tests evaluating the involvement of CCD on samples positive to pollen allergens.MethodsSerum samples from 322 subjects were tested for IgE antibodies to pollens and CCD. The research subjects were given questionnaires about pollen allergic symptoms to help assess the presence of allergies. Allergen IgE antibodies for Japanese cedar, Japanese cypress, orchard grass, ragweed, MUXF, bromelain, horseradish peroxidase (HRP), and ascorbate oxidase (ASOD) were analyzed.ResultsIt was observed that among individuals who tested positive to any of the pollen allergens, the positive ratio of CCD-specific IgE antibody was the highest for HRP (13.5%–50.0%). The results from the inhibition tests revealed that CCD was marginally present. Although IgE antibodies for cedar pollen did not react with CCD, IgE antibodies for Japanese cypress, orchard grass, and ragweed might be detected by the presence of CCD.ConclusionThe results of the inhibition tests revealed the obvious presence of CCD suggesting its involvement. Considering these findings, careful evaluation of patient IgE results should be performed for Japanese cypress, orchard grass, and ragweed.

Multi-parametric Point of Care Device for Allergen-specific IgE Detection in Veterinary Applications

This paper reports the first validation results of a promising point of care device based on an injected plastic microfluidic cartridge and commercial off the shelf components. An 8x8 spotted array is immobilized in the incubation chamber inside the cartridge, and once the chemical reactions have been finished, the colorimetric results are captured by a CMOS camera. The captured image of the array is analysed in terms of intensity levels at each spot location. This device is intended to detect allergen specific IgE in veterinary applications, but it can also be applied to any application based on ELISA like protocols.

Use of Humanized Rat Basophilic Leukemia Reporter Cell Lines as a Diagnostic Tool for Detection of Allergen-Specific IgE in Allergic Patients: Time for a Reappraisal?

The interaction between allergens and specific IgE is at the heart of the allergic response and as such lies at the center of techniques used for diagnosis of allergic sensitization. Although serological tests are available, in vivo tests such as double-blind placebo-controlled food challenges (DBPCFC) and skin prick test (SPT) associated to the patients' clinical history are still the main guides to clinicians in many practices around the world. More recently, complex protein arrays and basophil activation tests, requiring only small amounts of whole blood, have been developed and refined, but are yet to enter clinical practice. Similarly, the use of rat basophilic leukemia (RBL) cell lines for detection of allergen-specific IgE has been made possible by stable transfection of the human FcεRI α chain into this cell line more than 20years ago, but has not found widespread acceptance among clinicians. Here, we review the perceived limitations of diagnostic applications of humanized RBL systems. Furthermore, we illustrate how the introduction of reporter genes into humanized RBL cells is able to overcome most of these limitations, and has the potential to become a new powerful tool to complement the armamentarium of allergists. A demonstration of the usefulness of humanized RBL reporter systems for elucidation of complex IgE sensitization patterns against wheat proteins and a section on the use of fluorescence-based reporter systems in combination with allergen arrays close the review.

Molecular Allergy Diagnostics: Analytical Features That Support Clinical Decisions.

Application of purified native and recombinant allergenic molecules into IgE antibody assays can improve analytical sensitivity and specificity for selected allergen specificities. They enhance analytical sensitivity by allowing assays to detect IgE antibodies with a lower limit of quantification (LoQ) to missing or poorly represented allergens in diagnostic extracts that are commonly used in vivo and in vitro. Use of selected allergenic molecules can help improve the clinician's prediction of the risk of a serious allergic reaction to stable allergens. They can provide diagnostic information to determine if a provocation challenge (e.g., oral food challenge) is indeed mandatory or not necessarily needed to support the final diagnostic decision. Suspected cross-reactivity based on the clinical history can be adjudicated by analyzing IgE antibodies to allergenic molecules from cross-reactive protein families. Finally, genuine primary sensitization can be identified by IgE antibody responses that are measured to selected allergenic molecules which are present in only one particular allergen source. After allergen-specific IgE detection, careful interpretation is required by the physician who knows the patient's history. Applying single allergen molecules, positive IgE antibody results are still only relevant in the case of corresponding objective symptoms. Subsequently, clinical relevance of such an IgE antibody test result must be determined by the clinician and not by the test itself. Because of their comprehensive nature, allergen extracts will remain the principal allergen source for diagnostic in vivo and in vitro assays of IgE antibody for many years. Judicious use of individual allergenic molecules in serum IgE assays may provide their most cost effective and efficient application for establishing a definitive diagnosis of human allergic disease.

Allergen Specific IgE Detection Performance of Allergyq® System in Korean Allergy Patients

Allergen Specific IgE Detection Performance of Allergyq® System in Korean Allergy Patients