RU2633749C1 - Method for food allergy diagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: sample of biological material (coprofiltrate) is prepared followed by spectrophotometry at 405 nm. A morning fecal sample in an amount of 1 g is taken, dissolved in 9 ml of phosphate-buffered saline with 0.05% Tween 20 (PBST), pH=7.2. Then mixed to a homogeneous consistency. After that, it is centrifuged for 10-20 minutes at 3500 rpm. The supernatant is sampled and an enzyme immunoassay is carried out for the presence of allergen-specific IgE antibodies to food allergens followed by spectrophotometry. When an optical density of 0.15 units of optical density and more is determined, allergies to a specific allergen are determined. When an optical density of less than 0.15 units is determined, the absence of allergies to a particular allergen is diagnosed.

EFFECT: increased accuracy of food allergies diagnosis.

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Description

The invention relates to medicine, namely to allergology and immunology, and is used to diagnose food allergies.

A known method for diagnosing allergies ("Performance of the PROTIA ™ Allergy-Q® System in the Detection of Allergen-specific IgE: A Comparison With the ImmunoCAP® System." Allergy Asthma Immunol Res. 2015 Nov; 7 (6): 565-572 Published online, 2015. May 26. doi: 10.4168 / aair.2015.7.6.565 PMCID: PMC4605929), including the collection of 5 ml of whole blood in vacuum tubes, obtaining blood serum by centrifugation at 3000 rpm for 5 min, carrying out enzyme-linked immunosorbent analysis with the determination of the concentration of allergen-specific IgE antibodies, by which the presence or absence of allergies is judged.

The disadvantage of this method is the possible false-negative result in a number of studies in the presence of clinical symptoms of food allergy, due to the elimination of allergen-specific antibodies from the blood into the tissues of target organs (lungs, intestines, skin, etc.), as well as the invasive nature of the production of biomaterial.

A known method for the diagnosis of allergies to a specific allergen (RF Patent 2222814, IPC G01N 33/533, publ. 2004). The essence of the method: peripheral blood lymphocytes are added to strip tablets sensitized with MKAD, the plates are incubated at (37 ± 2) ° C for 1 h 30 min, washed from cells, then fluorescent immunoglobulins against human immunoglobulins G or immunoglobulins E are added to the wells they are kept for 30 min at (37 ± 2) ° C, the wells are washed three times, the washed strips are examined under a luminescent microscope and, when localized areas of MKAD fluorescence are detected, they are diagnosed with an allergy to a specific allergen.

The disadvantages of this method are the low accuracy of the study due to the lack of standardization of the content of lymphocytes in the patient’s blood, which does not exclude the possibility of obtaining false positive and false negative results in a number of physiological conditions, concomitant viral, bacterial and autoimmune diseases, as well as subjective assessment by the researcher of fluorescence and the invasive nature of the biomaterial .

A known method for the diagnosis of food allergies (RF patent, IPC G01N 33/48, publ. 2011), the essence of which is that before the diagnostic elimination test and on the 4th-5th day of the test, the secretory IgA in saliva is determined and its parameters are changed (in the direction of increasing or decreasing) more than 30% are diagnosed with food allergies.

The disadvantages of this method are the lack of specificity for the diagnosis of a specific causally significant food allergen, the mandatory elimination test and the need to exclude diseases of the ENT organs in the patient before the test, which can lead in some cases to a false-positive or false-negative result.

The closest method for diagnosing allergies to the proposed (Patent RF 2027193 G01N 33/68, publ. 1995) involves preparing a sample of biological material, followed by spectrophotometry at 450 nm. Take blood serum, dilute it with buffered saline in a ratio of 1: 4, then add 0.1 ml of diluted serum to 2 ml of 6% PEG-6000, the mixture is incubated for 1 h at room temperature, followed by spectrophotometry at 450 nm and at the optical density of the mixture is less than 1.2 units diagnosed with allergies.

The disadvantages of this method is the lack of accuracy in food allergies, since there is no specificity for the diagnosis of a specific causative food allergen, since the method reveals only immune complexes in the blood, as well as the invasive nature of the biomaterial.

The objective of the invention is to eliminate these drawbacks, improving the accuracy of the diagnosis of food allergies to a specific causative allergen by using coprofiltrate as a biomaterial, as a substrate reflecting the functional state of the intestine, which is the target organ in food allergies, with the exception of the invasive nature of the biomaterial.

To solve the problem in the diagnosis of allergies, including the preparation of a sample of biological material followed by spectrophotometry at 405 nm, it was proposed to study coprofiltrate as a biological material. In this case, a morning sample of feces is taken in an amount of 1 g, dissolved in 9 ml of phosphate-saline buffer with 0.05% Tween 20 (FSBT) pH = 7.2, stirred until a homogeneous consistency, centrifuged for 10-20 minutes at 3500 r / min, the supernatant is taken, enzyme-linked immunosorbent assay for the presence of allergen-specific IgE antibodies to food allergens, followed by spectrophotometry. When determining the optical density of 0.15 units. and more are diagnosed with an allergy to a specific allergen, and when determining the optical density of less than 0.15 units. they diagnose a lack of allergy to a particular allergen.

Preparing a sample of biological material for research in the proposed mode allows you to get a supernatant containing allergen-specific antibodies and at the same time having low levels of substances capable of non-specific binding and cross-reaction at the stage of enzyme-linked immunosorbent assay.

Due to the increasing prevalence and number of life-threatening cases, food allergies have become one of the main health problems. The classic immune response in food allergies is characterized by the production of allergen-specific IgE antibodies and the development of sensitization, and in the effector phase it manifests itself as a hypersensitivity reaction to food products with the development of clinical symptoms of allergic inflammation. According to modern concepts, immunoglobulin E (IgE) is a class of immunoglobulins that is normally found in small amounts in serum and secrets. According to the physicochemical properties, IgE is a glycoprotein with a molecular weight of 190,000 daltons and is synthesized mainly by plasma cells localized in the mucous membranes. The biological role of IgE is characterized by the ability to bind to the surface of mast cells and human basophils, causing them to degranulate at the moment of antigen attachment, which triggers a cascade of reactions leading to the release of inflammatory mediators. In case of food allergy, allergen-specific IgE antibodies to food products are synthesized, while on the one hand, allergen-specific IgE antibodies act as a component of the pathological process, and on the other, they can be a reliable diagnostic marker. Modern methods of diagnosing food allergies include the detection of allergen-specific IgE antibodies in blood serum, on the basis of which a conclusion is made on the presence of a causally significant allergen, however, conditions are possible in which systemic circulation of allergen-specific IgE antibodies is not detected due to their elimination in the target organs (intestines ) Thus, the detection of allergen-specific IgE antibodies in the material characterizing the functional and immune state of the intestine expands the possibilities of allergic diagnostics for identifying a causally significant food product.

The method is highly sensitive, has a non-invasive nature of obtaining the studied biomaterial, as a result of which it becomes possible to level the psycho-emotional stress in patients.

The method is as follows.

If you suspect a food allergy, the patient is assigned an allergy-diagnostic study according to the proposed method.

A fecal sample weighing 1 g is dissolved in 9 ml of phosphate-saline buffer with 0.05% Tween 20 (FSBT) with pH = 7.2. The resulting suspension is thoroughly mixed, mainly using a vortex, which allows to obtain a homogeneous consistency of the material, after which it is centrifuged for 10-20 minutes at 3500 rpm. The supernatant is taken in the required amount of 50 μl for each allergen.

An enzyme-linked immunosorbent assay is then carried out according to a well-known method: in 96 well microplates, in which chemically activated paper discs with covalently attached allergens are present as solid phase. 50 μl of coprofiltrate obtained by the proposed method are introduced into the wells of the microplate. Specific binding of antibodies to the allergen occurs within 1 hour, at 37 ° C, after which 4-hole washing of the FSBT microplate wells is carried out. The next incubation for 1 hour at 37 ° C occurs when 50 μl of a solution of antibodies to human immunoglobulin E labeled with alkaline phosphatase are added to the wells of the microplate, followed by a 4-fold washing of the wells of the FSBT microplate. Then, 200 μl of a solution of the chromogenic substrate of p-nitrophenyl phosphate (pNPP), which, having reacted with alkaline phosphatase, gives a characteristic staining, is added to the wells. The reaction is stopped after 45 minutes by adding 50 μl of a 1N NaOH solution to the wells of the microplate.

The reaction is recorded on a vertical spectrophotometer for each well containing a paper disk with a covalently attached allergen using a wavelength of 405 nm (reference wavelength of 620 nm).

Thus, in the presence of allergen-specific antibodies in the studied coprofiltrate to a specific causally significant allergen fixed on a paper disk, their complex forms, and the addition of antibodies to human immunoglobulin E labeled with alkaline phosphatase and a chromogenic substrate of p-nitrophenyl phosphate leads to an increase the optical density of the reaction mixture in the well of the microplate with the investigated allergen.

When determining the optical density of 0.15 units. and more are diagnosed with an allergy to a specific allergen, and when determining the optical density of less than 0.15 units. they diagnose a lack of allergy to a particular allergen.

Example 1

Patient K., 1 year 3 months, was admitted with erythematous plaques with irregularly shaped borders, with the presence of scales, vesicles, weeping, crusts and excoriations on the flexion surfaces of the arms and legs, and on the face. According to the mother of the patient, rashes appeared after eating adapted milk formula based on cow's milk, cottage cheese, turkey meat, apple and carrot puree.

The study was carried out using the proposed method, while a morning sample of the patient's feces was taken in an amount of 1 g, dissolved in 9 ml of phosphate-saline buffer with 0.05% Tween 20 (FSBT) pH = 7.2 using vortex and subsequent centrifugation in for 10 minutes at 3500 rpm. For the subsequent enzyme immunoassay, a supernatant was used, while in the wells of the microplate were chemically activated paper disks with covalently attached allergens of cow's milk protein, goat's milk protein, α-lactalbumin, β-lactoglobulin, casein, apple, turkey meat, carrots.

After incubation for 1 h at 37 ° C, followed by 4-fold washing of the wells of the FSBT microplate. The next incubation for 1 h at 37 ° С occurred when 50 μl of a solution of antibodies to human immunoglobulin E labeled with alkaline phosphatase were added to the wells of the microplate, followed by a 4-fold washing of the wells of the FSBT microplate. Then, 200 μl of a solution of the chromogenic substrate p-nitrophenyl phosphate (pNPP) was added to the wells, which, reacting with alkaline phosphatase, gave a characteristic staining. The reaction was stopped after 45 minutes by adding 50 μl of a 1N NaOH solution to the wells of the microplate. The reaction was taken into account on a vertical spectrophotometer at a wavelength of 405 nm (reference wavelength of 620 nm). The following results were obtained:

protein of cow's milk - 0.020 units. optical density;

goat milk protein - 0.018 units. optical density;

α-lactalbumin - 0.014 units. optical density;

β-lactoglobulin - 0.658 units. optical density;

casein - 0.018 units. optical density;

apples - 1,584 units. optical density;

turkey meat - 0,092 units. optical density;

carrots - 0.031 units. optical density.

An allergy to β-lactoglobulin (0.658> 0.150) and an apple (1.584> 0.150) was diagnosed. Apples, products based on cow's milk, which were replaced by milk mixtures based on goat's milk, were excluded from the baby's nutrition. After 6 months of diet therapy, the clinical manifestations of allergies disappeared.

Example 2

Patient B., 42 years old, complaints of dyspeptic symptoms after eating beef, sausages and ravioli during the year. After exclusion of gastrointestinal pathology, the patient was analyzed by the proposed method.

At the same time, chemically activated paper discs with covalently attached allergens of beef protein, mutton protein, pork protein, turkey meat and rabbit meat were in the wells. The following results were obtained:

beef protein - 1,890 units. optical density;

mutton protein - 0.054 units. optical density;

pork protein - 0, 168 units. optical density;

turkey meat - 0.020 units. optical density;

rabbit meat - 0.018 units. optical density.

Diagnosed with allergies to beef protein (1.890> 0.150) and pork protein (0.168> 0.150). Excluded from the patient’s diet are products based on beef and pork, which have been replaced with products from turkey, rabbit and lamb.

After 1 month, the patient disappeared dyspeptic manifestations.

Example 3

Patient Sh., 10 years old. Received complaints of skin itching of the hands and feet, dyspeptic symptoms in the form of bloating and loose stools for a month, loss of appetite. According to the parents, dyspeptic symptoms and itching intensified after eating cod, tomatoes and apples. The patient had a study of the level of total IgE in the blood, which amounted to 865 IU / ml (with a norm of up to 100 IU / ml), on the basis of which a food allergy was diagnosed. However, after 2 months of diet therapy with the exception of fish, tomatoes and apples, complaints remained. It was decided to conduct an allergy diagnosis using the proposed method to confirm the diagnosis of food allergy based on the identification of a causally significant food product. A feces sample was weighed 1 g in weight and investigated by the proposed method, while chemically activated paper disks with covalently attached allergens of cod meat, tomato, and apple were in the wells of the microplate. The following results were obtained:

cod meat - 0,023 units. optical density;

tomatoes - 0.011 units optical density;

apple - 0.065 units. optical density.

Thus, food allergies to these products have not been identified.

Based on persistent complaints and received data, the diagnosis of food allergy was withdrawn, a helminthiasis study was performed by enzyme-linked immunosorbent assay, in which antibodies to ascarids were detected. The diagnosis of ascariasis was confirmed by a coprological study. 2 weeks after anthelmintic therapy (levomizole, 75 mg, twice), the clinical symptoms disappeared.

The proposed method examined 126 children with clinical manifestations of food allergies, aged 2 months to 2 years, and 45 adult patients. The control group consisted of 15 healthy children and 16 adults. In children and adults suffering from food allergies, the disease was confirmed by anamnesis and skin tests (pri-tests). In these groups of patients, the sensitivity and specificity of the proposed diagnostic method (A) were compared with the enzyme immunoassay for the diagnosis of allergy based on the detection of allergen-specific IgE antibodies in blood serum (B).

Thus, the proposed method allows to increase the accuracy of diagnosis of food allergies by 10-12%, to ensure the timely identification of a causally significant allergen (causative food) to determine the tactics of diet therapy, safe for the patient, has the ability to use one sample for various combinations of allergens , has a non-invasive nature of obtaining biomaterial, which eliminates the complications of medical manipulations associated with blood sampling, as well as related psychoemotional Foot patient stress (including children) and personal presence of the patient.

Claims (1)

A method for diagnosing allergies, including preparing a sample of biological material, followed by spectrophotometry at 405 nm, characterized in that coprofiltrate is examined as a biological material, while a morning sample of feces is taken in an amount of 1 g, dissolved in 9 ml of phosphate-saline buffer with 0.05 % Tween 20 with pH = 7.2, mix until smooth, centrifuged for 10-20 min at 3500 rpm, supernatant is taken, enzyme-linked immunosorbent assay for allergen-specific IgE antibodies to specific schevym allergens followed by spectrophotometry, and determining the absorbance of 0.15 optical density units and more diagnose allergy to specific food allergen, and in determining the optical density of less than 0.15 units diagnosed absence of allergy to specific food allergen.