How to detect sensitization to allergens

 

(57) Abstract:

The invention relates to medicine, namely to Allergology, and can be used to identify sensitization to allergens in adults and children. The essence of the method is an indirect method of fluorescent antibodies using test systems containing purified protein food, household, pollen, epidermal allergens, and labeled anti-IgE antibodies, which are used monoclonalny antibodies to human IgE labeled with dye - isothiocyanato fluorescein. The resulting complex is examined in a fluorescent microscope and in the presence of erythrocyte-specific fluorescent ring detect sensitization to the relevant allergen. The technical result is the detection of sensitization to a wide range of allergens and increasing the specificity of the reaction.

The invention relates to medicine, namely, Allergology, and can be used to identify sensitization to allergens (hypersensitivity) in adults and children.

In Allergology to detect hypersensitivity to food allergens using different serological tests, of which the most up and food allergies to milk, chicken meat, whole eggs, yolk and albumen of eggs in the indirect method, a fluorescent antibody method for the diagnosis of food Allergy. Patent 2140085, registered in the state register of inventions of the 20th of October 1999 ).

The disadvantages of the method:

1. The claimed set of allergens provides diagnosis allergies only to a limited number of food allergens.

2. Poor specificity of the indirect method of fluorescent antibodies (nmfa) due to the use of polyclonal fluorescent antibodies against immunoglobulin E man.

The invention is aimed at solving problems: detection of sensitization in humans to a wide range of allergens (food, household, epidermal, pollen), increasing the specificity of nmfa.

These objectives are achieved through the use of NMF, which differs from that used diagnostic tools - formalisation Anisimovna the sheep erythrocytes sensitized with purified protein food (buckwheat, beef, pork, chicken, hake, Mandarin), household (library dust, house dust, house dust mite), epidermal (feather pillows, cat's fur, wool dogs, LHA) allergens and fluorescent anti-IgE antibody is produced by conjugation of monoclonal antibodies (MAB) to the immunoglobulin E person with dye - isothiocyanato fluorescein (FITZ).

Preparation of fluorescent anti-IgE antibodies carried out by the following method: in the MCA to immunoglobulin E man with a protein concentration of 10 mg/ml) add a solution of FITZ. For this sample FITZ, taken at the rate of 1.5 mg per 100 mg of protein was dissolved in 0.15 ml of carbonate-bicarbonate buffer solution, pH of 9.0, and added dropwise into a solution of the ICA. Conjugation was carried out under continuous stirring for 18 hours at 2-8oC. after the time of conjugation labeled MCA freed from unreacted dye by gel-filtration on Sephadex G-50. In the resulting fluorescent anti-IgE antibodies determine the molar ratio FITZ/protein, protein quality, coloring titer and working dilution, assess the specificity of nmfa.

When setting nmfa with the serum of the patient is allergic to the diagnosticum is formed complex (allergen + specific IgE in human serum + fluorescent anti-IgE antibody), which reveal in the study in fluorescent microscope in the form of fluorescent is saved to the allergen.

The method is as follows. On smears of allergens (8 allergens on the same slide) put 5 µl of test serum in the whole breeding. Glass slides are placed in a humid chamber and incubated for 20 min at a temperature of 37oC. Upon expiration of the incubation of slides with smears out and washed in 0.9% sodium chloride solution with the addition of 0.01 M phosphate buffer, pH 7.4 (wash solution), twice for 7 min, then dried. All strokes cause fluorescent anti-IgE antibodies, taken in a working dilution, the volume of 5 ál.

Working dilution of fluorescent anti-IgE antibody is a concentrated 2 times coloring titer, i.e., the maximum dilution that can provide specific fluorescence, not lower than 3+ complex: diagnosticum + human sera containing IgE to this diagnosticum. For example, coloring the title (maximum breeding) fluorescent anti-IgE antibody is 1:8, then working dilution of the drug will be 1:4. Working dilution is determined by the manufacturer and is always indicated on the label of the vial.

Glass slide with dabs again placed in the moist chamber and incubated for 20 min is a thief. Glass slides are dried in air and viewed under oil immersion lens fluorescent microscope.

In the presence of serum allergen specific IgE antibodies to relevant allergens see specific fluorescence of the circle of red blood cells.

Evaluation of fluorescence intensity rim of red blood cells and the assessment of the degree of sensitization of the people to the allergen is carried out on chetyrehrazovoe scale:

4+ emerald green fluorescence of the rim around the erythrocyte (very high degree of sensitization);

3+ bright green fluorescence of the bezel around the erythrocyte (high sensitivity);

2+ - green fluorescence of the rim around the erythrocyte (average degree of sensitization);

1+ weak, barely visible fluorescence of the rim around the erythrocyte (low degree of sensitization);

the absence of fluorescence, (no sensitization).

Setting reaction must be accompanied by the following controls:

1. Obviously, the positive control: diagnosticum + standard serum to the allergen + fluorescent anti-IgE antibodies. Having 4+ - 3+ fluorescence.

2. Obviously negative is I.

3. The control conjugate: diagnosticum + fluorescent anti-IgE antibodies. The absence of fluorescence.

The duration of reaction 1 hour 30 minutes Examples of specific performance.

Example 1.

Sick,, 24.12.90 year of birth. During the treatment no complaints.

From the anamnesis: itchy skin, redness, scratching in the popliteal fossae, elbow after consuming citrus fruits, chicken eggs, sea fish. Notes repeated sneezing with watery discharge from nose, itchy eyes, tearing, bronchospasm after contact with a cat.

Inspection: satisfactory condition. Zev calm, the lymph nodes are not enlarged. The vesicular breathing, wheezing no.

Examination.

1. Spirography: moderate obstruction at the level of the large bronchi. The time of expiration (IV) normal. Test with bronchodilator (BD) is positive.

2. Index local eosinophilia (PMM) of 10.4.

3 Scratch skin test (CRU): test with a control fluid (TKJ) negative. , histamine 3+, whole egg (CCA) 3+, the yolk of chicken eggs (GCA) 3+, protein chicken eggs (CJD) 1+, cod 3+, the cat's fur (HQ) 3+.

4. Total IgE 340 ME/ml.

5. Specific IgE: cod 3+, Caina sensitization.

Bronchial asthma atopic, intermittently, remission.

Allergic rhinitis, intermittently, remission.

Allergic conjunctivitis, intermittently, remission.

Atopic dermatitis, limited remission.

Example 2.

Patient B. 3.11.87 year of birth. Complaints about sneezing, itching of nose, eyes, shortness of breath after a visit to the circus, zoo, contact with cat. During the inspection condition is satisfactory. Zev calm, the lymph nodes are not enlarged. Breathing hard, no wheezing. The colours coz clear, rhythmic. The abdomen is soft, painless.

Examination.

1. Spirography: on a moderate decrease in FVC moderate generalized obstruction. BB increased. The sample database is positive.

2. PMM 4,3.

3. Total IgE 285 ME/ ml.

4. Specific IgE: library dust (BP) 2+, HQ-1+.

5. UPC: TKJ the negative, histamine 2+, house dust (DP) 2+, BP 2+, 2 SHK+, dandruff horses 2+.

6. Intradermal tests (tests): TKJ the negative., histamine 3+, house dust mite (PIC) 2+, feather pillows (PP) 2+, HQ 2+, PD 2+, BP 2+.

7. nmfa: SHK 2+, PD 2+, KDP 2+.

Diagnosis: domestic, epidermal sensitization.

Bronchial Allergic conjunctivitis, intermittently, remission.

Example 3.

Patient P. 25.04.96 year of birth. Complaints against the attacks of difficult breathing, stuffy nose in the morning.

From the anamnesis it became known that in the spring and summer, twice was bronchoobstructive syndrome.

Inspection. The condition is satisfactory. Internal organs without deviation.

Examination.

1. PMM (worsening) of 20.6.

2. Total IgE 640 ME/ml (exacerbation).

4. Specific IgE: wheat flour (PM) 3+, buckwheat (ha) 4+, the tree pollen (AP )2+, SHK -0.

5. UPC: TKJ the negative., histamine 3+, dandruff horses 1+, PD 4+.

5. nmfa. SHK-0, PD 3+ PM 4+ CC 4+.

Diagnosis: food, pollen sensitization.

Bronchial asthma, atopic remission.

Allergic rhinitis, persistent, subramania.

Example 4.

Patient P. 17.10.95 year of birth. At the time of treatment no complaints.

From the anamnesis it is known that after drinking cow's milk, citrus, egg, fish are skin rashes in elbow and knee pits, accompanied by severe itching. In spring (may) marked the During the inspection. The condition is satisfactory. Skin dry, light pigmentation of the skin of the eyelids. The vesicular breathing, wheezing no. The colours coz clear, rhythmic.

Examination.

1. UPC: TKJ the negative., histamine 3+, Mandarin 1+, cow's milk (KM) 1+, MK 1+, beef (MG)1+, IKA 1+, CJD 1+ fish (hake) 1+ fescue 1+, wormwood 1+, hazel 1+.

2. Total IgE 956 ME/ml.

3. nmfa: KM 1+, CCA 1+, IKA 1+, CJD 1+, 1 MG+, MK 1+, hake 1+, Mandarin 1+.

Diagnosis: food, pollen sensitization.

Bronchial asthma atopic, intermittently, remission.

Allergic rhinitis, intermittently, remission.

Allergic conjunctivitis, intermittently, remission.

Atopic dermatitis, limited remission.

Example 5.

Patient O. 01.06.89 year of birth. He complained of skin rashes, nasal congestion, sneezing.

Inspection. The condition is satisfactory. Zev calm. The lymph nodes are not enlarged. On the skin all over body rash. Nasal breathing is difficult. In the lungs vesicular breathing, wheezing no. The colours cor clear, rhythmic. The abdomen is soft, painless.

Examination.

1. PMM 32,2 (PMM in remission 5,5.

2. Total IgE 1075 MCPD 2+, BP 2+, GD 3+.

Diagnosis: domestic, epidermal sensitization.

Allergic rhinitis, intermittently, aggravation. Strophulus, aggravation.

Example 6.

Patient P., 30.08.98 year of birth. At the time of treatment no complaints.

From the anamnesis it is known that in the spring appear itchy eyes and nose, watery eyes, sneezing multiple times with profuse watery discharge.

During the inspection. The condition is satisfactory. Zev calm. The lymph nodes are not enlarged. The vesicular breathing, wheezing no. The colours coz clear, rhythmic. The abdomen is soft, painless.

Examination.

1. PMM lower than the 5.37.

2. UPC: TKJ-, histamine 3+, DK 0, birch 0, castor 0, ovsyannitsa 0.

3. Total IgE 975 IU/ml

4. nmfa: SHK 0, DP 0, SC 0, KM 0, PD 0, CCA 0, GA 0, BI 0.

Diagnosis: acute respiratory infection. Reaction to allergens is missing.

Positive effect: method allows to detect sensitization to a wide range of allergens: domestic, epidermal, pollen, as well as to the extended range of food allergens that can improve the specificity of the reaction due to the use of fluorescent monoclonal antibodies against human IgE.


 

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