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Human IgE antibody detection kit, and making method and detection method thereof

A technology of antibody detection and kit, applied in the field of enzyme-linked immunoassay, which can solve the problems of insufficient sensitivity of ELISA detection method, inability to be widely used, and inaccurate results, so as to improve binding efficiency, facilitate popularization and application, and improve sensitivity Effect

Inactive Publication Date: 2013-06-26
北京北方北方有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Because the content of IgE antibodies in the human body is very low, it is easily interfered by IgG and other antibodies. The conventional ELISA detection method is not sensitive enough, and the detection accuracy is poor, especially when detecting low-value IgE antibodies, the results are even more inaccurate.
At present, imported kits occupy the top three positions in the domestic market, and imported IgE antibody diagnostic kits are prohibitive for many small and medium-sized hospitals due to their high price.
The sales in the Chinese market are only limited to some large hospitals, and cannot be widely used, especially because the country has been relying on imports for a long time, and foreign products occupy an absolute monopoly and control position in the domestic market

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the component of human IgE antibody detection kit

[0031] The components of the human IgE antibody detection kit include: coated microwell plate or its preparation materials, as well as sample diluent, quality control serum, concentrated cleaning solution, substrate solution, stop solution, and biotin-labeled anti-IgE Antibody Conjugate Solution, Horseradish Peroxidase-labeled Avidin Conjugate Solution.

[0032] Wherein: the coated microwell plate is a microwell plate coated with anti-human IgE antibody. Sample diluent is carrier protein or animal serum dissolved in phosphate buffer; quality control serum is positive serum dissolved in phosphate buffer; concentrated cleaning solution is Tween dissolved in phosphate buffer; substrate solution is four Methylbenzidine and hydrogen peroxide were dissolved in citric acid buffer; the stop solution was a solution containing sulfuric acid.

[0033] The components of the above kit also include standard serum, whic...

Embodiment 2

[0035] Embodiment 2: the preparation method of human IgE antibody detection kit

[0036] 1. Dissolve anti-human IgE antibody at 1μg / ml in 50mM carbonic acid (or phosphoric acid, acetic acid) buffer and mix well.

[0037] 2. Put them into the microwell plate in a certain order, 100μl per well, incubate overnight at 4°C, wash the plate with Tween dissolved in phosphate buffer for 3 times, and then add blocking solution (containing 2% BSA in phosphate buffer), 200 μl per well, and incubated overnight at 4°C.

[0038] 3. Discard the liquid in the well, dry the microporous plate overnight at room temperature, and seal the microporous plate in an aluminum foil bag after drying.

[0039] 4. Weigh 10g of BSA and dissolve it in 1 liter of 50mM phosphate buffer solution to prepare sample diluent, and pack in 50ml bottles.

[0040] 5. SATA (N-Succinimidyl-S-acetylthioacetate) modification of the labeled antibody. Weigh the anti-human IgE antibody and dissolve it in 0.1M phosphate buff...

Embodiment 3

[0052] Embodiment 3: the preparation method of human IgE antibody detection kit

[0053] Prepare human IgE antibody detection kit by the method of embodiment 2, difference is:

[0054] In step 5, the pH is 7.0, and the antibody concentration is 1 mg / ml. Weigh SATA and dissolve it in DMSO to make the concentration 5mg / ml. Add 10 μl of SATA solution to each milliliter of antibody solution, and react at room temperature for 30 minutes. Unreacted SATA solution was removed by dialysis.

[0055] In step 6, the HPDP-LC-biotin was weighed and dissolved in DMSO to make the concentration 1 mg / ml. Add 50 μl of HPDP-LC-biotin solution to each ml of SATA-modified antibody solution, and react at room temperature for 60 min. The unreacted HPDP-LC-biotin solution was removed by dialysis.

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Abstract

The invention discloses a human IgE antibody detection kit. The kit comprises a coated micro-porous plate or its making raw material, a sample diluent, a quality control serum, a concentrated cleaning solution, a substrate solution, a stopping solution, a biotin-labeled anti-IgE antibody binding solution, and a horseradish peroxidase-labeled avidin binding solution. The kit has a high sensitivity and a high specificity because of the adoption of a biotin-avidin amplification system. The invention also discloses a making method of the kit. The labeling efficiency can be improved through the SATA modification of the Fc end of the invariable region of an antibody and the connection of HPDP-LC-biotin and the modified antibody. The invention further discloses a detection method of the human IgE antibody. The human IgE antibody detection kit or the kit made through the making method has the advantages of low cost, simple operation, use convenience, and easy popularization application when the kit is used in detection.

Description

technical field [0001] The invention relates to the field of enzyme-linked immunoassay detection, in particular to a human IgE antibody detection kit, a preparation method and a detection method. Background technique [0002] Allergic disease, also known as allergic disease, refers to a specific immune response that occurs when the body responds to certain antigens for the first time and is stimulated by the same antigen again, mainly due to physiological dysfunction or tissue damage. In 1966, Ishizaka et al. first discovered and proved that IgE antibody is the main antibody mediating type I allergic reaction. Clinical allergy mainly refers to type I allergic reaction, the main mechanism of which is that allergen (allergen) can stimulate specific B cells to produce IgE antibody after entering the body, and the Fc segment of specific IgE antibody can interact with mast cells and basophils The binding of FcεRI on the cell membrane puts the body in a sensitized state. When th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/558
Inventor 刘兴旺
Owner 北京北方北方有限责任公司
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