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Stabilized cholinesterase substrate solution

A technology of cholinesterase and substrate solution, applied in the field of determining cholinesterase activity and determining the solution field of cholinesterase activity, can solve problems such as inability to obtain, low efficiency, time-consuming and the like

Inactive Publication Date: 2007-06-13
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, this is only possible by reconstitution of BTC particles, which is complex and time-consuming
[0015] No reagents are available in the prior art which allow the storage of solubilized cholinesterase substrates such as BTC in the absence of any enzymes for longer periods of time without increasing the autohydrolytic activity
Detection of cholinesterase activity in parallel larger sets of samples would be complex and inefficient due to the need to provide freshly dissolved substrate to minimize reagent blank reactions in cholinesterase assays

Method used

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  • Stabilized cholinesterase substrate solution
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  • Stabilized cholinesterase substrate solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Determining cholinesterase activity

[0050] Cholinesterase (CHE) activity was determined based on the formation and detection of thiocholine according to the following reaction scheme:

[0051]

[0052] 2 Thiocholine Dithiobis(choline)

[0053] +2OH - +[Fe(CN) 6 ] 3- ——→+H 2 O+2[Fe(CN) 6 ] 4-

[0054] Thiocholine is specifically released from butyrylthiocholine depending on cholinesterase activity. Thiocholine rapidly reduces yellow hexacyanoferrate III to nearly colorless hexacyanoferrate II. The reduction in extinction can be measured spectrophotometrically at a wavelength of 405 nm and a temperature of 37°C. By the molar absorption coefficient of potassium hexacyanoferrate III (92.7+ / -0.4m 2 / mol) to calculate the concentration of cholinesterase.

[0055] Specifically, the cholinesterase activity was determined as follows:

[0056] Two reagent solutions, R1 and R2, are freshly prepared:

[0057] R1: 91.5mM pyrophosphate

[0058] 2.44mM potassiu...

Embodiment 2

[0102] Evaluation of the Stability of Butyrylthiocholine (BTC) Using Different Polar Organic Solvents

[0103]Insufficient stabilization of BTC can be seen in the variation of reagent aging results. To assess BTC stability, the catalytic concentration of cholinesterase obtained from a temperature-controlled substrate solution (at 35°C for 18-21 days) was compared with the catalytic concentration of cholinesterase obtained from a freshly prepared substrate solution. concentration for comparison.

[0104] Measurements were performed on a Roche / Hitachi 917 analyzer similar to the procedure described in Example 1.

[0105] The above "aging model" is suitable for rapid evaluation of different cholinesterase substrate solutions, because the reagents at 35°C for 18-21 days are equivalent to the reagents stored under normal conditions of refrigeration for 15-18 months.

[0106] Using this "aging model", the stability of different cholinesterase substrate solutions - / + certain polar ...

Embodiment 3

[0121] Determination of Limiting Concentrations of Different Polar Organic Solvents for the Stability of Butyrylthiocholine (BTC)

[0122] Based on the "aging model" described in Example 2, the minimum concentration of polar organic solvent (in volume percentage).

[0123] Table 2: Stability of butyrylthiocholine (BTC)

[0124] And determine the limit concentration of different polar organic solvents.

[0125] ethanol

[0126] Considering the criteria for stabilizing the cholinesterase substrate solution, i.e. the intercept is about less than + / -300 U / l and the slope is less than + / -3%, the lowest concentration with a stabilizing effect is 1% (vol. %), 2% (vol%) for THF and isopropanol, respectively.

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Abstract

The present invention provides a stabilized cholinesterase substrate solution, wherein at least one substrate is stabilized by at least one species of a polar organic solvent. The present invention also relates to the use of a stabilized cholinesterase substrate solution, wherein at least one substrate is stabilized by at least one species of a polar organic solvent. Also within the scope of the present invention is a stabilized cholinesterase substrate solution for determining the activity of a cholinesterase in a sample, wherein at least one substrate is stabilized by at least one species of a polar organic solvent. Furthermore, the invention relates to a method for determining the activity of a cholinesterase in a sample, as well as a kit for determining the activity of a cholinesterase in a sample and a kit for conducting a method for determining the activity of a cholinesterase in a sample.

Description

technical field [0001] The field to which the present invention relates is the measurement of enzyme activity in biological samples. More specifically, the present invention relates to the use of polar organic solvents for stabilizing cholinesterase substrate solutions, and the use of said solutions for determining cholinesterase activity in samples. In addition, methods and kits for determining cholinesterase activity are provided. Background technique [0002] Cholinesterase is the general name for an enzymatic activity that catalyzes the hydrolysis of acyl esters to alcohols and carboxylates. Cholinesterase acts on a variety of substrates with alcohol moieties (such as phenol (phenyl-alcohol), indoxyl-alcohol (indoxyl-alcohol) and preferably choline) and carboxylate moieties (such as carbonic acid, dicarbonic acid (dicarbonic acid) , benzoic acid) (Brown et al., Adv. Clin. Chem., 22: 1-123 (1981)). Cholinesterases are subdivided into two classes based on their specific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/46
CPCC12Q1/46
Inventor N·戈特沙尔克H·-J·凯特兹亚M·穆克克R·纳格尔F·韦伯
Owner F HOFFMANN LA ROCHE & CO AG
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