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Protein chip and kit for detecting abnormal DCP (Des--Carboxy-Prothrombin) in serum and preparation method thereof

A protein chip and carboxythrombin technology, applied in the field of protein detection, can solve the problems of difficulty in improving the detection sensitivity and specificity, inability to make a high-throughput chip, and large detection spot size, etc., to ensure the uniformity of spotting , the effect of reducing the amount of blood samples and antibodies, and the precise volume

Inactive Publication Date: 2018-02-23
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low-cost, fast, efficient, accurate, and high-throughput detection methods are the best choices, but there are currently no reports on high-throughput detection of DCP. Although the common Elisa method achieves sensitivity and specificity of 78.95% and 85.42%, it must Blood collection is 3-5ml, and the use of such a large amount of serum in high-throughput detection requires a correspondingly large amount of capture antibodies. The size of the detection spot is bound to be very large, which cannot be made into an integrated high-throughput chip, and the cost is very high. Popularity is not realistic
The content of DCP in serum is very low. It is difficult to improve the detection sensitivity and specificity if the amount of serum and antibody is reduced to make a high-throughput chip. The inventor speculates that this is the reason why there is no protein chip for detecting DCP at present.

Method used

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  • Protein chip and kit for detecting abnormal DCP (Des--Carboxy-Prothrombin) in serum and preparation method thereof
  • Protein chip and kit for detecting abnormal DCP (Des--Carboxy-Prothrombin) in serum and preparation method thereof
  • Protein chip and kit for detecting abnormal DCP (Des--Carboxy-Prothrombin) in serum and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Protein chip preparation and use process

[0047] Reagents and instruments used in the experiment: DCP antibody (Fuji Rebio Co., Ltd., Japan); aldehyde-based chip (Shanghai Biopro Company); HRP-labeled rabbit-derived antibody (Fitzgerald Company, USA); chemiluminescence scanner. (GE Corporation of America)

[0048] 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

[0049] PBST formula: PBS, 1L+Tween-20, 1ml

[0050] 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.

[0051] In each detection grid, spot the mouse DCP antibody on the chip in turn, spot four times, spot concentration DCP antibody 4mg / ml, spot four spots in a row; 10% bovine serum albumin (BSA) as negative For ...

Embodiment 2

[0059] Example 2 sample detection inspection result

[0060] Serum samples:

[0061] 8 liver cancer sera: from the specimen bank of You'an Hospital Affiliated to Capital Medical University, 1 normal healthy person's serum;

[0062] 1 blank control (blank control is 1×PBS).

[0063] The test results of this chip:

[0064] Abnormal prothrombin was not detected in the blank control and healthy control: indicating that the chip used in this experiment is effective. There is no abnormal prothrombin in normal people, and no prothrombin can be detected in healthy serum. It shows that the false positive detected by the chip and method provided by the present invention is 0, and the detection result can accurately distinguish liver cancer serum from normal serum.

[0065] The detection spots of 8 liver cancer sera were positive, indicating that there were abnormal decarboxylated prothrombin in 8 liver cancer sera, especially in patients with advanced liver cancer, and the detection...

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Abstract

The invention relates to a protein chip and a kit for detecting abnormal DCP (Des--Carboxy-Prothrombin) in serum and a preparation method thereof, and belongs to the technology of protein detection. The protein chip for detecting the abnormal DCP in the serum is characterized in that substrate slide glass of the protein chip at least comprises a detecting subarea, and one detecting subarea detectsone serum sample; a detecting spot area and a control spot area are formed in the detecting subarea; the detecting spot area has detecting spots formed by fixing a DCP specific antibody; the controlspot area has control spots formed by fixing bovine serum albumin; the concentrations of the substances on all detecting spots in the same detecting spot area are the same; the total amount of the DCPspecific antibody fixed on each of the detecting spot is 3nl; each detecting spot is formed by spraying 10 times, and 300pl is spotted each time. The protein chip and the kit disclosed by the invention can accurately detect the abnormal DCP; in clinical use, the protein chip and the kit have the advantages of high sensitivity, time conservation, convenience and quickness, economy and the like.

Description

technical field [0001] The invention relates to protein detection technology, in particular to a protein chip for detecting abnormal decarboxylated prothrombin in serum, a kit and a preparation method thereof. Background technique [0002] Des-r-carboxy-prothrombin (Des--carboxy-prothrombin, DCP) is an abnormal prothrombin produced by hepatocellular carcinoma. Compared with normal prothrombin, the molecular structure of DCP is characterized by its alanine domain ( One or more glutamic acid (Glu) residues in the Gla domain) are not fully carboxylated into Gla, thus losing the coagulation function. Normal prothrombin is in the microsomes of liver cells, with the participation of γ-glutamyl carboxylase, coenzyme and Vitamin Kreductase, which mainly depend on vitamin K, and the 6, 7, 14, 16, 19, 20 in the structure Gla Domain , 10 Glu residues at positions 25, 26, 29 and 32 are carboxylated to Gla to become an active prothrombin. Incomplete carboxylation of any or more of the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893C12Q1/56G01N33/521G01N33/543G01N33/86G01N2333/974G01N33/54386G01N33/6854G01N33/566G01N33/582G01N33/57438
Inventor 李宁张爱英王升启柯杨
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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