HU195580B - Method for detecting antibodies for thyroid antigens - Google Patents

Abstract

The present invention relates to a method for detecting antibodies against thyroid antigens from a patient's serum. The antigen fixed on the nitrocellulose filter is first incubated with test serum and then with a second antibody conjugated to peroxide gas specific for the first antibody. The color reaction after development refers to positivity. -1-

Description

The present invention relates to a method for the detection of antibodies against thyroid antigens.

In the diagnosis of thyroid diseases, the detection of antibodies against thyroid antigens is of diagnostic value, showing the activity and course of the diseases. The most commonly accepted and widely used methods for this purpose are radioimmunoassays. The disadvantage is that not all hospitals have an isotope laboratory where the test can be performed and the R1A kits used are expensive.

There are also known cheaper methods that can be performed in general, well-equipped laboratories. These include the detection of antibodies against thyroglobulin and microsomal antigen by passive haemagglutination. The sensitivity of these methods is low and difficult to standardize.

The aim is to develop a procedure that eliminates the above disadvantages and can be routinely used in hospital-clinical laboratories in the field of patient care. During my experiments, I discovered that one of the purified antigens of various thyroid antigens, such as TSH receptor, human thyroglobulin, thyroid microsome, was sprayed, fixed, dried and optimized to 100 micrograms depending on its purity. the other binding sites of the nitrocellulose filter are blocked with an indifferent protein, usually bovine serum albumin, to provide a filter suitable for diagnosing thyroid diseases.

The filter thus prepared is incubated with test serum or with serum dilutions. Serum diluted wood in the blocking solution can be titrated from 1: 10 to 1: 100,000. The excess unbound material is then rinsed.

Bound antibodies are peroxidase specific for human IgG or IgM. is detected with conjugated antihuman immunoglobulins and the reaction is finally eluted with hydrogen peroxide and a suitable dye, preferably 4-chloro-1-naphthol. In the case of specific antibodies present in the serum, the positivity of the reaction appears in the color of the substrate used on the white background in the area of the dripped antigen. A positive reaction is indicated by the appearance of a blue color on 4-chloro-1-naphthol.

If TSH receptor antigen is added to the filter, the presence of color is diagnostic for Basedow's disease in case of a positive reaction. Of particular importance is the ability to predict fetal Basedow disease in pregnant patients with Basedow's disease. In the clinical treatment of Basedow's disease, it is important to monitor the presence of the antibody for the purpose of discontinuation of treatment, choice of treatment, and detection of relapse.

If human thyroglobulin or thyroid microsomal antigen is added to the filter, the appearance of a color in a titre above 1: 100 serum dilution indicates chronic thyroid inflammation.

Advantage of the invention over the methods and methods used so far:

The test procedure does not require any special equipment, instruments, isotope laboratory.

The process is well reproducible.

Examination results allow the doctor to take quick and effective steps in the field of healing.

The method of the present invention also allows for individual assays, as opposed to a specific assay number of 50-100 RIA kits.

Errors due to storage of serum samples for different periods of time can be eliminated.

The process is concentration dependent, which results in the determination of the antibody titer and, optionally, disease activity.

The second reagent coupled to the peroxide can also determine the type of antibody: IgG or IgM.

Due to its sensitivity and specificity, the process is well suited for scientific and research work.

The following example illustrates the invention:

A 4 x 4 mm Millipore type nltrocellulose filter was washed with distilled water for 5 minutes and then dried at room temperature. Subsequently, 10 micrograms of thyroid TSH receptor antigen were added to the square.

The sputter on the filter is then stabilized by drying. The filter thus dried can be stored in a dry place and the reactivity of the antigen is not reduced.

The filter is then moistened in TRIS-Buffer-Saline (TBS), 50 mM TRIS-IICL, 200 milliliters NaCl, pH 7.4.

Blocking was done with a 5% solution of bovine serum albumin in TBS by applying 150 microliters of blocking solution to the color-facing filter and leaving for 15 minutes and drying the filter. Filters at this stage can be well stored for extended periods of time.

A first reaction is then performed in which the antibodies in the serum may bind to the antigen. The test solution is serum dilution 1: 10 or greater in blocking solution. 150 microliters of test solution was weighed onto the filter and incubated at room temperature with shaking. The incubation time is approx. 4 o'clock.

After suctioning the test page, the filter is washed with TBS-cl and the blocking is repeated.

Subsequently, a second reaction is performed in which the self-antibodies bound to the antigen are detected with a 1: 1000 diluted peroxidase-conjugated anti-human immunoglobulin specific for human IgG in 150 microliters of blocking solution.

After incubation for 2 hours, the solution is aspirated and the filter is washed with TBS.

The filtrate obtained above was developed with a solution of 4-chloro-1-naphthol and H ₂ O ₂ . The developing solution is 3 mg / ml 4-chloro-1-naphthol in methanolic alcohol solution, + 0,01% H ₂ O ₂ . The appearance of a blue color indicates that the patient's serum contains an antibody against the TSH receptor and the patient is suffering from Basedow's disease.

Claims (6)

PATENT CLAIMS A method for detecting antibodies to thyroid antigens, wherein the purified thyroid antigen is fixed at 0.1-10 micrograms or a complex antigen at 1-100 micrograms by drying on a nitrocellulose filter and blocking the other binding sites of the filter, followed by incubation with the patient's serum. the antibodies thus bound are detected with specific peroxidase-conjugated anti-human immunoglobulin and the reaction is induced by the addition of hydrogen peroxide solution and dye. A process according to claim 1, characterized in that: 195,580 that the TSH receptor, or human Ihyreoglobulin, or human thyroid microsome fraction antigen is conveniently attached to the nithiocelhilose filter. 3. The method of claim 1, wherein the antigen-free surface of the filter is blocked with an indifferent protein, preferably bovine serum albumin. 4. The method of claim 1, wherein the incubation is performed with a 1: ΙΟΙ: 10,000-fold dilution of patient serum at 20-24 ° C for 2 to 4 hours. 5. The method of claim 1, wherein the detection is human IgG or IgM 5 specific peroxidase-conjugated anti-human immunoglobulins are used. 6. Process according to claim 1, characterized in that 4-chloro-1-naphthol is suitably used as the dye for the reaction.