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Stable cyclic enzymatic detection kit for homocysteine

A technology of homocysteine ​​and detection kits, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as high detection costs, and achieve high accuracy, rapid detection, and good sensitivity

Active Publication Date: 2019-10-08
BIOSINO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the hydrolytic enzyme reagents on the market are monopolized by foreign companies, resulting in high detection costs. Therefore, it is urgent to develop a HCY detection reagent with strong anti-interference ability, high accuracy and low cost.

Method used

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  • Stable cyclic enzymatic detection kit for homocysteine
  • Stable cyclic enzymatic detection kit for homocysteine
  • Stable cyclic enzymatic detection kit for homocysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of Example 1 Homocysteine ​​(Double Reagent) Cycle Enzyme Detection Kit

[0032] The homocysteine ​​(dual reagents) cycle enzymatic detection kit provided in this example includes detection reagents R1 and R2, and HCY calibrator solution.

[0033]The formula of reagent R1 is: 50mM, pH8.5 Tris buffer, Triton X-100 1g / L, mannitol 5g / L, tert-butylhydroquinone 1mM, edetate disodium 0.5mM, Triton (2-Carbonylethyl) phosphate hydrochloride 2mM, S-adenosylmethionine 1mM, α-ketoglutarate 5mM, NADH 0.4mM, NaN 3 1g / L.

[0034] The formula of reagent R2 is: 50mM, pH9.0 HEPES buffer, Triton X-100 1g / L, mannitol 5g / L, S-adenosyl homocysteine ​​hydrolase 3KU / L, Hcy methyltransferase 5KU / L, adenosine deaminase 2KU / L, glutamate dehydrogenase 10KU / L, NaN 3 1g / L.

[0035] HCY calibrator solutions with concentrations of 2 μM, 8 μM, 15 μM, 30 μM, and 60 μM, respectively.

[0036] The preparation method of the kit is as follows:

[0037] Preparation of reagent R1: Weigh e...

Embodiment 2

[0041] Example 2 Application of Homocysteine ​​Detection Kit

[0042] 1. Instruments: HITACHI 7180, OLYMPUS AU400 and TOSHIBA 40FR automatic biochemical analyzer.

[0043] 2. Samples to be tested: Plasma or serum collected by clinical standard methods can be stored at 2°C to 8°C for 2 days, and at -25°C to -15°C for 1 month.

[0044] 3. Using the homocysteine ​​detection kit prepared in Example 1, the specific detection steps are shown in Table 1.

[0045] Table 1 detection operation steps

[0046]

[0047] The concentration of the calibrator is as follows: calibrator solutions with HCY concentrations of 2 μmol / L, 8 μmol / L, 15 μmol / L, 30 μmol / L, and 60 μmol / L.

[0048] 4. Calculation result: the concentration of HCY in the sample (μmol / L) = (ΔA 样品 / min-ΔA 空白 / min) / (ΔA 校准 / min-ΔA 空白 / min)×calibration solution concentration

[0049] Where: ΔA 样品 / min refers to the absorbance change rate of the sample tube per minute.

[0050] ΔA 空白 / min refers to the absorbance chan...

Embodiment 3

[0056] Example 3 The linear detection range test of the homocysteine ​​detection kit

[0057] The HCY sample with a concentration of 60 μmol / L was compared and diluted into different gradient concentrations, and each concentration was repeatedly measured 3 times with the kit prepared in Example 1, the average value was obtained, and the linear correlation coefficient was calculated with the theoretical value. The test results are shown in the table 2, the linear dependence is as figure 1 shown.

[0058] Table 2 HCY gradient concentration detection results

[0059]

[0060]

[0061] figure 1 The corresponding linear correlation equation is: y=0.967x+0.7348, the correlation coefficient R 2 =0.9988, the results show that the linear detection range of this kit is 2-60 μmol / L, and the detection limit can reach 2 μmol / L.

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Abstract

The invention provides a stable cyclic enzymatic detection kit for homocysteine. The kit comprises reagents R1 and R2. The reagent R1 is composed of a buffer solution, a surfactant, a protective agent, an antioxidant, ethylene diamine tetraacetic acid, tris(2-carbonylethyl) phosphine hydrochloride, S-adenosyl methionine, 2-ketoglutaric acid, a reduced coenzyme, and a preservative. The reagent R2 includes a buffer solution, a surfactant, a protective agent, S-adenosyl-L-homocysteine hydrolase, Hcy transmethylase, adenosine deaminase, glutamate dehydrogenase, and a preservative. According to thekit, because of the cooperative effect of the surfactants, the protective agents and the antioxidants, the provided kit has advantages of high precision, high sensitivity, wide linear range and highstability and the like by being compared with the commercially available HCY kit, thereby providing the important base for the auxiliary diagnosis of clinical HCY related diseases.

Description

technical field [0001] The invention relates to the field of preparation of biochemical diagnostic reagents, in particular to a stable homocysteine ​​cycle enzyme detection kit. Background technique [0002] Homocysteine ​​(HCY), also known as homocysteine, is a sulfhydryl-containing amino acid and is an important intermediate product produced during the metabolism of methionine and cysteine. HCY exists in two forms in human plasma, oxidized homocysteine ​​and reduced homocysteine. The free sulfhydryl group in reduced homocysteine ​​has high activity, and it is easily oxidized to form a disulfide form; the oxidized form is the main form of homocysteine ​​in plasma, in the form of disulfide or It exists in the form of protein binding. Under normal circumstances, homocysteine ​​in the blood can be catabolized in the body, and the concentration is maintained at a low level. However, under the influence of primary and secondary diseases, the concentration of homocysteine ​​in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6815G01N2333/914
Inventor 张勇
Owner BIOSINO BIO TECH & SCI
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