New kit for diagnosing hashimoto's disease (the fifth principle of elisa)

Abstract

The idea of the patent includes a new principle, which is the detection of immune complexes using ELISA technology, which is a new and modern principle that is added to the four principles of ELISA, the new principle (the fifth ELISA) depends on detection of immune complexes, which consist of two different parts of proteins (an antigen - an antigen). This complex can be detected through this new principle, and it was applied to the immune complex (thyroid-stimulating hormone receptors and antibodies formed against it) Hashimoto's patients. It was considered an example of applying this theory, the immune complex was actually detected, its concentration determined, and compared to other techniques. A standard solution specially designed for this purpose was used to prove this theory. In this patent, a new kit is specifically designed to detect these immune complexes. Immune complex, the group is considered successful. In terms of use.

Description

[0001] Description

[0002] New kit for diagnosing Hashimoto's disease (The Fifth Principle of ELISA) Technical Field

[0003] The present invention relates to the manufacture of a new test kit used to diagnose Hashimoto's disease and other autoimmune diseases, such as the Rheumatoid Factor (RF), by detecting the disease's immune complex (Antibody- Antigen).

[0004] Background Art

[0005] One of the most common problems that accompanies the diagnosis and differentiation between types of diseases is Hypothyroidism, which may be due to low iodine, damage to the gland, decreased secretion of the gland’s stimulating hormone, or it may be caused by Hashimoto's disease, which is the body’ s secretion of antibodies to the receptors for thyroid-stimulating hormone (TSHR). It is one of the diseases for which there is no specific confirmed examination for the disease, as the doctor relies on a set of clinical symptoms of the patient, a set of general tests, and an ultrasound of the thyroid gland. And there was a need to detect this disease through a single confirmatory examination, and the challenge in diagnosing this disease lies in detecting special antibodies that the body produces for the thyroid stimulating receptors (TSHR), but in a special way because there are two types of antibodies that the body may produce that cause one of the diseases, as for. Hashimoto's or Craves' disease. However, each antagonist differs in its binding to the thyroid stimulating receptor. If the antagonist binds to the TSHR accidentally, this binding causes the receptor to stop working and the secretion of thyroid hormone to decrease due to its covering of the TSH receptors and the development of Hashimoto’s disease. However, if the antigen binds vertically, it stimulates the receptor for the thyroid hormone, causing it to stimulate the thyroid gland and cause an exaggerated secretion of the hormone. In this case, the person develops Craves’ disease.

[0006] There are four basic types of ELISA technology, allowing a good deal of flexibility that can be modified based on the antibodies available, the results required, or the complexity of the samples:

[0007] 1. Direct ELISA

[0008] 2. Indirect ELISA

[0009] 3. ELISA Sandwich

[0010] 4. Competition ELISA

[0011] Summary of Invention

[0012] The new kit designed to detect the immune complex (thyroid-stimulating hormone receptor and its antagonist) consists of the following components:

[0013] 1- Extracted and purified thyroid-stimulating hormone receptor (from the American company Mybiosaurus), which is encoded with the letter S, at a concentration of 10 pl / mg.

[0014] 2- Purified thyroid-stimulating hormone receptor antibodies (from the American company Mybiosaurus), which are coded with the letter A, at a concentration of 10 pl / ml.

[0015] 3- The base material, tetramethylbenzidine TMB, is used at a concentration of 100 pl / ml.

[0016] 4- HRP horseradish peroxidase enzyme used at a concentration of 100 pl / ml

[0017] Technical Problem There are four basic types of ELISA technology, which are as follows:

[0018] 1. Direct ELISA

[0019] 2. Indirect ELISA

[0020] 3. ELISA Sandwich

[0021] 4. Competition ELISA

[0022] The lack of a technique through which to detect immune complexes, as the four principles of ELISA detect either the antigen or the antibody. Therefore, the need arose to design a new principle (a fifth principle for ELISA) that detects an immune complex formed inside the body.

[0023] Solution to Problem

[0024] Manufacturing a new kit for ELISA with a new technology that represents the fifth principle of ELISA, through which an immune complex (Ab-Ag) is detected for the first time. In the previous four principles, either an antigen or an antibody is detected. As for the new principle, an immune complex that is formed is detected. Of both antigen and antigen, that is, it detects the immune complexes that occur inside the human body due to infection with a specific disease. The example to which this kit was applied is the detection of immune complexes for a disease. Hashimoto's.

[0025] Advantageous Effects of Invention

[0026] Through this principle, the immune complexes that cause many immune diseases are accurately investigated. Which is considered a diagnostic kit for all tests that treat hepatic complex diseases, such as:

[0027] Rheumatoid Factor (RF), Hashimoto's thyroiditis, Graves' disease (Anti TSHR) and other diseases. Brief Description of Drawings

[0028] Figure No. (1) shows the new principle of ELISA (the fifth ELISA), where the plate pits are covered with antibodies (TSHR-Ab) specialized to bind to antigens that are TSHR, which represent the first part of the immune complex (TSHR Ab-TSHR Ag), and bind The conjugated antibodies are antibodies and an enzyme (HRP), which is specialized in binding to TSHR-Ab antibodies, which represent the second part of the antibody. The immune complex, and thus this principle explains the detection of the immune complex (Ag-Ab) that is formed inside the human body for Hashimoto’ s disease (Anti-Thyroid Stimulating Hormone Receptor-Thyroid-Stimulating Hormone Receptor Antigen).

[0029] Description of Embodiments

[0030] Preparing the examination kit (manufacturing a new examination kit):

[0031] □ 200 microliters of S are mixed with 200 microliters of A within 30 minutes at 37 degrees Celsius to form the standard complex, which is considered the standard solution for concentrations of the same complex inside the human body. Thus we create the positive control analyzer

[0032] □ Antibodies A are fixed to the base of the pits by adding 100 microliters of this antibody at a concentration of 10 pl / ml and incubating for 24 hours, after which the pits are washed with buffer phosphate solution once to get rid of the remaining unbound antibodies.

[0033] 1- Add 100 pl of the serum of patients who have been confirmed and diagnosed with Hashimoto's to the For pre-prepared well plate ELISA, and in which the antibodies have been fixed, and 100 pl of the standard solution prepared previously in the previous step is added, as well as 100 pl of the sera of healthy people as a negative control. Three models are used to test the quality of the new kit, which are as follows: A- Serum from infected patients previously diagnosed by doctors, which represents the examination sample

[0034] B- Serum from healthy people who have been diagnosed as safe by specialized doctors and represent a negative control.

[0035] C- The positive control analyzer, which was previously manufactured during the stages of preparing the test kit. The concentration of this solution is 200 pL / mg and is diluted with 5 half dilutions to obtain concentrations of 100 - 50 - 25 - 12.56.25. This is an approved international protocol.

[0036] 2- Incubate for one hour at 37°C, then wash the holes 2 times

[0037] 3- Placement of the HRP enzyme coupled to the antibody

[0038] 4- The holes are washed twice .

[0039] 5- Add the base material and incubate it for half an hour

[0040] 6- Add the stopping solution

[0041] 7- Reading etching using a wavelength of 450 nm.

[0042] Results:

[0043] The results obtained by using the new kit were: the sensitivity of the new kit was 97.2%, while the specificity was 98.1%, which is considered a very high percentage for diagnosing the immune complex (TSHR Ab-TSHR Ag), and the kit is considered successful in terms of use. The special equation was used to extract the value of specialization and sensitivity, as follows:

[0044] Sensitivity = TP / (TP + FN)

[0045] Specificity for manufacturing tools Specificity = TN / (TN + FP) *TP(true positive) represents the true positive value

[0046] *FP(false positive) represents a false positive

[0047] *FN (false Negative) represents false negative

[0048] *TN (True Negative) represents true negative value

[0049] Example:

[0050] Using technology to detect immune complexes (Ab-Ag), as there is no technology through which to detect immune complexes that form within the human body. Through this principle, the immune complexes that cause Hashimoto’ s disease are investigated through the detection of Anti-Thyroid Stimulating Hormone. Receptor-Thyroid-Stimulating Hormone Receptor Antigen), and this technique can also be used for many other immune diseases.

[0051] Deposited Biological Material

[0052] - Examples of immune complexes are Thyroid Stimulating Hormone Receptor (TSHR-Ab), Thyroid Stimulating Hormone Receptor (TSHR-Ag).

[0053] - Tetramethylbenzidine (TMB)

[0054] -Stop solution (HCL)

Claims

Claims1- New kit for diagnosing Hashimoto's disease (The Fifth Principle of ELISA) 2- 2- A new kit according to claim No. (1) Consisting of a standard immune complex preparation at a concentration of (10 pl / ml) and a volume of 100 microliters, through which the real immune complexes inside the human body were measured.3- A new kit according to claim No. (1) Consists of a drill bit inside which an anti-thyroid-stimulating hormone is fixed, while the coupling solution contains an enzyme loaded with an anti-thyroid- stimulating hormone, and this is the fundamental difference between the new principle and the Sandwich principle for ELISA.4- A new kit according to claim No. (1) Obtained results, where the sensitivity (Sensitivity) of the new kit was 97.2%, while the specificity (Specificity) was 98.1%, which is considered a very high percentage for diagnosing the immune complex (TSHR Ab-TSHR Ag) and the kit is considered Successful in terms of use.

Images