RU2140085C1 - Method for diagnosing alimentary allergy - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves applying indirect detection of fluorescent antibodies. Allergen-specific antibodies of blood serum are brought in contact with marked anti-IgE antibodies. Formalin- and tannin-treated sheep erythrocytes sensitized with nutrient protein antigens are used as alimentary allergens. Conjugates of anti-IgE antibodies and fluorescine isothiocyanate are used as marked anti-IgE antibodies. the so produced complex is examined through fluorescence microscope. Specific fluorescent rim being observed on erythrocytes, that means 1:40 or higher anti-IgE antibodies titer being determined in serum under investigation, alimentary allergy is considered to be diagnosed. EFFECT: accelerated high specificity test; low examination costs.

Description

 The invention relates to medicine, namely to allergology and immunology and can be used to detect food allergies in adults and children.

 In allergology, various serological reactions are used to diagnose food allergies, the most common is the enzyme-linked immunosorbent assay (ELISA) (Gervazieva V.B., Ovsyannikova I.G., Voronkin N.I., Sorochinskaya N.N., Raikis B.N. The use of enzyme-linked immunosorbent assay for the determination of allergen-specific IgE antibodies // ZhMEI, 1987. -N 9. -C. 33-35.).

 Disadvantages: the multi-stage method, time-consuming, requires the use of expensive test systems and the use of reagents containing potent or toxic substances. The use of a significant number of ingredients in ELISA affects the specificity of an enzyme-linked immunosorbent assay ("Optimization of some parameters that improve the sorption activity of a plate for enzyme-linked immunosorbent assay" Meshandin A.G., Minasyan B.O., Vaneeva L.I., Simonov V.P. , Agapkina N.P. ZhMEI, 1991, N 3, p. 60-61).

 The invention is aimed at solving the problem: increasing specificity; reduction in analysis time; cheaper method. This task is achieved through the use of an indirect method of fluorescent antibodies, which is characterized in that food allergens are formalized and tanized sheep erythrocytes sensitized with food protein antigens, and labeled anti-IgE antibodies are obtained by conjugation of anti-IgE antibodies with a dye - isothiocyanate fluorescein. The resulting complex is examined in a luminescent microscope and, in the presence of a specific fluorescent rim on red blood cells, food allergy is determined.

The method is as follows: on smears fixed on a glass slide from food allergens (8 smears from one allergen on one glass slide), 10 ml of successive dilutions of the test sera from 1:10 to 1: 1280 are applied. Glasses with smears are placed in a humid chamber and incubated for 20 minutes at 37 ^° C. At the end of the incubation period, the slides with smears are washed in PBS (buffered saline solution) twice for 7 minutes and the droplets of the solution are removed with filter paper. On all smears apply fluorescent anti-IgE antibodies in a volume of 10 ml, taken in working dilution. Slides are placed in a humid chamber and incubated for 20 minutes at 37 ^° C. Then the slides with smears are again washed twice in PBS for 7 minutes, dried in air and viewed under an immersion lens of a luminescent microscope.

 In the presence of allergen-specific IgE antibodies in serum to the corresponding food allergens, specific erythrocyte fluorescence in the form of a rim is observed.

Assessment of the intensity of fluorescence is carried out on a four-cross scale:
4+ emerald green rim fluorescence around the erythrocyte;
3+ bright green fluorescence rim around the red blood cell;
2+ green fluorescence rim around the red blood cell;
1+ weak, barely noticeable fluorescence of the rim around the red blood cell;
- lack of fluorescence.

 The titer of the test serum is taken to be its maximum dilution, in which fluorescence is observed no lower than 3+.

 The determination in the test serum of anti-IgE antibodies in a titer of 1:40 or more indicates the presence of an allergy to this food allergen.

 The reaction should be accompanied by the following controls.

 1) Knowingly positive control: allergen + standard serum to this allergen with a known titer + fluorescent anti-IgE antibodies. Serum titer should be confirmed.

 2) Obviously negative control: sheep's PTE (formalized and tanized red blood cells) + test serum + fluorescent anti-IgE antibodies. No fluorescence.

 3) Conjugate control: allergen + fluorescent anti-IgE antibodies in working dilution. No fluorescence.

 The duration of the reaction is 1 hour 10 minutes.

 Example 1. Patient Ch., 5 years. Diagnosis: Atopic dermatitis, limited.

 From the anamnesis: suffered from allergic diathesis from 1.5 months of age, for the first time changes in the skin appeared in connection with the use of milk mixtures in food. At the age of 1 year, allergic diathesis transformed into atopic dermatitis.

 Objectively: there is dry skin and excoriation on the extensor surfaces of the shoulders and forearms, in the popliteal fossae, on the neck and periorbital region, itching is expressed.

 Allergen-specific IgE antibodies were determined using the indirect method of fluorescent antibodies. Food allergens of milk, chicken, protein and chicken yolk were used. Examination result: allergen-specific IgE antibodies to milk allergen were detected in a titer of 1: 160, which indicates the presence of food allergy. The obtained result was confirmed by enzyme immunoassay.

 Example 2. Patient M., 10 years old. Diagnosis: Neurodermatitis, limited.

 From the anamnesis: for the first time, changes in the skin appeared at 6 months of age. Until 3 years of age, he suffered from allergic diathesis, then the disease was transformed into neurodermatitis.

 Objectively: dry skin, lichenization and excoriation in the area of elbow bends and in the popliteal fossae, skin itching are noted.

 Allergen-specific IgE antibodies were determined using the indirect method of fluorescent antibodies. Food allergens of milk, chicken, protein and chicken yolk were used. The examination revealed allergen-specific IgE antibodies to chicken allergen in a titer of 1: 640, which indicates the etiological role of this allergen in the development of the disease. The obtained result was confirmed by enzyme immunoassay.

 Example 3. Patient S., 7 years old, examined for bronchial asthma.

 From the anamnesis: from 3 months of age to 3 years, he suffered from allergic diathesis, at the age of 3 years, skin manifestations of allergies disappeared, episodes of difficulty breathing appeared. At the age of 4 years was diagnosed with bronchial asthma, mixed form, mild. Objectively: the skin is clean, physiological in color, moderately moist. The percussion sound over the entire surface of the lungs is clear, pulmonary, vesicular breathing in the lungs, no wheezing.

 Allergen-specific IgE antibodies in the blood serum were determined using the indirect method of fluorescent antibodies. Food allergens of milk, chicken, protein and chicken yolk were used. Allergen-specific IgE antibodies to the chicken yolk allergen in a titer of 1: 320 were detected in the patient's serum, which indicates the presence of an allergy to this food allergen. The obtained result was confirmed by enzyme immunoassay.

 Example 4. Patient A., 14 years old, underwent an allergological examination in connection with a suspicion of an allergic pathology of the respiratory tract.

 From the anamnesis: in February 1997 he fell ill with pneumonia, after which a dry cough persists for two months.

 Objectively: the chest is the correct form. Respiratory rate 18 in 1 min. Percussion sound over the entire surface of the lungs is clear, pulmonary. Hard breathing is heard in the lungs, no wheezing.

 Serum was tested for the content of allergen-specific IgE antibodies using the indirect method of fluorescent antibodies. Food allergens of milk, chicken, protein and chicken yolk were used. As a result of examination in the serum of the patient, allergen-specific IgE antibodies were not detected, which excludes the allergic nature of the disease. In the study of serum by enzyme immunoassay, a negative result was also obtained.

 There are no contraindications for this research method.

 The positive effect of this technique is to reduce economic costs, reduce the reaction time, more specificity of the study compared to the prototype method, since, firstly, the allergen sensitized on red blood cells is a highly purified protein preparation, and secondly, significantly reduced the number of reagents in the reaction.

Claims (1)

 A method for diagnosing food allergies by the interaction of allergen-specific blood serum JgE antibodies with food allergens and labeled anti-JgE antibodies, characterized in that formalized and tanizated sheep erythrocytes sensitized with food protein antigens are used as food allergens, and labeled anti-JgE antibodies use a conjugate of anti-JgE antibodies with dye fluorescein isothiocyanate, the resulting complex is examined in a luminescent microscope and in the presence of a specific flu restsiruyuschego rim on the red blood cells, namely, the determination in the test serum anti-JgE-antibody titer of 1:40 or more are diagnosed with a food allergy.