Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid

A protein chip and chemiluminescence technology, applied in the field of protein detection, can solve the problems of increasing the complexity of operations, high technical requirements of the i30 detection system, and limited popularization and application, so as to reduce the detection cost and expense, reduce the amount of blood samples and antibodies, The effect of improving detection efficiency

Pending Publication Date: 2015-06-03
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, plant lectin affinity immunoelectrophoresis technology and The i30 detection system has high technical requirements, cumbersome operation, and expensive reagents, which limit

Method used

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  • Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid
  • Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid
  • Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Protein chip preparation and use process

[0059] Reagents and instruments used in the experiment: mouse monoclonal antibody AFP (Shenzhen Faipeng Company); lentilin (Sigma Company); aldehyde-based chip (Shanghai Biopro Company); biotin-labeled rabbit-derived primary antibody (Abcam Company, USA) ; HRP-labeled avidin (abcam, USA), chemiluminescence scanner. (Developed by the laboratory of Professor Wang Shengqi, Academy of Military Medical Sciences)

[0060] PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g,

[0061] Potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH to 7.4, constant volume 1L

[0062] PBST formula: PBS, 1L+Tween-20, 1ml

[0063] The chip is an aldehyde-based chip (Shanghai BioPro Company), each chip contains 10 detection grids (detection sub-areas), each grid detects one serum, and 10 serums are tested at a time.

[0064] In each detection grid, mouse-derived monoclona...

Embodiment 2

[0072] Embodiment 2. Establishment of the detection method of the present invention

[0073] (1) Standard curve and regression equation a

[0074] Using the purchased AFP antigen (abcam company in the United States), set to different concentration gradients, (1-5) 80ng / ml, 40ng / ml, 20ng / ml, 10ng / ml, 5ng / ml, 6. Liver cancer serum, 7. Liver cancer serum, 8 blank controls, 9 healthy serum, 10 liver cancer serum ( image 3 , the chip antibody spotting antibody is AFP 2mg / ml, 1mg / ml, 0.5mg / ml, 0.25mg / ml).

[0075] The operation process and protein chip in Example 1 are used to detect each concentration gradient of AFP standard substance, and the detection scan results are shown in image 3 . The test results are drawn as a standard curve, with the concentration of the standard substance as the abscissa and the pixel value as the ordinate, and the standard curve is drawn on the coordinate paper. Find the corresponding concentration from the standard curve according to the pixel ...

Embodiment 3

[0082] Embodiment 3 sample detection checks the stability of the inventive method, accuracy and reliability

[0083] Serum samples:

[0084] 39 liver cancer sera: from the specimen bank of You'an Hospital Affiliated to Capital Medical University;

[0085] 32 normal healthy human serum;

[0086] 9 blank controls (blank control is 1×PBS).

[0087] The detection process is the same as in Example 1.

[0088] Substitute the sample's pixel values ​​into Figure 4 The regression equation a calculates the AFP concentration of the sample, and then multiplies it by the dilution factor, which is the total AFP concentration of the sample. Substitute the sample's pixel values ​​into Figure 7 The regression equation b of the sample AFP-L3 concentration is calculated, and then multiplied by the dilution factor, which is the total concentration of AFP-L3 in the sample.

[0089] Each chip has 10 detection sub-areas, including healthy serum samples, liver cancer serum samples, and blank ...

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Abstract

The invention relates to a chemical luminescent protein chip, a kit and a detection method for detecting the fucose index of seroglycoid, and belongs to a protein detection technology. The chemical luminescent protein chip is characterized in that a substrate slide glass of the protein chip at least comprises a detection subregion, wherein one detection subregion is used for detecting one serum sample; two detection spot regions and one line of contrast spot regions are arranged in the detection subregion; a detection spot formed by a specific antibody of fixed alpha fetoprotein is arranged in one detection spot region, and a detection spot formed by fixed lens culinaris agglutinin is arranged in the other detection spot region; a contrast spot formed by fixed bovine serum albumin is arranged in each contrast spot region; and the concentrations of substances on all the detection spots in the same detection spot region are equal. The protein chip, the kit and the method provided by the invention can be used for accurately detecting the fucose index of the seroglycoid with high throughput, and have the advantages of high sensitivity, time saving, convenience, economy and the like in clinical application.

Description

technical field [0001] The invention relates to protein detection technology, in particular to a chemiluminescent protein chip and a detection method for detecting serum glycoprotein fucose index. Background technique [0002] The sugar chain structure of alpha fetoprotein (AFP) produced by primary liver cancer and benign liver diseases such as hepatitis and cirrhosis is very different. The sugar index is much higher. Fucose has the property of binding to lentilin. AFP can be divided into AFP-L1, AFP-L2 and AFP-L3 according to its (fucosyl) affinity for lentil lectin. Among them, AFP-L1 mainly comes from benign liver diseases, AFP-L2 mainly comes from pregnant women, and AFP-L3 is the fucose glycosylated form of alpha-fetoprotein, which mainly comes from HCC. In 2005, FDA officially approved AFP-L3 as one of the markers of primary liver cancer. AFP-L3 has high specificity and sensitivity in early diagnosis, differential diagnosis, curative effect evaluation and prognosis...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
CPCG01N21/76G01N33/57438G01N33/68G01N2800/50G01N2800/7028G01N2800/52G01N2800/085B01J19/0046G01N33/5308G01N2333/42B01J2219/00533B01J2219/00605B01J2219/00621B01J2219/00662B01J2219/00693B01J2219/00725G01N33/54366G01N2333/471G01N2440/38G01N33/54353G01N33/57488
Inventor 李宁张爱英王升启柯杨张永宏
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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