Method for liquid chromatograp (LC) detection of cathepsin G in serum

A technology of cathepsin and liquid chromatography, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of expensive detection kits and achieve the effects of short detection time, high detection sensitivity and strong specificity

Inactive Publication Date: 2019-11-05
缪荣明
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, polyclonal or monoclonal antibody-coated microplates are used to capture cathepsin in specimens, and then double-sandwich EIJSA with enzyme-labeled anti-cathepsin antibody is used to detect the content of cathepsin in serum. The detection kit is expensive and urgently needed Development of new assays for cathepsins

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A method for detecting cathepsin G in serum by liquid chromatography, comprising the steps of:

[0022] 1) Preparation of the test sample: Centrifuge the blood sample at 3000~5000r / min for 10~20min to get the serum, add 15mg iodoacetamide and 150mg NH to 1mL serum 4 HCO 3 Mix well, avoid light for 45 minutes and mix thoroughly, then elute with 1.0mol / L sodium chloride solution and acetonitrile in sequence, and collect the eluate;

[0023] 2) Preparation of reference substance: Take 1 mg of cathepsin G standard substance, weigh it accurately, put it in a 50ml volumetric flask, dissolve it with mobile phase and dilute to the mark, shake well, accurately measure 10ml, put it in a 50ml volumetric flask, and use mobile phase Dilute to the mark and shake well;

[0024] 3) Determination: Accurately measure 20 μl each of the reference solution and the test solution, inject them into a high-performance liquid chromatograph, record the chromatogram, and calculate the content of...

Embodiment 2

[0027] Embodiment 2 precision experiment

[0028] The same sample (accurately prepared sample containing 80 µg / mL cathepsin G) was divided into 6 times for the same treatment and injection, and the results were measured. The results are shown in Table 1, and the precision is good.

[0029] Table 1

[0030] serial number 1 2 3 4 5 6 Concentration µg / mL 80.6 79.1 79.4 80.9 79.7 81.2

Embodiment 3

[0031] Embodiment 3 recovery rate experiment

[0032] Prepare spiked plasma samples with low, middle and high concentrations of 40, 100, and 200 µg / mL, 6 copies of each concentration, and measure according to the sample pretreatment and analysis method, and measure continuously for 3 days to calculate the average recovery rate and precision. As a result, the recoveries of high, medium and low concentration samples were 95.43%-103.66%, the intra-assay precision was 2.8%-5.5%, and the inter-assay precision was 1.8%-4.6%. The results are shown in Table 2. All conform to the requirements of "Guidelines for the Development of Analytical Methods for Biological Materials".

[0033] Table 2 The average recovery and relative standard deviation of the method (n=6), %

[0034] Fortified at 40 µg / mL Spike 100 µg / mL Spike 200µg / mL Recovery 95. 43 98. 29 103.66 RSD (within batch) 4.7 2.8 5.5 RSD (between batches) 4.6 1.8 4.5

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Abstract

The invention discloses a method for LC detection of concentration of cathepsin G in serum, which comprises steps of centrifuging a blood sample at a rate of 3000 - 5000r / min for 10 -20min to obtain serum, adding iodoacetamide inside for mixing with NH4HCO3, shading the mixture from light for 45min for full mixing, eluting the mixture sequentially with a sodium chloride solution and acetonitrile,and collecting the eluent; taking a cathepsin G standard substance, and dissolving the cathepsin G standard substance with a mobile phase for preparation into a solution; accurately measuring 10-20 microliters of each of a reference solution and a test solution, injecting the solutions into a high-performance liquid chromatograph, recording a chromatogram, and calculating the content of cathepsinG in serum according to an external standard method by peak area. The invention adopts LC detection of concentration of cathepsin G for the first time, and removes influence of other complex components in plasma on a detection result by simply processing a plasma sample. The processed sample can be directly injected and detected. Therefore, the detection sensitivity is high, the detection time isshort, the repeatability is good, and the method is simple, convenient, accurate, good in repeatability and strong in specificity.

Description

technical field [0001] The invention belongs to the field of protein detection, and in particular relates to a method for detecting cathepsin G in serum by liquid chromatography. Background technique [0002] Cathepsin is widely distributed and is a member of the cysteine ​​protein family. It is closely related to many serious diseases, such as malignant tumors and arthritis. Cathepsin G, found in the azurophilic granules of neutrophil polymorphonuclear leukocytes, encodes a protease with similar specificity to chymotrypsin C and may be involved in the digestion of pathogenic bacteria after phagocytosis, and at sites of connective tissue inflammation remodeling. The concentration of cathepsin G in the serum of silicosis patients increased relative to that of tuberculosis and lung cancer, but the trend in the serum of tuberculosis and lung cancer was basically the same. The expression changes of cathepsin G in silicosis and the latter two groups of diseases suggest that it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 缪荣明
Owner 缪荣明
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