Chemiluminescent protein chip, kit and detection method for detecting serum glycoprotein fucose index

一种化学发光试剂、蛋白芯片的技术，应用在检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片及检测领域，能够解决增加操作的繁琐性、i30检测系统技术要求高、限制推广应用等问题，达到降低检测成本和费用、血样及抗体量减少、提高检测效率的效果

Inactive Publication Date: 2018-02-13
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 its popularization and application
However, the glycosyl capture spin column increases the complexity of the operation because the sample processing and detection are carried out separately.

Method used

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  • Chemiluminescent protein chip, kit and detection method for detecting serum glycoprotein fucose index
  • Chemiluminescent protein chip, kit and detection method for detecting serum glycoprotein fucose index
  • Chemiluminescent protein chip, kit and detection method for detecting serum glycoprotein fucose index

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) ; Avidin HRP (US abcam company), chemiluminescent 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, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH to 7.4, constant volume 1L

[0061] PBST formula: PBS, 1L+Tween-20, 1ml

[0062] 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.

[0063] In each detection grid, mouse-derived monoclonal antibody A...

Embodiment 2

[0071] Embodiment 2. Establishment of the detection method of the present invention

[0072] (1) Standard curve and regression equation a.

[0073] 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 A: 1mg / ml; B: 0.5mg / ml; C: 0.25mg / ml).

[0074] 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 pixe...

Embodiment 3

[0080] Embodiment 3. Stability, accuracy and reliability of sample detection test method of the present invention

[0081] Serum samples:

[0082] 39 liver cancer sera: from the specimen bank of You'an Hospital Affiliated to Capital Medical University;

[0083] 32 normal healthy human serum;

[0084] 9 blank controls (blank control is 1×PBS).

[0085] The detection process is the same as in Example 1.

[0086] 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.

[0087] Each chip has 10 detection sub-areas, including healthy serum samples, liver cancer serum samples, and ...

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Abstract

The invention relates to a chemiluminescence protein chip, a kit and a detection method for detecting serum glycoprotein fucose index, belonging to protein detection technology. It is characterized in that: the matrix slide of the protein chip includes at least one detection sub-area, and one detection sub-area detects one serum sample; two detection spot areas and one row control spot area are set in the detection sub-area , wherein one detection spot area has a detection spot formed by a specific antibody immobilized to alpha-fetoprotein, the other detection spot area has a detection spot formed by immobilizing lentilin, and the control spot area has a control spot formed by immobilizing bovine serum albumin ; The concentration of substances on all detection spots in the same detection spot area is the same. The protein chip, kit and method of the present invention can accurately and high-throughput detect the fucose index of serum glycoproteins, and have the advantages of high sensitivity, time saving, convenience and economy in clinical use.

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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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
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
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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