A paper-based enzyme-linked immunosorbent assay based on covalent bonding to immobilize capture antibodies

A technology of capturing antibodies and immunoadsorption, applied in the field of immunoassay, can solve the problems of low sensitivity of covalent bonding technology, many steps of covalent bonding technology, complicated operation, etc., to avoid the formation of cross-linked network and the amount of reagent The effect of few, accurate quantitative results

Active Publication Date: 2022-04-22
XI AN JIAOTONG UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a paper-based enzyme-linked immunosorbent assay based on covalently bonded immobilized capture antibodies for detecting the content of allergy-related MRGPRX2 receptors in human blood, which can Solve the problems of many steps, complex operation, high cost and low sensitivity of covalent bonding technology in existing covalent bonding technology

Method used

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  • A paper-based enzyme-linked immunosorbent assay based on covalent bonding to immobilize capture antibodies
  • A paper-based enzyme-linked immunosorbent assay based on covalent bonding to immobilize capture antibodies
  • A paper-based enzyme-linked immunosorbent assay based on covalent bonding to immobilize capture antibodies

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Experimental program
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Effect test

Embodiment 1

[0050] The steps to detect MRGPRX2 content in human blood by paper-based ELISA dual antibody sandwich method based on covalent bonding legally fixed antibodies are as follows:

[0051] (1) Filter paper wax sealing: use a 6mm inner diameter punch and a small hammer to knock out a series of 6mm inner diameter holes in the wax paper, cover the wax paper with the hole on the filter paper; put the steel plate into a box-type resistance furnace at 300 ° C for 5 min, take out and press it on the wax paper for 5 min to prepare the wax sealed filter paper.

[0052] (2) Fixation of capture antibodies: aspirate 600 μM of BSA 25 μL, 100 μM pH 6.0 MES 50 μL, 100 μM of EDC 60 μL and 40 μg / mL of medulumab 25 μL in a centrifuge tube, take 5 μL drops on the wax-sealed filter paper holes, and dry at room temperature.

[0053] (3) Washing: Add 10 μL of PBST to the filter paper well after drying, and dry at room temperature after three times.

[0054] (4) Closed: Add 10 μL of 8% BSA per well to block ...

Embodiment 2

[0062] The steps to detect MRGPRX2 content in human blood by paper-based ELISA dual antibody sandwich method based on covalent bonding legally fixed antibodies are as follows:

[0063] (1) Filter paper wax sealing: use an 8mm bore punch and a small hammer to knock out a series of 8mm inner diameter holes in the wax paper, and cover the wax paper with the hole on the filter paper; put the steel plate into a box-type resistance furnace at 300 ° C for 5 min, take it out and press it on the wax paper for 5 min to prepare the wax sealed filter paper.

[0064] (2) Fixation of capture antibodies: aspirate 600 μM of BSA 50 μL, triple distilled water 80 μL, 100 μM of EDC 40 μL, 100 μM NHS 40 μL and 30 μg / mL of murine monoclonal antibody 50 μL in a centrifuge tube, take 8 μL drops on the wax-sealed filter paper holes, and dry at room temperature.

[0065] (3) Washing: Add 10 μL of PBST to the filter paper well after drying, and dry at room temperature after three times.

[0066] (4) Closed:...

Embodiment 3

[0074] The steps to detect MRGPRX2 content in human blood by paper-based ELISA dual antibody sandwich method based on covalent bonding legally fixed antibodies are as follows:

[0075] (1) Filter paper wax sealing: use a 10mm bore punch and a small hammer to knock out a series of 10mm inner diameter holes in the wax paper, and cover the wax paper with the hole on the filter paper; put the steel plate into a box-type resistance furnace at 300 ° C for 5 min, take it out and press it on the wax paper for 5 min to prepare the wax sealed filter paper.

[0076] (2) Fixation of capture antibodies: aspirate 300 μM of BSA 25 μL, 100 μM pH 6.0 PBS 50 μL, 100 μM NHS 50 μL and 10 μg / mL of murine monoclonal antibody 25 μL in a centrifuge tube, take 10 μL drops on the wax-sealed filter paper holes, and dry at room temperature.

[0077] (3) Washing: Add 10 μL of PBST to the filter paper well after drying, and dry at room temperature after three times.

[0078] (4) Blocking: Add 20 μL of 5% milk ...

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Abstract

The invention discloses a paper-based double-antibody sandwich method based on a covalent bonding method to fix and capture antibodies, belongs to the technical field of immune analysis, and is used for detecting the content of allergy-like related MRGPRX2 receptors in human blood. Using MRGPRX2 mouse monoclonal antibody as the capture antibody, without the need to chemically modify the surface of the filter paper, by introducing bovine serum albumin (BSA) molecules with both carboxyl and amino functional groups, using the amide condensation reaction between the antibody molecule and the amino and carboxyl groups in the BSA molecule The antibody-BSA network structure was formed to realize the immobilization of the capture antibody; the MRGPRX2 rabbit polyclonal antibody labeled with horseradish peroxidase HRP was used as the detection antibody, and the MRGPRX2 solution was used as the standard to establish a paper-based double-antibody sandwich method for MRGPRX2. The immobilization method of the capture antibody is simple, efficient, and low in cost. The technical indicators of the detection method are high in detection throughput, fast in speed, detection limit and precision, etc., and the accuracy and reproducibility of the quantitative results are good. It is suitable for For clinical examination and blood epidemiological investigation.

Description

Technical field [0001] The present invention belongs to the field of immunoassay technology, relates to a paper-based enzyme-linked immunosorbent method, in particular a covalent bond legally fixed capture antibody detection of human blood allergy-related MRGPRPRX2 receptor content of paper-based ELISA double antibody sandwich method. Background [0002] Enzyme-linked immunosorbent assay (ELISA) is a technology that uses the specific reaction of antigen antibodies to adsorb a known antigen or antibody on the surface of a solid-phase carrier, captures the analyte by a specific reaction, and then reacts with the enzyme-labeled antigen or antibody by the analyte, and then quantitatively detects the analyte by the color reaction generated by the enzyme and the substrate. This technology can be used to detect macromolecular antigens and specific antibodies, etc., with the advantages of rapidity, sensitivity, and simplicity, and can be used not only for clinical examination, but also ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/58G01N33/543
CPCG01N33/74G01N33/581G01N33/577G01N33/54393G01N2333/726G01N2800/24
Inventor 孔利云贺浪冲张涛韩省力王楠丁园园
Owner XI AN JIAOTONG UNIV
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