Method for detecting platelet factor-4 without depending on antibody

A platelet factor and antibody detection technology, applied in the biological field, can solve the problems of high antibody price, high price, and many factors affecting the test results, and achieve the effect of reliable test results and low price

Inactive Publication Date: 2010-04-21
ZHEJIANG CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method is more expensive, and the test results are affected by many factors
For example, the ELISA kits (96 persons) of BIOWIN Company of Canada and R&D Systems Inc of the United States need 2,800 RMB per box; since the ELISA method is inseparable from the antibody, and the quality of the antibody is affected by many factors, it will also affect the quality of the antibody. The test results of the method; and the price of the antibody is correspondingly expensive

Method used

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  • Method for detecting platelet factor-4 without depending on antibody
  • Method for detecting platelet factor-4 without depending on antibody
  • Method for detecting platelet factor-4 without depending on antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The specific steps of the method for detecting platelet factor-4 independently of antibodies of the present invention are as follows:

[0043]First, divide the selected magnetic beads (fluidMAG-ARA magnetic beads from Chemicell, USA) into polymerase chain reaction tubes (PCR tubes) according to 50ul / tube (the magnetic beads should be mixed evenly before dispensing), and PCR Store the tubes in a refrigerator at 4°C until use. Take a portion of the PCR tube packed with magnetic beads and place it on the magnetic processor, and remove the liquid in the PCR tube after 1-2 minutes. Take out the PCR tube from the magnetic processor, add 100 μL phosphate buffer saline (PBS) (8 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate to dissolve in water) to 1000 ml, the pH value is 7.3), mix well and place at room temperature for 3-5 minutes, then place the PCR tube on the magnetic processor, remove the liquid in th...

Embodiment 2

[0051] Example 2: Comparison of the elution effects of different eluents after fluidMAG-ARA magnetic beads capture platelet factor-4 in serum.

[0052] Adopt 4 kinds of eluents in this embodiment (the eluent that is 5% acetic acid by volume percentage respectively, the eluent that contains 50% acetonitrile and 0.5% trifluoroacetic acid by volume percentage, the eluent that contains 1% by volume percentage The eluent of trifluoroacetic acid and the eluent of 0.1 mol / L glycine) were compared for elution effect. In this embodiment, except that the eluent is different, other detection steps and conditions are the same as in Embodiment 1. The comparison of the elution effects of different eluents after fluidMAG-ARA magnetic beads capture platelet factor-4 in serum can be obtained by figure 2 It can be seen that the result shows that the volume percentage is 50% acetonitrile and 0.5% trifluoroacetic acid, the volume percentage is 1% trifluoroacetic acid, and the three eluents of 0...

Embodiment 3

[0053] Example 3: Comparison of the effects of magnetic beads with different diameters on capturing platelet factor-4 in serum.

[0054] In this embodiment, three kinds of magnetic beads with different diameters are used. The diameters of the magnetic beads are 50nm, 200nm and 1um respectively, and the surfaces of the magnetic beads are marked with -COOH functional groups. Except that the magnetic beads are different, other detection steps and conditions of this embodiment are the same as the specific steps of the method for detecting platelet factor-4 without relying on antibodies in Example 1. The effect of magnetic beads with different diameters on capturing platelet factor-4 in serum can be compared by image 3 It can be seen that the results show that the smaller the diameter of the magnetic beads labeled with the same group, the better the enrichment effect of platelet factor-4 in serum. image 3 The highest protein peak is 7769m / z protein peak.

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Abstract

The invention discloses a method for detecting platelet factor-4 without depending on antibody, which belongs to the technical field of biology. The method comprises the following steps: split-charging magnetic beads labeled with -NH2 or -COOH functional groups into a PCR tube, and then placing the PCR tube on a magnetic processor and removing liquid in the tube; taking the PCR tube out, then adding buffer solution into the PCR tube, and mixing the mixture evenly; placing the PCR tube on the magnetic processor and removing the buffer solution in the tube; taking the PCR tube out, adding serum or blood plasma sample into the PCR tube, and mixing the mixture evenly and then standing the mixture; placing the PCR tube on the magnetic processor and removing the liquid in the tube; taking the PCR tube out, adding the buffer solution into the PCR tube, and mixing the mixture evenly; placing the PCR tube on the magnetic processor and removing the buffer solution in the PCR tube; taking the PCR tube out, adding eluent into the PCR tube, and mixing the mixture evenly and then standing the mixture; placing the PCR tube on the magnetic processor and removing the liquid in the tube; and mixing the sucked liquid and saturated sinapic acid solution evenly, sucking the mixed solution to a target of matrix-assisted laser resolution ionization mass spectrometry, and putting the target into a mass spectrometer to detect the target after the solution on the target is crystallized. The method has the advantages of low price and reliable detection result.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular, the invention relates to a method for detecting platelet factor-4. Background technique [0002] Platelet factor-4 (Platelet factor 4, PF4) belongs to the chemokine a family, stored in platelet a granules, and has a high affinity with heparin. It has a wide range of biological functions: chemotaxis to monocytes and granulocytes; can inhibit angiogenesis and tumor growth; immune activation; regulate hematopoietic cell function; reduce the sensitivity of hematopoietic cells to chemotherapeutic drugs; Cell survival and induce their differentiation into macrophages and dendritic cells (DC); promote lipopolysaccharide (LPS) activated monocyte tissue factor (TF) activity, etc. Specifically, Heparin-induced thrombocytopenia (HIT) is caused by platelet activation of anti-platelet factor-4 and heparin complex antibodies. This process is different from other immune responses, and postop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64G01N1/28G01N1/38
Inventor 郑智国毛伟敏邓海腾凌志强牟瀚舟
Owner ZHEJIANG CANCER HOSPITAL
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