Kit for detecting alpha-1,3Gal antigen and application thereof

A kit and antigen technology, applied in the field of kits for the detection of α-1,3Gal antigens, can solve the problems of unavailability, difficulty in popularization and use, confusion of immunotoxicity, etc., and achieve the effect of high sensitivity and specificity

Active Publication Date: 2014-08-13
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since all wild-type lower animals express α-Gal antigen, it is impossible to use wild-type lower animals to evaluate the immunotoxicity related to α-Gal antigen, and only baboons can be used as recipients to investigate α-Gal antigen-positive or allogeneic organs Immunotoxicity reaction, and this kind of animal is rare and difficult to popularize
Therefore, how to evaluate the immunotoxicity of animal-derived biological materials or xenogeneic organ tissues has become perplexing. The testing and regulatory agencies cannot conduct effective safety and quality monitoring because there are no methods and standards to base on, and it also limits the development and quality of such products. Industrialization development

Method used

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  • Kit for detecting alpha-1,3Gal antigen and application thereof
  • Kit for detecting alpha-1,3Gal antigen and application thereof
  • Kit for detecting alpha-1,3Gal antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: α-1,3Gal Antigen Detection Kit

[0031] The test kit of the present invention comprises:

[0032] 1. 96-well plate coated with solid-phase α-Gal antigen: coated with Gal1, 3Gal-BSA antigen (Dextra Laboratories, UK), coated buffer is 0.2M, pH9.5 carbonate buffer, coated The amount is 100μl (50ng / ml);

[0033] 2. Blocking solution: human serum albumin with a mass concentration of 1%;

[0034]3. Mouse M86 antibody: commercially available product (Enzo, ALX-801-090-1, α-Gal Epitope, mAb)

[0035] 4. Secondary antibody enzyme conjugate: horseradish peroxidase-labeled goat anti-mouse IgM antibody;

[0036] 5. TMB chromogenic solution: 1% TMB

[0037] 6. Chromogenic stop solution: 1mol / L concentrated sulfuric acid (diluted 10 times when used);

[0038] 7. Tissue lysate: directly use the commercially available product, Biyuntian "P0013B RIPA Lysis Solution (Strong)";

[0039] 8. Concentrated lotion: 5% Tween-20 (diluted 100 times when used);

[0040] 9. Sample...

Embodiment 2

[0043] The preparation of embodiment 2 kit

[0044] 1. Preparation of α-Gal antigen-coated ELISA plate: prepare Gal1, 3Gal-BSA (Dextra Laboratories, UK) solution, 50ng / ml, the buffer is pH9.5 carbonate buffer (0.2M); take 100μl Add to 96-well plate (Nunc.Rochester, NY), incubate overnight at 4°C; block with 1% human serum albumin (4°C, 2 hours), then wash with 0.05% Tween-20 / PBS Wash the plate three times, add 250 μl / well of washing solution, soak for 30 seconds, shake and wash three times with a microplate shaker, the first time is 5 minutes, and the last two times are 3 minutes; after removing the washing solution, place the coated plate in a biological safety cabinet Ventilate for 2 minutes to make it air-dry, seal the package together with a desiccant, and store at 4°C (use within one week).

[0045] 2. Preparation of other solutions:

[0046] 2.1 Sample diluent: phosphate buffer solution with pH 7.2 to 7.5;

[0047] 2.2 Concentrated lotion: prepare 5% Tween-20 concentr...

Embodiment 3

[0056] Embodiment 3: kit quality detection

[0057] 1. Check the coating effect: Dilute the M86 antibody (1:100 as the initial concentration) in a gradient manner, react with the solid phase antigen on the solid-phase antigen-coated plate, and then detect the OD, draw the regression curve equation, and analyze the coating effect. The recommended qualification criteria are: R 2 ≥0.95.

[0058] 2. Specific detection: respectively detect α-Gal antigen positive and α-Gal antigen negative reference products with the kit of Example 1

[0059] 2.1 Add 2mg each of α-Gal antigen positive reference substance and α-Gal antigen negative reference substance (lyophilized powder) into 1ml lysate for tissue lysis for 5-10min; centrifuge at 14000rpm, 4°C for 30min, and take the supernatant of the positive reference substance Perform serial doubling dilution to 5 concentrations for later use; take the supernatant of the negative reference product and use it directly.

[0060] 2.2. Preparatio...

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Abstract

The invention relates to a detection kit, specifically to a kit for detecting alpha-1,3Gal antigen and application thereof. According to the invention, a solid phase antigen coated plate is prepared from Gal1,3Gal-BSA antigen in advance, and then the antibody competitive ELISA inhibition method detection kit is prepared through combination with the specific antibody M86 of the alpha-1,3Gal antigen. The kit provided by the invention is applicable to qualitative and quantitative detection of alpha-Gal antigen of a variety of biomaterials, especially to detection of residual alpha-Gal in animal-derived biomaterials. The specificity of the kit reaches 100%, and the sensitivity of the kit is embodied by capacity of detection of rabbit blood corpuscles with a concentration of 3.9 * 10<4> / ml and Gal antigen in a Gal antigen positive reference with a concentration of 6.25 mu g / ml.

Description

technical field [0001] The invention relates to a detection kit, in particular to a detection kit for alpha-1,3Gal antigen and its application. Background technique [0002] Biogenic materials composed of mammalian extracellular matrix are widely used in surgical wound repair, tissue reconstruction, and tissue engineering scaffold materials due to their good biocompatibility. Xenogeneic organs have also become one of the potential ways to solve the serious shortage of donor organs in organ transplantation. However, the immunological problems brought about by the application of animal-derived biological materials or xenogeneic organ tissues to the human body directly affect the safety and effectiveness of the use of such materials. The biggest obstacle to xenotransplantation is the hyperacute immune rejection (Hyperacute rejection, HAR), which occurs within a few days after xenotransplantation. From minutes to several hours, the donor organ fails in a short period of time a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/78
CPCG01N33/6803
Inventor 徐丽明邵安良柯林楠单永强陆艳杨昭鹏
Owner NAT INST FOR FOOD & DRUG CONTROL
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