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Ki67 and p16<INK4a> detection kit based on glucan signal amplification

A detection kit and signal amplification technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex process flow and long operation time, achieve high detection sensitivity, improve signal strength, and avoid non-specific signal interference Effect

Inactive Publication Date: 2020-04-28
江苏美克医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently widely used double-staining kit uses the immunoassay method of primary antibody-secondary antibody, which has complicated process and long operation time.

Method used

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  • Ki67 and p16&lt;INK4a&gt; detection kit based on glucan signal amplification
  • Ki67 and p16&lt;INK4a&gt; detection kit based on glucan signal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The main steps of immunohistochemical experiment of ki-67 / p16 double staining detection kit are as follows:

[0020] 1. Select TCT-positive cervical brush samples, centrifuge them, fix them with 95% ethanol for 30 minutes, and dry them after antigen retrieval;

[0021] 2. Add ki-67 and p16 dropwise INK4a For antibodies, wash 5 times with PBS after incubating at 37°C for 1 hour;

[0022] 3. Add the HRP secondary antibody and AP secondary antibody of the corresponding species dropwise, incubate at room temperature for 30 minutes, and wash 5 times with PBS;

[0023] 4. Use DAB chromogenic agent ABC solution to add pure water to configure brown chromogenic agent, develop color at room temperature for 6 minutes, then rinse with pure water 5 times;

[0024] 5. Use AP chromogenic agent ABC solution to configure red chromogenic agent, develop color at room temperature for 30 minutes, then rinse with pure water 5 times;

[0025] 6. After counterstaining with hematoxylin, moun...

Embodiment 2

[0027] Dextran signaling amplifies ki-67 / p16 INK4a Double-staining detection kit immunohistochemical experiment, the steps are as follows:

[0028] 1. The preparation method of ki-67 dextran poly AP-labeled antibody is as follows: dextran activation, AP enzyme reagent activation, polymer ki-67 antibody coupling, washing and storage. The preparation method of the ki-67 dextran poly AP labeled antibody is as follows: dissolve dextran (DeX) in 20 mM phosphate buffered saline (PBS) with pH=7.0, add appropriate amount of sodium periodate (NaIO 4 ) was oxidized, dialyzed overnight with the same PBS as the DeX matrix, water-soluble alkaline phosphatase (AP), and an appropriate amount of NaIO was added 4 Oxidation, use ethylene glycol to terminate the reaction, and dialyze overnight; mix and react the oxidized DeX, ki-67 antibody and activated AP, dialyze overnight, and dilute to the working solution with the preservation solution;

[0029] 2.p16 INK4a The preparation method of dex...

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Abstract

The invention discloses a ki67 and p16<INK4a> detection kit based on glucan signal amplification. The kit comprises a ki-67 glucan poly-AP labeled antibody, a p16<INK4a> glucan poly-HRP labeled antibody, a DAB color developing solution and an AP color developing solution. According to the kit disclosed by the invention, an enzyme-labeled poly-primary antibody is used for immunodetection; specificconditions for preparing a glucan polyantibody are disclosed; and meanwhile, glucan molecules serve as a supporting framework, the glucan polyantibody is formed by a composite antibody and signal markers, the large number of signal markers are carried so that signal intensity is greatly improved, a use concentration of the antibody is reduced, and non-specific signal interference is avoided. Meanwhile, a macromolecular compound and enzyme are combined at multiple sites so that stable support is provided for natural three-dimensional conformation of the enzyme, stability of the enzyme and the antibody is greatly maintained, and efficient bonding between macromolecules is realized. According to the kit, a use amount of the antibodies is greatly reduced, an operation process and an operationtime are shortened, and the kit has higher color development efficiency and detection sensitivity.

Description

technical field [0001] The invention relates to the field of biochemical detection, in particular to a dextran-based signal amplification method for ki-67 and p16 INK4a Double stain detection kit. Background technique [0002] In the immunological detection method based on the basic principle of antibody-antigen reaction, the strength of the signal carried by the bound antibody is the focus of attention during the detection process. Generally, enzyme oxidation color development, chemiluminescent substances, colored markers, and fluorescent markers are used as signal sources. Differences in the type and quantity of antibody-bound signal sources directly affect the sensitivity of signal detection. [0003] At present, the incidence of cervical cancer ranks first in the malignant tumors of the female reproductive system in China, and there are about 100,000 new cases of cervical cancer every year. Cervical cancer is one of the most common malignant tumors in gynecology. Early...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/541G01N33/535
CPCG01N33/541G01N33/535
Inventor 李文成汪骏青孙康俊黄宝福
Owner 江苏美克医学技术有限公司
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