Human carboxylesterases 2 detection kit and application method and application thereof

A human carboxylate and kit technology, applied in the field of medicine, can solve the problems of expensive antibodies, complicated and time-consuming processes, and achieve the effect of simple and easy synthesis process

Inactive Publication Date: 2016-12-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above methods have good specificity, they all require expensive antibodies, peptides or specialized instruments, and the process of sample preparation is complicated and time-consuming. More importantly, these methods all measure the protein content of hCE2 rather than its real activity

Method used

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  • Human carboxylesterases 2 detection kit and application method and application thereof
  • Human carboxylesterases 2 detection kit and application method and application thereof
  • Human carboxylesterases 2 detection kit and application method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Selectivity in Recombinant Expression of Human Monozyme

[0039] (1) Contains carboxylesterase 1b, carboxylesterase 1c, carboxylesterase 2, trypsin, pepsin, carbonic anhydrase, а-chymotrypsin (10μg / mL), acetylcholinesterase (5U / L) , butyrylcholinesterase (20U / L), paraoxonase 1, paraoxonase 2 (5μg / mL), human serum albumin (500μg / mL), bovine serum albumin (500μg / mL) Solution A 198μL, shake pre-incubation at 37℃ for 5 minutes;

[0040] (2) Add 2 μL of solution B (the final concentration of NCEN is 10 μM) to start the reaction, and incubate with shaking at 37 degrees;

[0041] (3) After 90 minutes, add 200 μL of solution C, shake vigorously, and terminate the reaction;

[0042] (4) Detect the substrate molecule NCEN (λ ex =354nm,λ em =452nm) and hydrolyzate (λ ex =430nm,λ em=542nm) at the fluorescence intensity value at the collection wavelength, calculate the ratio of the fluorescence intensity of the substrate to the product (see figure 2 ).

Embodiment 2

[0044] Drawing of Carboxylesterase 2 Quantitative Standard Curve

[0045] (1) Use 5 mg / mL standard solution of human carboxylesterase 2 (hCE2), first use solution A to dilute to 100 μg / mL, and then dilute to standard working solutions of different concentrations (0, 0.5, 1, 2, 3,4,5,6,7,8,9,10μg / mL), pre-incubated at 37°C for 5min;

[0046] (2) Add 2 μL of solution B (final concentration of NCEN is 10 μM) to each solution sample (198 μL), incubate with shaking at 37°C for 60 min, add 200 μL of solution C and shake vigorously for 15 seconds to terminate the reaction;

[0047] (3) Detect the probe molecule NCEN (λ ex =354nm,λ em =452nm) and the hydrolysis product NAH (λ ex =430nm,λ em =542nm) at the fluorescence intensity value at the collection wavelength place, the fluorescence intensity ratio of NCEN and NAH is carried out linear fitting graph to hCE2 concentration, establishes the quantitative standard curve of human carboxylesterase 2; Curve equation is Y=0.1653*X- 0.0...

Embodiment 3

[0049] Inhibition test of loperamide

[0050] (1) 198 μL each of hCE2 recombinant expression single enzyme (5 μg / mL), human liver microsomes (10 μg / mL), and human intestinal microsomes (10 μg / mL), were shaken and pre-incubated at 37 ° C for 5 minutes;

[0051] (2) Add 1 μL of loperamide, a positive inhibitor of carboxylesterase hCE2, to the reaction system at a final concentration (0-100 μM), and continue to incubate for 5 minutes;

[0052] (3) Add 2 μL of solution B (the final concentration of NCEN is 10 μM) to initiate the reaction;

[0053] (3) After 60 minutes, add 200 μL of solution C, shake vigorously, and terminate the reaction;

[0054] (4) Fluorescence detection (λ ex =354nm,λ em =452nm;λ ex =430nm,λ em =542nm); Calculate the fluorescence intensity ratio, according to the ratio of the fluorescence intensity ratio of each group at 542nm and 452nm to the ratio of the DMSO group to calculate the inhibitory intensity of hCE2 (see Figure 5 ).

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Abstract

The invention discloses a human carboxylesterases 2 detection kit and application method and application thereof. The kit comprises liquid A, liquid B, liquid C and a quality control standard substance, and the liquid A refers to a 100mM phosphate buffer solution with pH (potential of hydrogen) ranging from 5.5 to 10; the liquid B refers to a 0.1-10mM NCEN solution with an solvent of any optional one of methyl alcohol, acetonitrile or dimethyl sulfoxide; the liquid C refers to the acetonitrile or the methyl alcohol; the quality control standard substance refers to a 5mg / ml hCE2 solution. The kit is used for rapid quantitative detection of activity of the human carboxylesterases 2 and rapid screening and evaluation of enzyme inhibitors. The human carboxylesterases 2 detection kit and application method and application thereof have the advantages of simpleness in operation, low cost, flexibility and rapidness for per unit detection, and high-throughput detection can be achieved.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a kit for detecting human carboxylesterase 2 and its use method and application. Background technique [0002] Carboxylesterases (Carboxylesterases, CE) are one of the important members of the folded protein family of α / β hydrolase, widely distributed in the body. The enzyme is involved in the detoxification and metabolism of various drugs, prodrugs, environmental poisons and carcinogens, and can effectively catalyze the hydrolysis of exogenous substances such as carboxylates, carbamates, amides, and thioesters. In the human body, carboxylesterase mainly includes two isoforms, human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2), which have significant differences in tissue distribution and substrate specificity. hCE2 is highly expressed in human intestinal and tumor tissues, plays an important role in increasing the oral bioavailability of many prodr...

Claims

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Application Information

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IPC IPC(8): C12Q1/44
Inventor 杨凌崔京南金强葛广波冯磊王平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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