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Rapid-detection kit for alpha-Gal antigens, use method and application thereof

A detection kit and the technology of the kit, which are applied in the biological field, can solve the problems of inability to objectively count the number of antigens, difficult to detect, and inability to meet the needs of α-Gal antigen detection.

Inactive Publication Date: 2018-11-23
BEIJING SANYAO SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recognition of Gal antigen by GS-IB4 is not completely specific; in addition, the number of antigens contained in each cell is less than 5×10 4 One is difficult to detect, it cannot objectively reflect the number of antigens in the tissue, and cannot meet the needs of higher quality α-Gal antigen detection

Method used

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  • Rapid-detection kit for alpha-Gal antigens, use method and application thereof
  • Rapid-detection kit for alpha-Gal antigens, use method and application thereof
  • Rapid-detection kit for alpha-Gal antigens, use method and application thereof

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preparation example Construction

[0031] In the present invention, the coating concentration of the α-Gal antigen in the solid-phase α-Gal antigen coating well plate is preferably 0.5-2 μg / ml, more preferably 1 μg / ml; the solid-phase α-Gal antigen The preparation method of coated hole plate comprises the steps:

[0032] ① Add the α-Gal antigen solution to the well plate, shake at 20-37°C for 1-3 hours, and then overnight at 2-6°C;

[0033] ②The next day, block with serum albumin, and then incubate at 37±2°C for 1-2 hours;

[0034] ③Wash the plate 3-5 times with lotion, dry at 20-30°C, and store in a sealed container at 2-6°C.

[0035] In the present invention, the α-Gal antigen solution in step ① is preferably prepared with a carbonate buffer, the pH of the carbonate buffer is preferably 9-10, more preferably 9.5; the α-Gal antigen The concentration of the solution is preferably 0.5-2 μg / ml, more preferably 1 μg / ml; the amount of the α-Gal antigen solution added to the orifice plate is preferably 95-105 μl p...

Embodiment 1

[0070] A rapid detection kit for α-Gal antigen, comprising the following components:

[0071] 1) 96-well plate coated with solid-phase α-Gal antigen: coated with α-Gal antigen, the coating buffer is 0.2M, pH 9.5 carbonate buffer, and the coating amount is: the concentration is 1-2 μg / ml of α-Gal antigen solution, 100 μl per well;

[0072] 2) Anti-Gal antibody: mouse anti-Gal IgM antibody

[0073] 3) Secondary antibody enzyme conjugate: horseradish peroxidase-labeled goat anti-mouse IgM antibody;

[0074] 4) Chromogenic solution A: sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water to 500ml;

[0075] 5) Chromogenic solution B: 0.2g disodium edetate, 0.95g citric acid, 50ml glycerin, dissolve 0.15g TMB in 3ml DMSO, add distilled water to 500ml;

[0076] 6) Chromogenic stop solution: 10% concentrated sulfuric acid (volume ratio);

[0077] 7) Tissue lysate: directly use the commercially available product, Biyuntian "P0013B RIPA Lysis Solution...

Embodiment 2

[0085] A rapid detection kit for α-Gal antigen, the preparation method of which comprises:

[0086] 1) Preparation of α-Gal antigen-coated ELISA plate: prepare α-Gal antigen solution, 1-2 μg / ml, buffer solution is pH9.5 carbonate buffer (0.2M); take 100 μl into 96-well Plate (Nunc. Rochester, NY), incubate with shaking at room temperature for 2 hours, turn to 4°C overnight; use 1% human / bovine serum albumin to block (37°C, 2 hours), and then use 0.05% Tween-20 / Wash the plate with PBS washing solution for 3-5 times, add 200 μl / well of washing solution, soak for 30 s, shake and wash with a microplate shaker for 3-5 times, the first time is 5 minutes, and the second time is 3 minutes; after removing the washing solution, put The coated plate was placed in a biological safety cabinet and exposed to ventilation for 2 minutes to air-dry, sealed and packaged with a desiccant, and stored at 4°C.

[0087] 2) Preparation of other solutions:

[0088] Sample diluent: Phosphate buffer a...

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Abstract

The invention relates to the technical field of biology and particularly relates to a rapid-detection kit for alpha-Gal antigens, an use method and application thereof. The invention provides a rapid-detection kit for the alpha-Gal antigens. The kit comprises a solid-phase alpha-Gal antigen coating porous plate, an alpha-Gal antigen standard substance, anti-rat-Gal IgM antibodies, goat-anti-rat IgM antibodies labeled with horseradish peroxidase, a hydrogen-peroxide developing solution, a TMB (Tetramethyl Benzidine) developing solution, a developing stopping solution, a tissue lysis solution, phenylmethylsulfonyl fluoride, concentrated washing liquor, a sample diluting solution, a Gal antigen positive biological material reference and a Gal antigen negative biological material reference. The rapid-detection kit provided by the invention has the beneficial effects that the detecting specificity to the alpha-Gal antigens reaches 100%, and 31.25ng / ml Gal antigen standard substance (the lowest detection limit) can be detected by sensitivity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rapid detection kit for α-Gal antigen and its use method and application. technical background [0002] Xenotransplantation, as one of the potential ways to solve the serious shortage of donor organs in organ transplantation, has received extensive attention from the scientific community. Animal-derived biomaterials have been widely used in surgery for tissue repair, replacement, and trauma therapy. However, the main barrier directly affecting the safety and efficacy of the use of such materials is the issue of heteroimmunology. [0003] Scientific research has confirmed that α-galactosyl antigen (α-1,3-galactosyle, α-Gal) is the main target antigen of hyperacute immune rejection in xenotransplantation. α-Gal antigen is widely present in lower animals such as pigs, cattle, and horses, while humans, apes, and old century monkeys do not express α-Gal antigen. However, there are hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6854
Inventor 王金恒邵安良徐丽明吴勇
Owner BEIJING SANYAO SCI & TECH DEV
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