New technologies in molecular allergodiagnostics

The article presents the characteristics of the ALEX2 (MacroArrayDX, Wien, Austria). It is designed for simultaneous detection of IgE total and specific IgE-aB to 120 extracts and 180 molecules by solid-phase enzyme immunoassay. Extracts and allergen molecules combined with nano-particles are sorbed on a solid-phase substrate, forming a macroscopic multiplex matrix - the immune allergy chip. The Institute of Clinical and Laboratory Standards (CLSI) conducted research on the verification and validation of the ALEX2 in relation to the ImmunoCAP macroarray test system (ThermoFisher Scientific, Uppsala, Sweden), which is often used in allergodiagnostics. The results obtained on the two test systems were comparable. One of the most important features of the ALEX2 test system is that unique allergen molecules and allergenic extracts are included in its composition, and a method has been found to inhibit cross-reactive hydrocarbon determinants (CCDs), which cause frequent non-specific binding of IgE-aT. The use of this test system makes it possible to carry out component allergy diagnostics with the determine of the dominant sensitizing factor in cases of mono- and polyvalent sensitization. The test results affect the determination of indications and the effectiveness of ASIT, allow assessing the risk of anaphylaxis and predicting further treatment tactics for the patient.

Kinetics of Dermatophagoides pteronyssinus and Dermatophagoides farinae growth and an analysis of the allergen expression in semi-synthetic culture medium

Currently, several mite growth culture media used in the production of allergenic extracts contain animal-derived components that limit their use in diagnostic and/or therapeutic applications. The aim of this study was to evaluate the growth of D. pteronyssinus and D. farinae mites in a semi-synthetic medium without animal-derived proteins to produce highly reproducible allergenic extracts for diagnostic and therapeutic purposes to be more consistent with the regulations of health authorities. Both species of mites showed optimal growth in the semi-synthetic culture medium. The highest expression of allergens Der p 1 and Der f 1 was observed at the last phases of mite growth. Semi-synthetic media without animal-derived proteins facilitated excellent growth rates of house dust mites in cultures. Adjusting the cultivation time to decide the optimal time point for the processing of the extracts is decisive.

Urticaria, Anaphylaxia and Asthma from Contact with Work Air in Farmers and Agronomists Due to Bruchus Pisorum

Background: There are few reports of contact urticaria from the inhalation of allergens from legume pests. Objective: To study the origin of an outbreak of contact urticaria, asthma and anaphylaxis in farmers and agronomists who work handling dried peas. Method: Allergenic extracts composed of Bruchus lentis and B. pisorum, healthy peas, peas treated with aluminum phosphide and parasitized peas were used for in vivo tests (prick-test, oral challenge and bronchoprovocation) in affected patients and in five controls. with a history of atopy from other legumes. In addition, patch testing with live Bruchus pisorum, patch testing with more common insecticides, molecular component analysis, and Ig-E immunodetection were carried out. Results: Positive responses were found for the prick-test and the bronchoprovocation test to extracts of parasitized peas and B. pisorum, but the oral challenge was negative. A common 25 kDa band for infested peas and a 30 kDa band for infested pea and B. pisorum body was detected in all patients. The response for pea allergens was negative for all patients, unlike controls with a history of allergy to lentils and peanuts. Conclusion: It was determined that B. pisorum is a cause of symptoms of immediate hypersensitivity mediated by Ig E by inhalation of the allergen or by puncture of spicules or mushrooms of B. pisorum.

Allergenic Sensitization to pollen extracts in allergic patients. General “Calixto García” University Hospital, 2017.

Background: Nowadays, the allergic diseases are affecting the population at worldwide level and Cuba does not escape to this, inside the aeroallergens that trigger the crises we can find the pollens. The main goals of this study were to determine the allergenic sensitization to pollens in allergic patients and their relationship with the presence of allergic diseases.Methods: A cross-sectional descriptive observational study was performed in patients suffering from asthma, rhinitis, allergic rhino-conjunctivitis, atopic dermatitis and allergic conjunctivitis. All patients underwent an allergic clinical history and skin prick test with allergenic extracts of Helianthus annus, Cosmos bipinnatus, Cynodon dactylon, Quercus sp, Eucalyptus sp . Frequencies and percentages were determined for its analysis.Results: Thirty-three patients were studied. The average age was found in the third decade of life, with a predominance in women. More than half of the patients were sensitized to pollens and 24.24% of them were polysensitized; the most frequent pollen was the cynodon dactylon . The patients with rhinitis were the most sensitized with the pollens.Conclusions: There is sensitization to pollen in our patients.

Allergenic extracts from natural and genetically modified soybean

Background. The wide spread of soybeans both natural and genetically modified (GM) in agriculture and food industry arises a question about the safety of its use, as soy is the most common food allergen among leguminous plants. Meanwhile, there are no registered domestic diagnostic allergens from soybeans in Russia. Objective. The aim of this study was to obtain allergenic extracts from natural and GM soybeans resistant to the herbicide «Roundup» and evaluate their biochemical and allergenic properties. Methods. Soybean extracts were obtained by the Evans-Kok method. The amount of protein nitrogen was determined by the Nessler method. The protein composition of the soybean was determined by the SDSPAGE. Specific activity was assessed in the reaction of NDTK. Allergenic activity was assessed in ELISA according to the sIgE levels to soy in the sera of patients with food allergy. Results. The protein fractions corresponding to known allergens weare revealed by SDS-PAGE in the samples of extracts: natural soybeans - Gly m 3, Gly m 5, and Gly m 6, while GM soybeans - Gly m Bd 30k. In addition to those proteins, in both extracts the 20 kD protein was clearly detected, which can correspond to the inhibitor of trypsin Kunitsa (Hor v 1, CMb, BDR). Allergenic soybean extracts bind sIgE and sIgG in the sera of patients with allergies. Conclusion. The obtained data confirm the high allergenic potential of extracts from natural soybeans, whereas the allergenic activity of GM soybeans extracts is reduced.

Rapid in vitro assessment of hypersensitivity with whole blood leukocyte histamine release assay

Background. Clinical application of leukocyte histamine release assay (HRA) can be enhanced through the development of automated reversed-phase high-performance liquid chromatography with mass spectrometry (RP-HPLC-MS/MS) employing of whole blood (WB) samples. The purpose of this study was to evaluate the possibility to use WB-HRA technology for the in vitro diagnosis of hypersensitivity employing RP-HPLC-MS/MS. Methods. Method principle: heparinized whole blood samples after substitution of plasma with PIPES buffer («reconstituted» blood) are incubated at 37 °C with different concentrations of substances (allergens, drugs, chemicals, food etc.) suspected in relation to hypersensitivity reactions. Release of histamine is occured mainly from basophilic granulocytes depending on their sensitivity to stimulating substances (allergens etc.). The released histamine is subsequently direct determined in the supernatant using RP-HPLC-MS/MS technology. Heparinized whole blood (8 ml) was drawn from patients with sensitivity to D. pteronyssinus (D1), birch pollen (T3) and peach (F95) confirmed by case history and results of skin prick tests or detection of specific IgE. After removing plasma and substitute it with PIPES buffer aliquots of «reconstituted» blood were put into separate tubes (300-450 μl for macro-method) or to wells of U-shape 96-well micro-titer plate (150-200 μl for micro-method) already contained different concentrations (dilutions) of histamine standard, anti-IgE and allergenic extracts D1, T3, F95 including their chemically modified forms sD1 and sF95. After 1 h incubation at 37 °C tubes or plates were centrifuged and supernatants from each tube (macro-method) or well of the plate (micro-method) were directly analyzed for histamine content by RP-HPLC-MS/MS. Results. It is shown that the levels of histamine released from leukocytes of whole blood of patient sensitized to D. pteronyssinus upon stimulation with non-modified D1 extract are much higher than that of upon stimulation with modified sD1 that means that chemical modification of allergen leads to suppress of B-cell epitopes. It seems that this method is suitable for evaluation of hypersensitivity to allergen, as well as for detection of allergenicity of modified allergens (allergoids). We also found a significant reduction (in 60%) of histamine release from blood leukocytes upon stimulation with T3 extract in patient with sensitivity to birch pollen after allergen-specific immunotherapy (ASIT) in compare to the level of histamine before ASIT. These data indicate that our method of histamine release assay can be convenient as in vitro test for monitoring and evaluation of ASIT efficacy. It was also studied histamine release from blood basophils of patient with sensitivity to peach, confirmed by case history and detection in serum specific IgE. Incubation of patient's whole blood with peach extract (F95) resulted a histamine release at the level comparable to that of stimulated with anti-IgE. This high level of histamine is correlated with the level of allergen-specific serum IgE. Besides the incubation patient's blood with modified peach extract sF95 induced histamine release two times less than that of non-modified F95 indicating substantial reduction of sF95 allergenicity. So this methodology for detection of histamine released from leukocytes of whole blood may be successfully applied for diagnosis of food allergy employing of laboratory made allergenic extracts. Conclusion. Speed and simplicity of performance including the requirement of small quantities of blood makes the WB-HRA employing RP-HPLC-MS/MS a useful laboratory tool not only of scientific interest (detection of allergenicity of modified allergens, peptides etc.) but also of practical significance for evaluation the degree of sensitivity of patients to allergens (including those who received anti-allergic medication or ASIT), analysis of pathophysiological responses to drugs, chemicals and other compounds suspected for their adverse side effects.