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A kind of elisa reaction method based on nitrocellulose membrane

A nitrocellulose membrane and reaction technology, applied in the field of immunobiology, can solve the problems of increasing the enzyme-labeled secondary antibody, increasing, and increasing the background value of the enzyme-labeled secondary antibody, so as to achieve a sufficient antigen-antibody reaction and improve the detection signal strength. Effect

Active Publication Date: 2015-08-26
上海昌润生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the shaking speed of the antigen-antibody incubation reaction is 100rpm. Under this speed condition, the antigen-antibody reaction is not sufficient, so it is necessary to increase the amount of enzyme-labeled secondary antibody to improve the detection signal value, which not only increases the cost of raw materials but also increases the enzyme The amount of labeled secondary antibody will bring some negative effects such as increasing the background value

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Take the protein chip of a tumor marker monoclonal antibody AFP primary antibody sample spotted on the nitrocellulose membrane, and add 100ul of AFP antigen of concentration A and batch number B to the reaction well;

[0030] (2) Place on a shaker (model: HDYC-2004C) at 37°C, rotating speed: 150rpm, shake and incubate for 30min;

[0031] (3) wash 4 times with washing liquid;

[0032] (4) Add 100ul of enzyme-labeled secondary antibody AFP-HRP of concentration C and batch number D to the reaction well;

[0033] (5) Place on a shaker at 37°C, rotating speed: 150rpm, shake and incubate for 30min;

[0034] (6) Wash 4 times with washing liquid;

[0035] (7) Add 20ul of chemiluminescent substrate to the reaction well and let stand for 1min;

[0036] (8) Place the chip in a chip reader to read the optical signal value.

Embodiment 2

[0038] (1) Take the protein chip of a tumor marker monoclonal antibody AFP primary antibody sample spotted on the nitrocellulose membrane, and add 100ul of AFP antigen of concentration A and batch number B to the reaction well;

[0039] (2) Place on a shaker (model: HDYC-2004C) at 37°C, rotating speed: 200rpm, shake and incubate for 30min;

[0040] (3) wash 4 times with washing liquid;

[0041] (4) Add 100ul of enzyme-labeled secondary antibody AFP-HRP of concentration C and batch number D to the reaction well;

[0042] (5) Place on a shaker at 37°C, rotating speed: 200rpm, shake and incubate for 30min;

[0043] (6) Wash 4 times with washing liquid;

[0044] (7) Add 20ul of chemiluminescent substrate to the reaction well and let stand for 1min;

[0045] (8) Place the chip in a chip reader to read the optical signal value.

Embodiment 3

[0047] (1) Take the protein chip of a tumor marker monoclonal antibody AFP primary antibody sample spotted on the nitrocellulose membrane, and add 100ul of AFP antigen of concentration A and batch number B to the reaction well;

[0048] (2) Place on a shaker (model: HDYC-2004C) at 37°C, rotating speed: 250rpm, shake and incubate for 30min;

[0049] (3) wash 4 times with washing liquid;

[0050] (4) Add 100ul of enzyme-labeled secondary antibody AFP-HRP of concentration C and batch number D to the reaction well;

[0051] (5) Place on a shaker at 37°C, rotating speed: 250rpm, shake and incubate for 30min;

[0052] (6) Wash 4 times with washing liquid;

[0053] (7) Add 20ul of chemiluminescent substrate to the reaction well and let stand for 1min;

[0054] (8) Place the chip in a chip reader to read the optical signal value.

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Abstract

The invention discloses an ELISA reaction method based on a cellulose nitrate membrane. Specifically, the method comprises the following steps of (1) taking a protein chip which is applied by a first antibody on the cellulose nitrate membrane, adding a to-be-detected sample; (2) putting the protein chip with the to-be-detected sample on a shaker with a temperature of 37 DEG C and a rotate speed of 150-350 rpm, oscillation-incubating for 30 min; (3) washing the chip; (4) adding an enzyme-labeled second antibody; (5) putting the protein chip with the to-be-detected sample on a shaker with a temperature of 37 DEG C and a ratate speed of 150-350 rpm, oscillation-incubating for 30 min; (6) washing the chip; (7) adding a chemiluminescent substrate; and (8) detecting optical signal intensity with a chip reading instrument. An effect of thorough reaction of antigen and antibody is achieved by increasing the rotate speed of the shaker in the incubating reaction of the antigen and antibody, appearing in that chip experiment detection results show that signals are increased significantly after the rotate speed of the shaker during the incubating reaction of the antigen and antibody is improved.

Description

technical field [0001] The invention relates to the field of immunobiological technology, in particular to a reaction method for forming a protein chip based on immobilizing a special protein on a nitrocellulose membrane. Background technique [0002] The basic principle of protein chip technology is to orderly immobilize various proteins on various carriers such as titer plates, filter membranes and glass slides to become detection chips. ELISA is the abbreviation of Enzyme-Linked Immunosorbnent Assay. It is an immunoenzyme technique developed after immunofluorescence and radioimmunoassay techniques. Since the advent of this technology in the early 1970s, it has developed very rapidly and has been widely used in many fields of biology and medicine. [0003] The double-antibody sandwich method, which is the most commonly used ELISA for detecting antigens, is suitable for detecting multivalent antigens with at least two antigenic determinants in the molecule. Its basic wor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/544
Inventor 曹友洪张秋平赵雷姚涌
Owner 上海昌润生物科技有限公司
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