Detect which antigen binds to IgE

Detect which antigen binds to IgE

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Assume that a patient has chronic hives (itchy) on the skin. I understand that he has an antigen, which binds to IgE and eventually triggers histamine release, causing an allergic reaction. So is there any method to detect which molecule/antigen has bound to his IgE? (can we capture it with like what we do to obtain a 3d protein/molecule structure?)

From what I understand most allergies are detected by a scratch test. from the link below:

"A skin prick test, also called a puncture or scratch test, checks for immediate allergic reactions to as many as 40 different substances at once… After cleaning the test site with alcohol, the nurse draws small marks on your skin and applies a drop of allergen extract next to each mark. He or she then uses a lancet to prick the extracts into the skin's surface. A new lancet is used for each allergen… About 15 minutes after the skin pricks, the nurse observes your skin for signs of allergic reactions. If you are allergic to one of the substances tested, you'll develop a raised, red, itchy bump (wheal) that may look like a mosquito bite"

More information on scratch tests can be found here:

After a quick google search,I found a paper from 2014 [1] that seems to speak to the essence of what you are asking, link below. [1]

In the paper above they propose using a protein microarray technology to find allergies.

I am not sure if there is any way to extract the igE-antigen complex from the patient. But I think it could probably be done with the microarray technology. Although it would be cool to see a picture of the complex. I hope I have helped answer your question.

[1] Harwanegg, Christian, and Reinhard Hiller. "Expert Review of Molecular Diagnostics." Taylor and Francis Online. Expert Review of Molecular Diagnostics, 09 Jan. 2014.


The enzyme-linked immunosorbent assay (ELISA) ( / ɪ ˈ l aɪ z ə / , / ˌ iː ˈ l aɪ z ə / ) is a commonly used analytical biochemistry assay, first described by Engvall and Perlmann in 1971. [1] The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.

In the most simple form of an ELISA, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate is added. If there was binding the subsequent reaction produces a detectable signal, most commonly a color change.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes, only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g., a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase, which is part of the plate, and so are not easily reusable.

Enhanced sensitivity of capture IgE‑ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens

The detection of allergen‑specific immunoglobulin (Ig)E is an important method for the diagnosis of IgE‑mediated allergic diseases. The sensitivity of the indirect IgE‑ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgE‑ELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28‑Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgE‑ELISA. Capture‑ELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidase‑conjugated streptavidin and 3,3,5,5‑tetramethylbenzidine. The relative sensitivities of indirect‑ELISA and capture‑ELISA for HDM allergen‑specific IgE binding were determined sera from non‑allergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgE‑specific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDM‑allergic sera. Furthermore, in a random sample of HDM‑allergic sera (n=71), capture‑ELISA (71/71, 100%) was more sensitive than indirect‑ELISA (68/71, 95.8%) for the detection of HDM‑specific IgEs (P<0.01), indicating that this novel method may be useful for the diagnosis of HDM allergy.


Reported linear B cell epitopes…

Reported linear B cell epitopes located in Der f 1 and Der f…

Expression, purification and IgE binding…

Expression, purification and IgE binding activity of rDer f 1/2. (A) Schematic diagram…

Analysis of the binding of…

Analysis of the binding of specific IgE to rDer f 1, rDer f…

Schematic diagrams of direct capture…

Schematic diagrams of direct capture IgE-ELISA and indirect IgE-ELISA. (A) Direct capture and…

Capture-ELISA detection of specific IgEs…

Capture-ELISA detection of specific IgEs in HDM sera. (A) Identification of optimal Der…

A single glycan on IgE is indispensable for initiation of anaphylaxis

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.

Biology and dynamics of B cells in the context of IgE-mediated food allergy

An increasing number of people suffer from IgE-mediated food allergies. The immunological mechanisms that cause IgE-mediated food allergy have been extensively studied. B cells play a key role in the development of IgE-mediated food allergies through the production of allergen-specific antibodies. While this particular function of B cells has been known for many years, we still do not fully understand the mechanisms that regulate the induction and maintenance of allergen-specific IgE production. It is still not fully understood where in the body IgE class switch recombination of food allergen-specific B cells occurs, and what processes are involved in the immunological memory of allergen-specific IgE responses. B cells can also contribute to the regulation of allergen-specific immune responses through other mechanisms such as antigen presentation and cytokine production. Recent technological advances have enabled highly detailed analysis of small subsets of B cells down to the single-cell level. In this review, we provide an overview of the current knowledge on the biology of B cells in relation to IgE-mediated food allergies.

Keywords: B cells IgE antibodies food allergy.

© 2020 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.


The cells of the adaptive immune system communicate in many ways. They can come into physical contact and exchange signals through receptors within the contact area or immunologic synapse. Examples include the contact between T cells and dendritic cells or between effector T cells and their targets. Immune cells can also signal nearby cells by secreting signaling proteins called cytokines. Several hundred different cytokines have been identified. Signaling cells secrete a mixture of cytokines that then bind to receptors on nearby cells. The target cell receives multiple signals that it must integrate to respond appropriately. Cytokines, acting through their specific receptors, can turn the synthesis of specific proteins on or off. They can cause the target cell to divide or differentiate, and they may trigger apoptosis. With hundreds of different cytokines acting in complex mixtures it is sometimes difficult to predict exactly how a specific target cell will respond. Major families of cytokines include the interleukins that mediate signaling between leukocytes, interferons that mediate interactions between cells and have significant antiviral activity, growth factors that regulate growth and differentiation of many different cell types, and tumor necrosis factors that modulate inflammatory responses.

The IgE–mast-cell axis in chronic allergic inflammation

Mast cells activated by IgE and specific antigens produce mediators that drive early phase reactions ( Fig. 2 ) and contribute to late phase reactions (Box 2), but these mast cells also secrete diverse cytokines, chemokines and growth factors that have the potential to influence airway remodeling 4𠄷,63,66 ( Fig. 3 ). Compared to wild-type mice, mice lacking the FcεRI α chain showed diminished airway inflammation, indicating decreased eosinophil concentrations, in an asthma model 67 . Studies in wild-type, genetically mast-cell�icient and mast-cell–knockin mice indicated that activation of mast cells through the FcR γ chain (which in mice is required for mast cell activation by antigen-IgG1 immune complexes through FcγRIII, as well as for signaling through FcεRI) is required for the full development of many features of allergic inflammation and tissue remodeling in a model of chronic asthma 68,69 . However, many other effector cells also have the potential to contribute to these features of asthma, including antigen-specific effector T cells 70 , and the relative roles of mast cells compared to other effector cells in some of these settings is not fully resolved, particularly in human subjects.

In chronic allergic inflammation, repetitive or persistent exposure to allergens can result in both the production of IgE against multiple antigen epitopes of several different antigens ( Fig. 1 , right) and the development of long-term changes in the involved tissues (Box 2), including changes in mast cell number, tissue distribution (with mast cells in the epithelium and the smooth muscle layer, not shown here) and phenotype. Moreover, repetitive epithelial injury caused by chronic allergic inflammation can be exacerbated by exposure to pathogens such as viruses or bacteria or environmental factors, and the consequent repair response results in epithelial and mesenchymal changes that are thought to sustain TH2 cell𠄺ssociated inflammation, promote sensitization to additional allergens or allergen epitopes (for example, epithelial-cell�rived TSLP can upregulate the expression of co-stimulatory molecules such as OX40, CD40 and CD80 by dendritic cells, not shown here) and regulate the airway remodeling process. These processes in turn result in many functionally relevant changes in the structure of the affected tissue. There is evidence that many of these changes can be influenced by IgE and mast cells, either acting in concert through the IgE–mast-cell axis or independently. For example, both soluble factors, such as INFγ, S1P, adenosine and IL-33, and cells present at the site, such as TH2 cells and Treg cells (which can interact with OX40L on mast cells) can modulate, or tune, IgE-dependent mast cell activation, and some pathogen-associated molecular patterns (PAMPs) and cytokines, including TSLP and IL-33, can activate mast cells independently of IgE to produce different spectra of cytokines or chemokines. Studies in mast-cell–knockin mice have indicated that some actions of mast cells, such as increasing the number of epithelial goblet cells, can occur in a model of chronic asthma by mast-cell�pendent mechanisms that do not require mast cell signaling through the FcεRI γ chain, whereas mast cells must express both the FcεRI γ chain and the IFN γ receptor 1 (IFN-γR1) to mediate increases in lung eosinophils, neutrophils and collagen (not shown here). Amplification of the IgE response by IgE, for example, by FAP ( Fig. 1 , right) and IgE- and antigen-dependent activation of basophils after their recruitment to the airways can occur independently of mast cells. PRR, pattern recognition receptor GM-CSF, granulocyte-macrophage colony-stimulating factor

Box 2 Late phase reactions and chronic antigen exposure lead to persistent allergic inflammation and tissue remodeling

Mast cells previously activated during the early phase reaction secrete mediators that can orchestrate the recruitment, tissue infiltration and functional activation of circulating leukocytes, including granulocytes such as eosinophils, basophils and neutrophils, as well as monocytes and T cells 66,80,163 ( Fig. 2 ), which substantially increases the diversity of the cellular drivers of inflammation at the site of antigen challenge. Mast cells may therefore be a crucial source of mediators contributing to the initiation of late phase reactions 164,165 . Other sources include antigen-specific T cells, as well as myeloid cells activated by antigen-containing immune complexes if these cells are present at the site of antigen challenge, such as in individuals with ongoing airway inflammation 163 . Indeed, low levels of ongoing inflammation in the airways, as can be observed in individuals with asthma even during treatment, together with exposure to antigen peptides that are recognized by effector T cells𠅋ut not IgE antibodies—may account for the development of late phase responses in individuals without previous detectable early phase reactions163 , 166 . As people afflicted with allergic asthma are usually exposed repeatedly to the antigens that elicit their allergic reactions, and this may occur over periods of years, their airways have experienced many antigen-induced early and late phase reactions. However, in addition to developing inflammation, the airways of individuals with asthma also show structural changes, called airway remodeling, which include increased numbers of mucus-producing goblet cells in the epithelium, evidence of repair responses at sites of epithelial injury, thickening of the smooth muscle layer and changes in connective tissues, blood and lymphatic vessels, mucus glands and nerves 66,167 . Persistent airway inflammation, a key feature of both allergic and non-allergic forms of asthma, probably contributes in a crucial way to tissue remodeling in asthma 167 , 168 . Evidence suggests that IgE and mast cells can substantially contribute to chronic airway inflammation and tissue remodeling in asthma by functioning both in a single pathway—interdependently through antigen- and IgE-dependent mast cell activation𠅊nd independently ( Fig. 3 ).

In addition to possible redundancy in the roles of mast cells and other cell types in the chronic changes associated with asthma, there is evidence that many factors can modify mast cell function in this setting. For example, in vitro 58,71� and in vivo 69,76,77 evidence indicates that the extent of antigen- and IgE-dependent mast cell activation may be influenced substantially (or ‘tuned’) by microenvironmental factors that affect the expression or function of surface receptors or signaling molecules that contribute to the positive or negative regulation of such responses 4,6,7,78,80 . Tuning factors that can be present locally at the sites of allergic inflammation, such as in the airways and other anatomical sites, include adenosine 75 , sphingosine-1 phosphate (S1P) 76 , certain chemokines 77 and a variety of cytokines, such as IL-4 (refs. 58 , 71 ), IL-33 (refs. 72 – 74 ) and interferon γ (IFN-γ) 69 . Tuning also can be accomplished by cell-cell interactions. For example, interactions of mast cells and T cells can be bidirectional and complex 79,80 and include the ability of IgE-activated mast cells to enhance proliferation and cytokine production in multiple T cell subsets 81,82 and the ability of CD4 + CD25 + regulatory T (Treg) cells to suppress IgE-dependent mast cell activation through interactions between tumor necrosis factor receptor superfamily, member 4 (OX40), either as expressed by Treg cells 83 or in a soluble form 84 , and the OX40 ligand, OX40L expressed on mast cells ( Fig. 3 ).

In addition to stabilizing expression of FcεRI on the mast cell surface and sensitizing mast cells to respond to specific antigens, IgE can have effects on mast cell survival or function 59,85 that seem to be independent of the presence of the antigen for which the IgE has specificity. Such findings suggest that certain IgE antibodies might favor the expansion of mast cell numbers or have effects on mast cell function in vivo, including increasing their secretion of cytokines and chemokines, even in the absence of a specific antigen. In addition to binding to FcεRI or CD23, IgE, and FcεRI itself, can be bound by β-galactose𠄼ontaining olilgosaccharide chains of galectin-3, permitting galectin-3 to activate mast cells and basophils through carbohydrate interactions that crosslink receptor-bound IgE, FcεRI or both 86 ( Table 1 ). Studies in mice lacking galectin-3 support the notion that galectin-3 can amplify the pathology that is observed in models of asthma 87 or atopic dermatitis 88 .

Rocket Electrophoresis

  • It is a one-dimensional single electroimmunodiffusion test.
  • It is mostly used for the quantitation of antigens.
  • In this case, the antibody is mixed with the agarose gel and this mixture will be used to form a layer on the glass slide.
  • Wells will be formed on the surface of the gel and antigens are added in those wells in increasing concentration.
  • Electrophoresis will be performed.
  • As a result, cone-like precipitation bands (rocket-like structure) will be observed.
  • The length of the rocket-like structure is directly connected with the concentration of antigens.

Hypersensitivity Pneumonitis

Some disease caused by hypersensitivities are not caused exclusively by one type. For example, hypersensitivity pneumonitis (HP), which is often an occupational or environmental disease, occurs when the lungs become inflamed due to an allergic reaction to inhaled dust, endospores, bird feathers, bird droppings, molds, or chemicals. HP goes by many different names associated with various forms of exposure (Figure (PageIndex<9>)). HP associated with bird droppings is sometimes called pigeon fancier&rsquos lung or poultry worker&rsquos lung&mdashboth common in bird breeders and handlers. Cheese handler&rsquos disease, farmer&rsquos lung, sauna takers' disease, and hot-tub lung are other names for HP associated with exposure to molds in various environments.

Pathology associated with HP can be due to both type III (mediated by immune complexes) and type IV (mediated by TH1 cells and macrophages) hypersensitivities. Repeated exposure to allergens can cause alveolitis due to the formation of immune complexes in the alveolar wall of the lung accompanied by fluid accumulation, and the formation of granulomas and other lesions in the lung as a result of TH1-mediated macrophage activation. Alveolitis with fluid and granuloma formation results in poor oxygen perfusion in the alveoli, which, in turn, can cause symptoms such as coughing, dyspnea, chills, fever, sweating, myalgias, headache, and nausea. Symptoms may occur as quickly as 2 hours after exposure and can persist for weeks if left untreated.

Figure (PageIndex<9>): Occupational exposure to dust, mold, and other allergens can result in hypersensitivity pneumonitis. (a) People exposed daily to large numbers of birds may be susceptible to poultry worker&rsquos lung. (b) Workers in a cheese factory may become sensitized to different types of molds and develop cheese handler&rsquos disease. (credit a: modification of work by The Global Orphan Project)

Explain why hypersensitivity pneumonitis is considered an occupational disease.

Figure (PageIndex<10>) summarizes the mechanisms and effects of each type of hypersensitivity discussed in this section.

Figure (PageIndex<10>): Components of the immune system cause four types of hypersensitivities. Notice that types I&ndashIII are B-cell/antibody-mediated hypersensitivities, whereas type IV hypersensitivity is exclusively a T-cell phenomenon.


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