Unlock instant, AI-driven research and patent intelligence for your innovation.

GSTs quantitative detection device and detection method and application thereof

A quantitative detection method and quantitative detection technology, applied in the biological field, can solve the problems of inability to represent and provide, achieve accurate and stable global detection, and realize the effect of global detection

Inactive Publication Date: 2020-05-22
SHENZHEN HUADA GENE INST
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are two commonly used methods for measuring GSTs, one is to use enzymatic means to measure GSTs activity, and use spectrophotometry to measure GST activity in rat liver lysates (Habdous M, Vincent-Viry M, Visvikis S, et al. al.Rapid spectrophotometric method for serum glutathione S-transferasesactivity.Clinica Chimica Acta 326,131-42(2002).); The other is immunochemical determination, using ELISA method to measure 121 cases of non-small cell lung cancer samples (Hida T, Kuwabara M, Ariyoshi Y, et al. Serum glutathione S-transferase-pi level as a tumor marker for non-smallcell lung cancer. Potential predictive value in chemotherapeutic response. Cancer 73, 1377-82(1994)), but these two commonly used The main disadvantages of the method for measuring GST are: first, when enzymatic means use spectrophotometry, the change of absorbance value at 340nm wavelength cannot represent the activity of GSTs in the sample to be tested, the main reason is that the active part of the measured serum GSTs comes from The non-enzymatic adducts of GSH and CDNB two substrates; the second is immunochemical determination, as far as ELISA is concerned, the main problems are due to antibody specificity, experimental repeatability and limited GSTs members being detected. come, and this method cannot provide consistent conclusions: whether plasma GSTs can be used as a standard for pathological diagnosis

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • GSTs quantitative detection device and detection method and application thereof
  • GSTs quantitative detection device and detection method and application thereof
  • GSTs quantitative detection device and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Preparation of 47 isotope-labeled GSTs family protein peptides

[0059] Forty-seven stable isotope-labeled GSTs family protein peptides with known quantitative information were synthesized.

[0060] Table 1

[0061]

[0062]

[0063]

[0064] Note: [+1] indicates that the detected peptide has natural deamination at this amino acid, so the molecular weight increases by 1;

[0065] Table 2

[0066]

[0067]

Embodiment 2

[0068] Example 2: Sample preparation of low-abundance GSTs in plasma

[0069] The low-abundance GSTs sample preparation method in the plasma comprises the following steps:

[0070] 1) Preparation of plasma samples

[0071] (1) Collect whole blood samples into 5mL vacuum EDTA-K2 blood collection tubes, slowly invert and mix well and let stand;

[0072] (2) Place the blood collection tube in a pre-cooled centrifuge, centrifuge at 2000g for 30min at 4°C, and separate the plasma;

[0073] (3) Carefully take out the blood collection tube and place it on ice to keep the temperature low, and observe whether the centrifuged sample has hemolysis. If the sample has hemolysis, the sample cannot be used for subsequent experiments;

[0074] (4) Carefully draw the upper layer of plasma and transfer it to a clean 1.5mL EP tube, and fill each tube with 600μL of plasma;

[0075] (5) The aliquoted plasma samples were frozen and stored at -80°C for later use.

[0076] 2) Affinity enrichment ...

Embodiment 3

[0104] Embodiment 3: the preparation of standard curve

[0105] Add 47 stable isotope-labeled GSTs family protein peptides with known quantitative information according to the concentration of Example 1 (wherein the concentration is the average concentration of the target peptide in the sample mixed with all samples when the standard curve is prepared) In the prepared mixed sample of plasma GSTs enzymatic hydrolysis, the obtained mixed sample was quantitatively detected by using the PRM mode to detect 47 stable isotope-labeled peptides and endogenous peptides in the sample. The specific detection conditions are as follows: after injection, the sample Under the condition of mobile phase A of 5 μL / min, the trap column was first enriched for 5 minutes, and then the peptides were eluted and separated according to the procedures in Table 3. The ions entering the mass spectrometer were detected in PRM mode. For basic PRM parameter settings, see Table 4 shows.

[0106] table 3

[0...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a GSTs quantitative detection device and a detection method and application thereof. The detection method comprises the following steps: (1) adding in-vitro synthesized isotope labeled GST family protein peptide fragments with quantitative information into a plasma GSTs enzymolysis mixed sample according to different proportions; (2) quantitatively detecting the isotope labeled GST family protein peptide fragments in the step (1) by adopting parallel reaction monitoring, drawing an absolute quantitative standard curve of a single peptide fragment, and calculating to obtain the average concentration of the corresponding target peptide fragment in the mixed sample; and (3) adding the isotope labeled GST family protein peptide fragments into a peptide fragment sampleof the to-be-detected plasma GSTs according to the average concentration of the corresponding peptide fragments, detecting, filtering, and calculating the concentration of the GSTs peptide fragments.The detection method comprises enriching a sample of the low-abundance GSTs, detecting the enriched GST superfamily members in the blood in a targeted manner and reflecting the concentration difference of the GSTs in the biological sample.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a quantitative detection device and its detection method and application, in particular to a GSTs quantitative detection device and its detection method and application, especially to a GSTs quantitative detection device and its detection method And in the diagnosis or treatment of tumor efficacy evaluation applications. Background technique [0002] Glutathione (GSH), as a scavenger for electrophilic substances and their reactive metabolites, is a host defense mechanism ubiquitous in the biological world. Glutathione Sulfur Transferases (GSTs) are a group of protein enzymes with various physiological functions. They convert active oncogenic metabolites into inactive metabolites by combining them with GSH, and are widely involved in cellular detoxification and antioxidant processes. . GSTs are considered as potential biomarkers for judging tumorigenesis, and different resea...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/89
CPCG01N30/02G01N30/06G01N30/89
Inventor 任艳梁莉媛林志龙李思奇张可人侯桂雪刘斯奇
Owner SHENZHEN HUADA GENE INST