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Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb)

A technology of thyroglobulin and chemiluminescence immunity, which is applied in the direction of chemiluminescence/bioluminescence, analysis of materials through chemical reactions, analysis of materials, etc., can solve the problem of low titer, achieve high-throughput detection, and best performance parameter, the effect of a wide linear range

Inactive Publication Date: 2013-01-30
JIANGSU ZECEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As an autoantibody of thyroglobulin, TGAb has a high positive rate in autoimmune thyroid diseases (such as Hashimoto's thyroiditis and Graves' disease). For example, the frequency of thyroiditis is about 80%, and the positive rate of TGAB in Graves' disease is about 60% (Rosenbaum D and Davies TF., 1992; Nordyke RA et al. , 1993; Jean Ruf et al. , 1994), it can also be detected in the serum of other thyroid diseases and healthy people, but the titer is low

Method used

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  • Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Operating procedures for preparing magnetic bead buffer: Take the preparation of 1L as an example:

[0041] 1. According to the preparation amount, select a suitable container and add 700ml of pure water;

[0042] 2. Weigh 6.02g of Tris, NaN 3 0.99g in a container, mix well, and stir at room temperature for 2 hours;

[0043] 3. Check the pH of the solution, it should be around 10;

[0044] 4. Adjust the pH value to 8.0;

[0045] 5. Measure 2.76ml of Tween-20; weigh 0.99g of neomycin sulfate; 0.03g of tetracycline; 4.97g of BSA in a container, stir for 1 hour, and adjust the pH value to 8.0±0.05;

[0046] 6. Set the volume to 1L, adjust the pH value to 8.0±0.05;

[0047] 7. Filter with a 0.22um filter and store at 4°C.

[0048] 2. Preparation process of magnetic particle reagent

[0049] Activate ferric oxide microspheres with a diameter of 0.1 micron with glutaraldehyde, mix at room temperature for 4 hours, wash with 0.01mol / L PBS pH7.4 buffer solution three t...

Embodiment 2

[0051] 1. Coupling of alkaline phosphatase AP and human IgG antibody

[0052] Antibodies are immunoglobulins containing amino groups (-NH 2), using SMCC activator for activation to generate maleimide-immunoglobulin; maleimide-immunoglobulin contains maleimide groups that can react with -SH groups, adding alkaline phosphatase, which can link antibodies to alkaline phosphatase.

[0053] 2. Coupling of FITC with TG antigen

[0054] The conjugate of FITC molecule and TG antigen is labeled with the antibody by the common alkaline solution labeling method. When FITC is mixed with antigen in alkaline solution protein when reacting, protein In nature, the r amino group of lysine is combined with the sulfocarbamine bond of fluorescein to form FITC- protein Substance conjugates, that is, fluorescent antibodies or fluorescent conjugates. Generally, it can bind up to 15-20 molecules, and one IgG molecule can bind 2-8 molecules of FITC. The reaction formula is as follows: FITC-N=C...

Embodiment 3

[0056] Preparation of calibrators / quality controls:

[0057] 1. The highest point: the highest concentration point is X, and the concentration of the target point is A, B, C, D, E, F. When preparing a V-volume solution, the volumes of raw materials to be added are as follows: Table 1

[0058] concentration Add calibrator diluent volume Add X volume A V-A*V / X A*V / X B V-B*V / X B*V / X C V-C*V / X C*V / X D V-D*V / X D*V / X E V-E*V / X E*V / X F V-F*V / X F*V / X

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Abstract

The invention relates to a magnetic particle chemiluminescence immunoassay method for human thyroglobulin antibodies (TGAb) and belongs to the technical field of immunoassay. TG antigens marked by fluorescein isothiocyanate (FITC) and human IgG antibodies marked by alkaline phosphatase are combined to form an antigen-antibody-second enzyme-labeled antibody sandwich immune complex with a sandwich-like structure. Then, magnetic particles connected with FITC antibodies are added, the antigen-antibody complex is connected to the magnetic particles through specific combination of the FITC antibodies and the FITC and directly deposited in an external magnetic field, and the complex formed by immune reaction can be separated from the other non-combined substances without centrifuging. A kit combines chemiluminescence with the magnetic particles to provide a reaction system close to a homogeneous phase. Compared with the prior art, the kit has the advantages of higher sensitivity, wide linear range, quickness and the like; the cost of a product is greatly reduced; and the kit has a broad application prospect on the aspects of clinical examination and the like.

Description

technical field [0001] The invention belongs to the technical field of immune detection and analysis, and relates to a magnetic particle chemiluminescence immunoassay kit for detecting the content of human thyroglobulin antibody (TGAb) in serum and a test method thereof Background technique [0002] Thyroglobulin (TG) is a glycoprotein in thyroid follicular columnar cells with a molecular weight of 660KD and plays an important role in the synthesis and storage of thyroid hormones. In the body, thyroglobulin contains T3 and T4 in the form of colloid, which is stored in the follicle cavity. Under the action of enzymes, T3 and T4 are released into the cytoplasm and enter the blood circulation. At the same time, a small amount of TG is also released into the blood, and normal people can also detect TG with a concentration of 3-40ug / l. However, when the thyroid gland is damaged due to pathological reasons, a large amount of TG is released into the blood, and TG acts as an autoan...

Claims

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Application Information

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IPC IPC(8): G01N33/538G01N33/577G01N21/76
Inventor 汪丹丁建华
Owner JIANGSU ZECEN BIOTECH CO LTD
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