Anti-human IgE Research Articles

Microfluidic Chip Coupled with MALDI-TOF MS for Multitarget Detection of Allergens in Crucian Carp (Carassius auratus).

The goal of the present study was to establish a rapid, simple method for simultaneous allergy testing of sera from multiple fish-allergic patients. Sera from fish-allergic patients were pooled and used for capturing allergens in fish muscle of crucian carp (Carassius auratus), which was studied as a fish model. Sarcoplasmic proteins of crucian carp (Carassius auratus) were extracted for the analysis of allergens. Anti-human IgE antibody-functionalized magnetic beads were utilized to collect IgE antibodies from human pooled sera. The isolation of allergenic proteins was immunomagnetically performed in microfluidic channels, and the elution of the captured allergenic proteins was done with 5% (v/v) acetic acid aqueous solution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting were used for the analysis of tryptic digests of eluted proteins. Ten potential allergenic proteins were identified from crucian carp (Carassius auratus). The present protocol provides a rapid, efficient, and simple method for simultaneous detection of multiple allergens, based on multitargeted antibodies from pooled sera of allergic patients. The constructed multiple antibody-modified MBs can be applied for the deallergenicity of food matrices. The efficiency of allergen detection can be greatly improved, with promising application in allergen discovery and filtration for other muscle-based foods.

Prevention of allergic reactions during oxaliplatin desensitization through inhibition of Bruton tyrosine kinase

BackgroundAcute infusion reactions to oxaliplatin, a chemotherapeutic used to treat gastrointestinal cancers, are observed in about 20% of patients. Rapid drug desensitization (RDD) protocols often allow the continuation of oxaliplatin in patients with no alternative options. Break-through symptoms including anaphylaxis can still occur during RDDs. ObjectiveTo evaluate if pretreatment with acalabrutinib, a Bruton’s tyrosine kinase inhibitor, can prevent anaphylaxis during a RDD in a patient sensitized to oxaliplatin. MethodsA 52-year-old male with locally advanced gastric carcinoma developed anaphylaxis during his 5th cycle of oxaliplatin. As he required 6 additional cycles to complete his curative-intent treatment regimen, he underwent RDD to oxaliplatin but still developed severe acute reactions. The risks and benefits of adding acalabrutinib prior to and during a RDD, were reviewed and he elected to proceed. ResultsWith acalabrutinib taken prior to and during the RDD, the patient was able to tolerate oxaliplatin RDDs without complication. Consistent with its mechanism of action, acalabrutinib completely blocked the patient’s positive skin prick response to oxaliplatin. Acalabrutinib did not alter the percentage of circulating basophils (1.24% versus 0.98%) prior to the RDD but protected from basopenia (0.74% versus 0.09%) after the RDD. Acalabrutinib was associated with a drastic reduction in the ability of basophils to upregulate CD63 in vitro following incubation with oxaliplatin (0.11% versus 2.38%) or polyclonal anti-human IgE antibody (0.08% versus 44.2%). ConclusionsFive doses of acalabrutinib 100mg orally twice daily starting the evening 2 days before and continuing through a RDD allowed a sensitized patient to successfully and safely receive oxaliplatin.

Combined analytical assays for the characterization of drugs binding to human IgE: Applicability to omalizumab-bearing biosimilar candidates assessment

Analytical and functional comparison is key for substantiating the level of convergence (essential sameness) or divergence between versions or variants of a given biological medicine. Accordingly, an overlapping biological activity between products meant to be equal probably reflects a highly similar structure and anticipates a comparable pharmacodynamic behavior. We developed an orthogonal approach to compare the human IgE binding features of different lots and versions of Xolair® (omalizumab), an anti-human IgE monoclonal antibody. The IgE binding affinity and kinetics were measured by surface plasmon resonance. Ability to prevent mast cell activity was assessed in vitro and in vivo in mast cell-based models. The variability of monoclonal antibodies with identical amino acid sequences produced either in Chinese hamster ovarian cells or in human HEK293 cells, was compared. Monoclonal antibodies from the two sources exhibited slightly different human IgE binding and neutralizing features. A known variant exhibiting a three amino acid replacement in the Fab region had lower IgE binding affinity than the original omalizumab. The lower binding affinity translated into reduced IgE neutralizing capacity and, in turn, a difference in the ability to prevent mast cell activation in vitro and in vivo. The proposed set of analytical and functional assays was sensitive enough to detect Fab-linked differences between anti-IgE antibody versions exhibiting an identical aminoacid sequence. In addition to add value to the comparative assessment of biosimilar candidates bearing omalizumab, these methods can aid pre-assessments of new anti-IgE agents that aim to improve therapeutic performance.

Pollen allergen products: current standardisation issues

Scientific relevance. Pollen allergen medicines are in high demand, and their therapeutic benefits directly correlate with their standardisation. Better diagnosis and treatment of allergic diseases require state-of-the-art procedures for assessing the allergenic activity of pollen allergen products using reference standards and physicochemical testing methods.Aim. The study aimed at developing methodological approaches to the standardisation of pollen allergen products in order to shift to measuring their potency in allergenic activity units (AAU) and bring their quality in line with the requirements of the State Pharmacopoeia of the Russian Federation.Materials and methods. The study used pollen allergen reference standards by Microgen, the WHO International Standard for timothy grass (Phleum pratense) pollen extract, a gel filtration standard kit of molecular weight markers ranging from 1.35 to 670 kDa, bovine serum albumin, serum samples with specific IgE obtained from donors sensitised to the study pollen allergens, labelled anti-human IgE antibodies, and reference standards for determining residual volatile solvents by gas chromatography. The identification of in-house reference standards for the potency of pollen allergens involved Western blotting (for allergenic components). The total protein content was determined by Bradford’s assay. In addition, the authors used high-performance liquid chromatography to study protein fractions and gas–liquid chromatography to determine the content of residual organic solvents.Results. To substitute the existing method of non-specific characterisation of allergenic activity in protein nitrogen units (PNU), the authors developed and tested a new method to control allergenic activity in allergenic activity units (AAU) based on an in vitro competitive enzyme-linked immunosorbent assay (ELISA) procedure developed and validated in this study. Furthermore, the authors developed and certified 15 primary in-house reference standards with allergenic activity established in AAU/mL using skin tests in vivo. The experimental data were analysed to standardise the allergenic activity of the pollen allergens manufactured by Microgen. The authors developed physicochemical methods for the certification of in-house reference standards and validated these methods in accordance with the State Pharmacopoeia of the Russian Federation. The study involved selecting chromatographic separation conditions for residual organic solvents (acetone and diethyl ether) and establishing system suitability criteria for the chromatographic system. The allergenic activity of secondary in-house reference standards was certified against that of primary in-house reference standards using competitive ELISA. Thus, the authors managed to shift to the standardisation of pollen allergen products in vitro.Conclusions. The authors developed their competitive ELISA-based method to standardise pollen allergen products by comparing the inhibition of immune responses to a product and a standard. The study demonstrated the feasibility of substituting allergenic activity quantification (in AAU) for protein nitrogen content determination (in PNU) and showed the first example of using AAU for the certification of in-house reference standards. Additionally, the authors developed and validated an analytical procedure for determining the content of residual organic solvents in pollen allergen products by gas–liquid chromatography.

Glutamine and lysine as common residues from epitopes on α-lactalbumin and β-lactoglobulin from cow milk identified by phage display technology

Cow milk is an important source of food protein for children; however, it could lead to allergy, especially for infants. α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) from whey protein make up a relatively high proportion of milk proteins and have received widespread attention as major allergens in milk. However, few studies have identified the epitopes of both proteins simultaneously. In this study, ImmunoCAP and indirect ELISA were first used for detection of sIgE to screen sera from allergic patients with high binding capacity for α-LA and β-LG. Subsequently, the mimotopes was biopanned by phage display technology and bioinformatics and 17 mimic peptide sequences were obtained. Aligned with the sequences of α-LA or β-LG, we identified one linear epitope on α-LA at AA 11-26 and 5 linear epitopes on β-LG at AA 9-29, AA 45-57, AA 77-80, AA 98-101, and AA 121-135, respectively. Meanwhile, the 8 conformational epitopes and their distributions of α-LA and β-LG were located using the Pepitope Server. Finally, glutamine and lysine were determined as common AA residues for the conformational epitopes both on α-LA and β-LG. Moreover, we found the addition of mouse anti-human IgE during the biopanning process did not significantly affect the identification of the epitopes.

Establishment of a homogeneous chemiluminescence immunoassay for cat dander sIgE antibody detection

Background In vitro specific IgE (sIgE) testing has become an important tool for the diagnosis of IgE-mediated allergic diseases. Current methods used to detect allergen sIgE are time consuming and/or expensive. Therefore, a new method was developed for rapid quantitative detection of cat dander-sIgE antibody based on homogeneous chemiluminescence immunoassay. Methods Selection of chemibeads with different chemical groups, and the best Light-initiated chemiluminescence assay (LiCA) analytical mode for cat dander-sIgE detection. To validate and eliminate the interference of IgE on the detection of cat dander-sIgE, concentration of biotinylated anti-human IgE antibody was optimized. For quantification of cat dander-sIgE, a calibration curve was established, and the performance of the assay was evaluated according to clinical guidelines. Results Indirect LiCA is the best mode of analysis and biotinylated anti-human IgE antibody at a dilution ratio of 1:250 minimizes IgE interference. The coefficient of variation of the developed LiCA was 1.49% to 4.66%, with an intermediate precision of 6.90% to 8.21%. The LoB, LoD, and LoQ of the assay were 0.023 kUA/L, 0.056 kUA/L and 0.185 kUA/L. The coefficient of correlation (r) between LiCA and ImmounoCAP was 0.9478. Conclusions A cat dander-sIgE quantitation assay based on homogeneous chemiluminescence immunoassay was established, which could be a new reliable analytical tool for the determination of cat dander-sIgE.

Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies.

Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.

Evaluation of Allergic Cross-Reactivity Among Fishes by Microfluidic Chips and MALDI-TOF MS.

Fish is one of the most common foods that cause allergic reactions. The study of cross-reactivity among fishes using mass spectrometry (MS) is still limited. We developed a strategy using microfluidic chips coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to evaluate cross-reactivity among fishes. The protocol employed commercial magnetic beads functionalized with anti-human IgE antibodies to carry out the IgEs immunomagnetic separation in blood samples, followed by the capture of allergens from seafood protein extracts in a single-straight microfluidic channel. After elution, the captured allergens were digested and identified by MALDI-TOF MS and high-performance liquid chromatography-tandem mass spectrometry and validated by enzyme-linked immunosorbent assay (ELISA). An investigation of the reproducibility revealed that the protocol can sense well the allergens in a food matrix. Seven fish species were analyzed to evaluate the allergic cross-reactivity among fishes. The commercial ELISA test gave consistent results with the presently developed strategy when the same allergenicity test was performed. Parvalbumins were detected from five of the seven analyzed fishes. The sequence alignment of parvalbumins revealed that the similarity of parvalbumins identified from the analyzed fishes is larger than 64%. Boiling may reduce the allergenicity of fish, as demonstrated by a marginal diminish in the parvalbumin content of crucian carp (Carassius carassius) muscle when boiling with water. The method can potentially be used to predict allergic cross-reactivity among fish species, provide advice and guidance to individuals with a history of seafood allergy, and ensure food safety in the food allergy community.

A Novel Method for Total IgE Purification from Human Serum.

For Ab purification, high-affinity chromatography is commonly used. This technique results in high-purity Abs, but it requires highly specific knowledge and equipment. Commercial kits for purification of IgE are not available. Therefore, we established a (to our knowledge) novel method for the purification of total IgE from human serum. Sera from 19 allergic and nonallergic patients were included. After depletion of polyclonal IgG, total serum IgE was captured using anti-human IgE Abs coupled to beads, eluted from the beads, and incubated with protein G-coupled beads to increase the final purity. Purity analysis and Ab detection were performed by Western blot. Total serum IgE and purified IgE concentrations were analyzed using ELISA. To determine their functionality, primary human mast cells were sensitized with purified IgE and activated with anti-IgE or a relevant allergen. CD63+ expression and histamine release were used as readout parameters. Concentrations of purified total IgE corresponded with the levels of total serum IgE. Minor fractions of IgE remained attached to the beads, confirming an effective elution of IgE Abs. Only minimal amounts of IgG were found in the purified IgE fractions, confirming a high purity of IgE. Mast cells sensitized with purified IgE and subsequent activation with anti-IgE Ab or a relevant allergen showed increased expression of CD63+ and increased histamine release. This (to our knowledge) novel method represents a highly effective and widely accessible approach for purification of human serum IgE, which can improve the use of IgE-based in vivo and in vitro models and contribute to allergy research.

Development of a light-initiated chemiluminescence assay for the quantitative detection of cow's milk allergen-specific IgE

BackgroundCow's milk allergy is a common food allergy in children. Clinically, cow's milk-specific IgE (CM-sIgE) antibody test is often used to diagnose milk allergy. An inexpensive light-initiated chemiluminescence assay (LICA), with fast detection speed and small sample volume demand, has application prospects in the field of quantitative detection of CM-sIgE. MethodsChemibeads coated with five major milk allergens, serum samples, biotinylated anti-human IgE antibodies, and streptavidin-coated sensibeads constitute a system to establish a LICA method for the quantitative detection of CM-sIgE. A series of experiments were performed to optimize its reaction conditions and evaluated its performance. ResultsThe optimal conditions for LICA were 10:4 mass ratio of chemibeads to milk allergen, 20 μg/mL chemibeads, 1.0 μg/mL biotinylated anti-human IgE antibodies and a 1/10 dilution of serum for 30-min incubation. The limit of Quantitation (LoQ) was 0.22 kUA/L. For repeatability, the CV ranged from 3.71% to 8.11%. For intermediate precision, the CV ranged from 4.08% to 14.71%. It was linear within 0.20–18.20 kUA/L. This method did not interfere with common interfering substances and total IgE in serum, and there was no obvious cross-reaction with milk-specific IgG and non-milk-specific IgE. ConclusionWe have established a method to quantitatively detect CM-sIgE based on light-initiated chemiluminescence assay, which has good analytical performance and could meet the needs of clinical laboratories.

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E

Background: Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of Artemisia-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. Methods: The assay was established after optimizing the first incubation time and the dilutions of Artemisia-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate Artemisia-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. Results: The developed LICA’s coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kUA/L, and the limit of quantitation was 0.11 kUA/L. The range of linearity was from 0.27 kUA/L to 97.53 kUA/L (r = 0.9968). The correlation coefficient (r) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). Conclusion: An Artemisia-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

MALDI-TOF MS and Magnetic Beads for Rapid Seafood Allergen Tests.

We developed a strategy using immunomagnetic separation (IMS) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to test seafood allergens. The protocol employed commercial magnetic beads (MBs) functionalized with anti-human IgE antibodies to carry out the IMS of IgEs in blood samples, followed by capture of allergens from seafood protein extracts for allergy analysis. After elution, the captured allergens were identified by MALDI-TOF MS and HPLC-MS/MS. The non-specific adsorption of MBs to biomolecules, the reproducibility and sensitivity of the protocol were investigated. The method shows consistent results with enzyme-linked immunosorbent assay tests. The false positive rate of the present method for the allergy test is 0%. The protocol was applied to detect the allergens in greasy-back shrimp for checking the allergenicity of patients' serum. Cooking fish as soup may effectively decrease the allergenicity. The method can be potentially used to identify unknown allergens of seafood to ensure the safety of allergic patients.

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.

Study of Human IgG and IgE Antibodies Against Bee (Apis mellifera) Venom

BACKGROUND: Bee venom contains various biomolecules, such as enzymes, peptides, and amines. The immune sys-tem produces IgG antibodies against bee venom proteins. However, IgE antibodies may also be developed in allergic individuals. OBJECTIVES: In this study, immune responses, including IgG and IgE reactions to bee venom were assessed in vari-ous individuals, using the immunoblotting technique. METHODS: Serum samples were collected from 20 people of three major groups, namely beekeepers, allergic individu-als, and normal people. Venom samples of honey bees and wild bees were collected from the suburbs of Tehran, Iran. Furthermore, commercial honey bee venom samples extracted from Apis mellifera and samples of wild bees extracted from Polistes and Vespula were purchased from France. Immunoblotting was carried out using the sera of subjects and anti-human IgG and IgE coupled to horseradish peroxidase. RESULTS: The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed similar protein bands in Iranian and European honey bee venoms, including α-glucosidase (170 kDa), Api m (100 kDa), acid phosphatase (49 kDa), hyaluronidase (43 kDa), phospholipase A2 (17 kDa), and melittin (2 kDa). In wild bees, two bands were found with the molecular weights of 35 and 25 kDa belonging to antigen 5 and phospholipase A1, respectively. These were not observed in honey bee venoms. Immunoblot analysis revealed that all the mentioned proteins were immunogenic and al-lergenic in different individuals. Hyaluronidase, as well as phospholipases A1 and A2, were the major allergens in most individuals, while IgE reaction to melittin was only reported in one person. CONCLUSIONS: In conclusion, studies on antibodies against bee venoms can be useful in immunotherapy. Different people indicated distinct allergenic patterns. Therefore, further similar assays are recommended before, during, and after immunotherapy.

Protein microarrays: The future of allergy diagnosis? Optimization of coating and printing of allergen arrays used with fluorescent humanised basophil reporter cell line NFAT-DsRed RBL

Up-to-date, in vitro allergy diagnosis techniques rely on the detection of allergen-specific Immunoglobulin E (sIgE). Protein microarrays enable the simultaneous screening of hundreds of sIgEs using only a small amount of serum. However, they do not always report clinically relevant sIgE. Combining protein arrays with humanised rat basophilic leukaemia (RBL) reporter cells that provide a biological readout, we aim to develop a high-throughput amenable, clinically relevant allergy diagnosis platform. Here, we demonstrate the effectiveness of different extracellular matrix (ECM) proteins to facilitate RBL cell attachment on various polymer-coated slide surfaces. We also determine printed protein stability using time-of flight secondary ion mass spectrometry (ToF-SIMS). Lastly, we demonstrate the ability of this system to detect an allergic response. Aldehyde-, epoxy- and amine-coated glass slides were coated with 10 μg/mL vitronectin, fibronectin or laminin for 48 hours at 37 ͦ C and with 10 μg/mL collagen for 48 hours at 4 ͦ C. After incubation, NFAT-DsRed RBL cells were cultured on each surface for 72 hours. The number of adherent cells was assessed every 24 hours using 1 μg/mL Hoechst 33342 solution. Next, the following proteins diluted in PBS to a final concentration of 1mg/mL, goat anti-human IgE, timothy grass pollen extract, human serum albumin, k-casein from bovine milk or the above mentioned proteins diluted with 10% fibronectin, were printed in triplicate onto the different polymer-coated slides using a XYZ3200 dispensing workstation (Biodot). Printed proteins were characterized by ToF-SIMS before and after washing the surfaces with deionized water. Finally, cells were sensitized overnight with human IgE. Cell activation was assessed using fluorescence microscopy after 24h incubation onto the protein printed surfaces. Treatment of the aldehyde- and epoxy-coated slides with different ECM proteins had no significant effect on cell adhesion compared to control (cells only). The amine coated surfaces showed higher printed protein stability compared to aldehyde and epoxy ones. Lastly, cell activation was higher on the epoxy slides after printing 10% fibronectin containing protein solution compared to control (protein only). The epoxy-coated slides would consist the optimum platform to assess cell activation and potentially detect an allergic response.

IL-4 enhances interferon production by virus-infected human mast cells

IL-4 enhances interferon production by virus-infected human mast cells

Inhibition of Allergic Reactivity through Targeting FcεRI-Bound IgE with Humanized Low-Affinity Antibodies.

Options for effective prevention and treatment of epidemic allergic diseases remain limited, and particularly so for IgE-mediated food allergies. We previously found that mouse low-affinity anti-human IgE mAbs with KD in the 10-6-10-8 M range were capable of blocking allergic reactivity without triggering immediate allergic mediator release. In this study, we humanized three parent low affinity allergic response inhibitor (LARI) mouse anti-human IgE mAbs and characterized their biological and immunological features, refined the lead candidate for further clinical development, examined their safety profiles, determined their therapeutic efficiency, and explored the mechanism of action potentially responsible for their therapeutic effects. LARI profoundly blocked cat- and peanut-allergic IgE-mediated basophil activation, inhibited acute release of both prestored and newly synthesized mediator from human mast cells, suppressed peanut-specific IgE-mediated passive cutaneous anaphylaxis, and attenuated dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mice. Safety testing demonstrated that concentrations of LARI well above therapeutic levels failed to trigger immediate release of prestored and newly synthesized allergic mediators, failed to promote robust cytokine/chemokine production from allergic effector cells, and did not elicit allergic reactivity in an animal model of cutaneous and systemic anaphylaxis. Mechanistic studies revealed that LARI downregulated surface FcεRI receptors and IgE via internalization of the IgE/FcεRI, promoted a partial mediator depletion pathway leading to slow release of small amount of mediators, and functioned as a partial antagonist to inhibit FcεRI signaling phosphorylation of Syk, Akt, Erk, and p38 MAPK. These studies demonstrate that targeting surface-bound IgE with LARI profoundly suppresses human allergic reactivity while displaying an excellent safety profile.

An indirect ELISA method for quantitative detection of anti-IgE autoantibodies

Objective The accurate measurement of anti-IgE levels in patients′ serum helps for make diagnosis of chronic urticaria (CU). An indirect ELISA method for quantitative detection of IgG anti-IgE, were established. Methods The serum samples and the clinical data of 75 first-diagnosed CU patients and 120 healthy controls were collected at Shanghai General Hospital during the year of 2018. In the indirect ELISA system to measure the IgG (anti-human IgE), the antigen, Human IgE Myeloma, was coated on the plate; Omalizumab (a humanized anti-human IgE antibody) was the standard, and horse radish peroxidase (HRP)-labeled anti-human IgG was the tracer. The optimum concentrations of serum and HRP-labeled anti-human IgG were determined by chessboard titration, and method evaluation was conducted. The comparison of serum anti-IgE levels in CU patients and healthy people were analyzed by Mann-Whitney U test and chi-square test. Receiver operating characteristic (ROC) curves were applied to establish the diagnostic performance of serum anti-IgE for CU. Results The coating concentration of IgE was 5.0×10-4 mg/ml; serum dilution was 1∶300; enzyme-labeled antibody dilution was 1∶100 000. Standard curve was in good linearity with R2=0.996. The intra-and inter-assay coefficient of variation were 3.9%-7.5% and 6.0%-8.2%, and the recovery rate of low and high concentration samples were 95.9% and 108.4%, respectively. When hemoglobin≤1.3 g/L, triglyceride≤4.6 mmol/L, bilirubin≤171 μmol/L, no interference were observed. The limit of blank, limit of detection, and limit of quantitation were 5.8×10-4, 1.8×10-3, and 2.0×10-3 mg/ml. The linearity range was from 2.0×10-3 to 354.4 mg/ml. No Hook effect was found until anti-IgE reached 354.4 mg/ml. The anti-IgE remained stable after serum storage at 4 ℃ overnight or treated with 6 repeated freeze-thaw cycles. The anti-IgE levels in CU patients [19.0(1.9, 58.6)mg/ml] were significantly higher than those in healthy controls [0.7(0.002, 11.1)mg/ml] with P<0.001. When cut-off value was set as 15.3 mg/ml, in this method, the positive rate of CU patients (54.7%) was significantly higher than these of healthy controls (18.3%) (P<0.001, AUC=0.736). Conclusions The indirect ELISA method for serum anti-IgE measurement was successfully established. Anti-IgE autoantibodies in serum would be used in the diagnosis of CU. Key words: Immunoglobulin E; Autoantibodies; Enzyme-linked immunosorbent assay; Urticaria

Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy

BackgroundClinical efficacy of allergen-specific Immunotherapy (AIT) towards Japanese cedar (JC) pollen allergy is firmly established but JC pollen-specific biomarker assays are lacking. Treatment-related increase of allergen-specific antibodies is a robust biomarker of successful AIT. Allergen-specific non-IgE antibodies are believed to reduce the effects of allergen exposure by competing with IgE for allergen binding, and in-vitro assays quantifying the effects of AIT-induced IgE-blocking antibodies are advantageous. A cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay of JC pollen was established. MethodsSerum IgE–allergen complexes were captured by immobilized recombinant CD23, and allergen–IgE–CD23 complexes were detected by a biotin-conjugated anti-human IgE antibody. Sera from JC pollen-allergic subjects without or with subcutaneous immunotherapy (SCIT) with JC pollen extract were used (n = 11/group). ResultsOptimal assay conditions were established at 20 μg/mL CD23 and 0.3 μg/mL JC pollen extract, and the dependency on CD23 and IgE was verified. The data show that the JC pollen ELIFAB assay is fit for purpose and demonstrates that the IgE-blocking activity is significantly increased in the JC pollen SCIT group compared with the non-treated group. ConclusionThe JC pollen ELIFAB assay represents a simple, cell-free biomarker assay for monitoring the development of IgE-blocking antibody activity during JC pollen AIT.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.