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Method for evaluating quality of blood samples

A blood sample and molecular technology, applied in chemical instruments and methods, biochemical equipment and methods, measurement devices, etc., can solve the problem that the catalytic activity and structural integrity of protease are easily affected by environmental factors, and the physiological state of the sample donor is unavoidable. It can be easily unified and standardized, easy to popularize, and moderately stable.

Active Publication Date: 2021-05-04
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, the molecular indicators tested by the above methods are all endogenous molecules in the blood sample, and their content is inevitably affected by the physiological state of the sample donor
Existing studies have shown that the catalytic activity and structural integrity of proteases are easily affected by environmental factors
When the blood sample is cryopreserved in a low temperature environment, the structure of the protease remains intact and its activity can be maintained for a long time; Degradation and other reasons, the fine catalytic structure of protease molecules is easily destroyed, leading to its inactivation

Method used

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  • Method for evaluating quality of blood samples
  • Method for evaluating quality of blood samples
  • Method for evaluating quality of blood samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Feasibility Analysis of Plasma Protease Activity Measurement by Addition of Graphene Oxide (GO) and Fluorescently Labeled Peptides. Proceed as follows:

[0049] 1) GO was prepared into a 5 mg / mL solution with pure water. The fluorescently labeled polypeptide is: a peptide segment DKSKLKKTETQEKNPLP (SEQ ID NO: 2) labeled at the N-terminus by the fluorescent group MCA. Fluorescence-labeled polypeptide was prepared into a 5 mg / mL solution with pH 7.4 phosphate buffer solution (PBS).

[0050] 2) Take 110 μL of plasma sample from a healthy person, add 2.3 μL of GO solution and 2.3 μL of fluorescent peptide solution. At the same time, take 110 μL of PBS, add 2.3 μL of GO solution and 2.3 μL of fluorescent peptide solution, as a negative control. The above solutions were all placed in a fluorescent 96-well plate.

[0051] 3) Immediately put the 96-well plate into a multifunctional microplate reader (Perkin Elmer Company, model EnSpire), incubate at 37°C, and start monitori...

Embodiment 2

[0055] To verify the response of protease activity in plasma to plasma sample storage temperature. Proceed as follows:

[0056] 1) Take five fresh plasma samples and mix equal volumes into one pooled plasma sample.

[0057] 2) Take three 1.5mL EP tubes, add 150μL of mixed plasma, and mark them as samples 1, 2, and 3 respectively. Sample No. 1 was placed in a constant temperature heater at 25°C; Sample No. 2 was placed in a refrigerator at 4°C; Sample No. 3 was placed in an ultra-low temperature refrigerator at -80°C. The storage time is 24 hours.

[0058] 3) GO was prepared into a 5 mg / mL solution with water. The fluorescently labeled polypeptide is: a peptide segment DKSKLKKTETQEKNPLP (SEQ ID NO: 2) labeled at the N-terminus by the fluorescent group MCA. Fluorescence-labeled polypeptide was prepared into a 5 mg / mL solution with pH 7.4 phosphate buffer solution (PBS).

[0059] 4) After the three plasma samples were stored for 24 hours, 3 μL of GO and fluorescent peptide s...

Embodiment 3

[0063] To verify the response of protease activity in plasma to plasma sample storage temperature and storage time. Proceed as follows:

[0064] 1) Take five fresh plasma samples and mix equal volumes into one pooled plasma sample.

[0065] 2) Take ten 1.5mL EP tubes, add 150μL of mixed plasma, and mark them as samples 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 respectively. Among them, samples No. 1-3 were placed in a constant temperature heater at 25°C; samples No. 4-6 were placed in a refrigerator at 4°C; samples No. 7-9 were placed in an ultra-low temperature refrigerator at -80°C. Storage time: samples 1, 4, and 7 were stored for 24 hours; samples 2, 5, and 8 were stored for 48 hours; samples 3, 6, and 9 were stored for 6 days. Sample No. 0 was directly tested in a fresh state without storage.

[0066] 3) GO was prepared into a 5 mg / mL solution with water. The fluorescently labeled polypeptide is: the peptide segment DKSKLKKTETQEKNPLP (SEQ ID NO: 2) labeled with the fluorescent g...

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Abstract

The invention provides polypeptide molecules as shown in SEQ ID NO: 1-4 and fluorescently-labeled polypeptides, and provides application of the polypeptide molecules and the fluorescently-labeled polypeptides. The invention also provides a method for evaluating the quality of blood samples. The activity of the protease in the blood samples is used as a quality parameter for evaluating the blood samples, and the polypeptide molecules or the fluorescently-labeled polypeptides are used as a protease substrate to detect the activity of the protease. The method is beneficial to improving the quality of blood samples applied in the fields of scientific research, clinical medicine, biological sample libraries and the like, thereby avoiding errors in laboratory or clinical medical data caused by reduction in sample quality.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a polypeptide molecule and a fluorescent marker thereof. The present invention also relates to a method for evaluating the quality of a blood sample through the polypeptide molecule, the method comprising adding a fluorescently labeled polypeptide and a fluorescent quencher to the sample, and obtaining the natural protease activity in the sample by detecting the continuous change of fluorescence within a certain period of time The technology also includes measuring the value of protease activity in the blood sample by this technology, comparing the difference between the measured value and the reference value of the standard fresh blood sample, and evaluating the quality of the tested blood sample. Background technique [0002] Plasma and serum samples are an important source of clinical information. Molecular indicators and biomarkers from blood samples are widel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K14/00C12Q1/37G01N21/64
CPCC07K7/08C07K14/001C12Q1/37G01N21/6428G01N2333/95G01N2021/6432
Inventor 梁锴李岩赵克力孙青
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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