CN110308282B - Stable homocysteine circulating enzyme method detection kit - Google Patents

Abstract

The invention provides a stable homocysteine cyclic enzyme detection kit, which comprises reagents R1 and R2, wherein a reagent R1 consists of buffer solution, surfactant, protective agent, antioxidant, disodium ethylene diamine tetraacetate, tris (2-carbonyl ethyl) phosphate, S-adenosylmethionine, alpha-ketoglutaric acid, reduced coenzyme and preservative, and a reagent R2 consists of buffer solution, surfactant, protective agent, S-adenosylhomocysteine hydrolase, Hcy methyltransferase, adenosine deaminase, glutamate dehydrogenase and preservative. Due to the synergistic effect of the surfactant, the protective agent and the antioxidant, the kit provided by the invention has the advantages of good precision, high sensitivity, wide linear range, good stability and the like compared with a commercially available HCY kit, and provides an important basis for auxiliary diagnosis of clinical HCY-related diseases.

Description

Stable homocysteine circulating enzyme method detection kit

Technical Field

The invention relates to the field of preparation of biochemical diagnostic reagents, in particular to a stable homocysteine circulating enzyme method detection kit.

Background

Homocysteine (HCY), also known as homocysteine, is a sulfhydryl-containing amino acid and belongs to an important intermediate product produced in the metabolic process of methionine and cysteine. HCY exists in human plasma in two forms, oxidized homocysteine and reduced homocysteine. Free sulfydryl in the reduced homocysteine has high activity, and is easy to be oxidized to form a disulfide form; the oxidized form is the predominant form of homocysteine present in plasma, either in the disulfide form or in a protein-bound form. Normally, homocysteine in blood can be catabolized in vivo, and the concentration is maintained at a low level. However, under the influence of primary and secondary diseases, the concentration of blood homocysteine is increased, HCY with high concentration can generate a large amount of oxidative free radicals to cause endothelial cell injury, increase lipid peroxidation and low density of oxidation type to cause injury and damage of blood vessels, increase damaged blood vessel walls to cause plaques, and cause diseases such as cardiovascular diseases, atherosclerosis diseases, stroke and the like. A large number of studies have shown that hyperhomocysteinemia plays an important role in the pathogenesis of atherosclerosis and thrombotic diseases such as cerebrovascular diseases, coronary heart diseases and venous thrombosis.

The homocysteine can be determined by High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), enzyme immunoassay (ELA), fluorescence polarization immunoassay (FPLA) and cycling enzyme method. The chromatography has high accuracy and stability, however, has high requirements on the processing of the sample, the maintenance of operators and equipment, and is usually used as a homocysteine reference method and cannot be used for detecting conventional samples; the enzyme-linked immunosorbent assay has high sensitivity and strong specificity, but has the disadvantages of complex operation, poor precision, long time consumption and unsuitability for detecting a large amount of samples; the fluorescence polarization immunoassay method needs a special detection instrument, and the single detection cost is higher; the circulating enzyme method can be used for large-scale biochemical analyzers, is simple and convenient to operate, has high precision and is suitable for clinical detection at present.

The current enzymatic recycling method comprises two methods, namely a hydrolytic enzyme method and a cystathionine method. Cystathionine method, which cannot eliminate normally metabolized β -cystathionine, produces particularly high β -cystathionine especially in renal patients, resulting in inaccurate test results. The hydrolase method has high specificity and strong anti-interference capability, can eliminate in vivo metabolism beta-cystathionine, and has more accurate detection result. Most of hydrolase reagents in the market are monopolized by foreign companies, which causes high detection cost, so that development of an HCY detection reagent with strong anti-interference capability, high accuracy and low cost is urgently needed.

Disclosure of Invention

The invention aims to provide a stable homocysteine circulating enzyme method detection kit.

In order to realize the purpose of the invention, the invention provides a stable homocysteine (double-reagent) circulating enzyme method detection kit, which comprises reagents R1 and R2, and is a liquid double reagent used on a biochemical analyzer;

the formula of the reagent R1 is as follows: 20-200mM buffer solution with pH 7.5-9.5, 0.5-5g/L surfactant, 0.5-20g/L protective agent, 0.05-50mM antioxidant, 0.2-20mM disodium ethylene diamine tetraacetic acid (EDTA-2Na), 0.2-10mM tris (2-carbonylethyl) phosphate (TCEP), 0.05-5mM S-adenosylmethionine (SAM), 1-20mM alpha-ketoglutarate (alpha-KG), 0.1-10mM NADH (reduced coenzyme I), and 0.5-2g/L preservative;

the formula of the reagent R2 is as follows: 20-200mM buffer solution with pH value of 7.5-9.5, 0.5-20g/L surfactant, 0.5-5g/L protective agent, 1-10KU/L S-adenosyl homocysteine hydrolase (SAHase), 2-20KU/L Hcy methyltransferase (HMTase), 1-10KU/L Adenosine Deaminase (ADA), 1-20KU/L glutamate dehydrogenase (GLDH), and 0.5-2g/L preservative;

wherein the buffer solution is at least one selected from boric acid buffer solution, triethanolamine buffer solution, Tris buffer solution, MES buffer solution, HEPES buffer solution, PIPES buffer solution, CAPSO-Na buffer solution, PBS buffer solution and the like.

The surfactant is at least one selected from Tween-20, Triton X-100, TX-10, ON-870, F68, Brij35, hexadecyl ammonium chloride, etc.

The protective agent is at least one selected from BSA, sucrose, lactose, trehalose, sorbitol, mannitol and the like.

The antioxidant is at least one selected from ascorbic acid, tert-butyl hydroquinone, thiodipropionic acid, butyl hydroxy anisol, dibutyl hydroxy toluene and the like.

The preservative is NaN3 and/or Proclin-300.

The detection kit also comprises HCY calibrator solution, wherein the concentrations of the HCY calibrator solution are respectively 2 mu M, 8 mu M, 15 mu M, 30 mu M and 60 mu M; the reagent used to prepare the HCY calibrator solution was 50mM PBS buffer containing 10g/L BSA and 1g/L Proclin-300.

In one embodiment of the invention, the test kit consists of reagents R1, R2, and HCY calibrator solution;

the formula of the reagent R1 is as follows: 50mM Tris buffer solution with pH8.5, Triton X-1001 g/L, mannitol 5g/L, tert-butyl hydroquinone 1mM, disodium ethylene diamine tetraacetate 0.5mM, Tris (2-carbonyl ethyl) phosphate 2mM, S-adenosylmethionine 1mM, alpha-ketoglutarate 5mM, NADH 0.4mM, NaN₃ 1g/L；

The formula of the reagent R2 is as follows: 50mM HEPES buffer solution with pH of 9.0, Triton X-1001 g/L, mannitol 5g/L, S-adenosylhomocysteine hydrolase 3KU/L, Hcy methyltransferase 5KU/L, adenosine deaminase 2KU/L, glutamate dehydrogenase 10KU/L, NaN₃ 1g/L；

HCY calibrator solutions at concentrations of 2. mu.M, 8. mu.M, 15. mu.M, 30. mu.M, and 60. mu.M, respectively.

The detection kit can be prepared according to the following method:

preparing a reagent R1: weighing the components in proportion, firstly preparing 20-200mM buffer solution, stirring for dissolving, adjusting the pH value of the solution to 7.5-9.5, then sequentially adding a surfactant, a protective agent, an antioxidant, disodium ethylene diamine tetraacetate, tris (2-carbonyl ethyl) phosphate, S-adenosylmethionine, alpha-ketoglutaric acid, reduced coenzyme I and a preservative, uniformly mixing, stirring for dissolving, then fixing the volume to a pre-prepared volume with purified water, filtering with a 0.45 mu m filter membrane, uniformly mixing, and storing at 2-8 ℃;

preparing a reagent R2: weighing the components in proportion, firstly preparing 20-200mM buffer solution, stirring for dissolving, adjusting the pH value of the solution to 7.5-9.5, then sequentially adding a surfactant, a protective agent, S-adenosyl homocysteine hydrolase, Hcy methyltransferase, adenosine deaminase, glutamate dehydrogenase and a preservative, uniformly mixing, after stirring for dissolving, fixing the volume to a pre-prepared volume by using purified water, filtering by using a 0.45 mu m filter membrane, uniformly mixing, and storing at 2-8 ℃;

preparing a calibrator solution: first, 50mM PBS buffer containing 10g/L BSA and 1g/L Proclin-300 was prepared, and HCY calibrator solutions were prepared at concentrations of 2. mu.M, 8. mu.M, 15. mu.M, 30. mu.M, and 60. mu.M, respectively, using these as reagents.

Subpackaging the prepared reagent according to the proportion of R160 mL/bottle and R220 mL/bottle; and subpackaging the HCY calibrator solution according to 1mL per unit, and labeling and boxing the subpackaged reagent and calibrator to obtain the homocysteine cyclic enzyme method detection kit.

The detection principle of the detection kit is as follows: the sample is converted into free Hcy by the oxidized homocysteine under the action of tris (2-carbonylethyl) phosphate (TCEP), and the free Hcy reacts with S-adenosylmethionine (SAM) under the catalysis of Hcy methyltransferase (HMTase) to generate methionine and S-adenosylhomocysteine (SAH). SAH is hydrolyzed by S-adenosyl homocysteine hydrolase (SAHase) into adenosine and Hcy, and the formed Hcy can be circularly added into the reaction (the amount of homocysteine is circularly amplified), so that the detection signal is amplified. And further coupling dehydrogenation reaction of NADH. The product adenyl is hydrolyzed into hypoxanthine and ammonia, and the ammonia converts NADH into NAD + under the action of glutamate dehydrogenase (GLDH), and the Hcy content in the sample is in direct proportion to the change of NADH. The HCY content of the samples was determined by comparison with a similarly treated calibrator at a wavelength of 340 nm.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the homocysteine detection kit provided by the invention has the advantages of rapid detection, wide linear range, high accuracy, good sensitivity and low cost. The lower detection limit can reach 2 mu mol/L, the linear range is 2-60 mu mol/L, and the accurate clinical diagnosis is guaranteed.

Due to the synergistic effect of the protective agent and the antioxidant, the prepared detection kit has better stability: the kit is stored for 12 months at the temperature of 2-8 ℃, and the detection accuracy is still good; the reagent uncapping stabilization period is as long as 14 days, and the uncapping stability is prolonged, so that the time and the energy of a clinical examination operator for frequently replacing the reagent are saved.

And thirdly, the detection kit can be used for directly determining the content of HCY in the sample, and compared with an indirect method, the detection kit has strong anti-interference capability, the determination result is not easily interfered by normally metabolized beta-cystathionine and other detection reagents, and the measurement result is more accurate.

Drawings

FIG. 1 shows the results of the linear detection range test of the homocysteine detection kit in example 3 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 preparation of homocysteine (double reagent) cycling enzyme assay kit

The homocysteine (two-reagent) cyclic enzyme detection kit provided by the embodiment comprises detection reagents R1 and R2 and an HCY calibrator solution.

The formula of the reagent R1 is as follows: 50mM Tris buffer, pH8.5, Triton X-1001 g/L, mannitol 5 g-L, tert-butylhydroquinone 1mM, disodium ethylenediaminetetraacetate 0.5mM, tris (2-carbonylethyl) phosphate 2mM, S-adenosylmethionine 1mM, alpha-ketoglutarate 5mM, NADH 0.4mM, NaN₃ 1g/L。

The formula of the reagent R2 is as follows: 50mM HEPES buffer solution with pH of 9.0, Triton X-1001 g/L, mannitol 5g/L, S-adenosylhomocysteine hydrolase 3KU/L, Hcy methyltransferase 5KU/L, adenosine deaminase 2KU/L, glutamate dehydrogenase 10KU/L, NaN₃ 1g/L。

HCY calibrator solutions at concentrations of 2. mu.M, 8. mu.M, 15. mu.M, 30. mu.M, and 60. mu.M, respectively.

The preparation method of the kit comprises the following steps:

preparing a reagent R1: weighing the components in proportion, firstly preparing 50mM Tris buffer solution, stirring for dissolving, adjusting the pH value of the solution to 8.5, and then sequentially adding Triton X-100, mannitol, tert-butylhydroquinone, disodium ethylenediamine tetraacetate, Tris (2-carbonyl ethyl) phosphate, S-adenosylmethionine, alpha-ketoglutaric acid, NADH and NaN₃Mixing, stirring for dissolving, adding purified water to constant volume to pre-prepared volume, filtering with 0.45 μm filter membrane, mixing, and storing at 2-8 deg.C.

Preparing a reagent R2: weighing the components according to the proportion, firstly preparing 50mM HEPES buffer solution, stirring for dissolving, adjusting the pH value of the solution to be 9.0, and then sequentially adding Triton X-100, mannitol, S-adenosyl homocysteine hydrolase, Hcy methyltransferase, adenosine deaminase, glutamate dehydrogenase and NaN₃Mixing, stirring for dissolving, adding purified water to constant volume to pre-prepared volume, filtering with 0.45 μm filter membrane, mixing, and storing at 2-8 deg.C.

Preparing a calibrator solution: first, 50mM PBS buffer containing 10g/L BSA and 1g/L Proclin-300 was prepared, and HCY calibrator solutions were prepared at concentrations of 2. mu.M, 8. mu.M, 15. mu.M, 30. mu.M, and 60. mu.M, respectively, using these as reagents.

Subpackaging the prepared reagent according to the proportion of R160 mL/bottle and R220 mL/bottle; and subpackaging the HCY calibrator solution according to 1mL per unit, and labeling and boxing the subpackaged reagent and calibrator to obtain the homocysteine cyclic enzyme method detection kit.

Example 2 application of homocysteine detection kit

1. The instrument comprises the following steps: HITACHI 7180, OLYMPUS AU400 and TOSHIBA 40FR full-automatic biochemical analyzers.

2. A sample to be detected: the plasma or serum collected by the clinical standard method can be stored for 2 days at the temperature of between 2 and 8 ℃ and can be stored for 1 month at the temperature of between 25 and 15 ℃.

3. The homocysteine detection kit prepared in example 1 was used, and the specific detection steps are shown in table 1.

TABLE 1 detection procedure

The concentrations of the calibrants were as follows: the HCY concentration is 2 mu mol/L, 8 mu mol/L, 15 mu mol/L, 30 mu mol/L, 60 mu mol/L.

4. And (3) calculating the result: concentration of HCY in sample (. mu. mol/L) (. DELTA.A)_{Sample (I)}/min-ΔA_{Blank space}/min)/(ΔA_Calibration/min-ΔA_{Blank space}Concentration of/min) x calibration solution

In the formula: delta A_{Sample (I)}And/min refers to the rate of change of absorbance of the sample tube per minute.

ΔA_{Blank space}The/min indicates the rate of change of absorbance per minute of the blank tube.

ΔA_CalibrationThe/min refers to the rate of change of absorbance per minute of the calibration tube.

5. Accuracy: the relative deviation should not exceed. + -. 10% for determination of SRM 1955.

6. Analysis sensitivity: the absolute value of the absorbance change (. DELTA.A/min) of 10. mu. mol/L should be 0.01A or more.

7. Precision: CV in the batch is less than or equal to 3 percent; the CV among batches is less than or equal to 5 percent.

8. Stability: and storing for 18 months at 2-8 ℃ in a sealed and dark place.

Example 3 Linear detection Range test of homocysteine detection kit

HCY samples at a concentration of 60. mu. mol/L were diluted in contrast to different gradient concentrations, and the measurement was repeated 3 times for each concentration using the kit prepared in example 1, and the average value was calculated as a linear correlation coefficient with the theoretical value, and the measurement results are shown in Table 2, and the linear correlation is shown in FIG. 1.

TABLE 2 HCY gradient concentration assay results

The linear correlation equation for fig. 1 is: y is 0.967x +0.7348, correlation coefficient R²0.9988, the result shows that the linear detection range of the kit is 2-60 mu mol/L, and the lower limit of detection can reach 2 mu mol/L.

Example 4 accuracy testing of homocysteine detection kit

The HCY International Standard substance SRM 1955 was assayed in duplicate using the kit prepared in example 1, and each concentration level was assayed in triplicate, and the mean value was taken, according to the formula: the relative deviation (measured mean-indicated value)/indicated value × 100% was calculated, and the accuracy of the kit was judged from the relative deviation from the standard substance.

TABLE 3 HCY accuracy test results

Detecting the concentration	LevelⅠ	LevelⅡ	LevelⅢ
				Repeat 1(μmol/L)	4.16	8.75	17.05
Repeat 2(μmol/L)	4.25	9.13	16.52
				Repeat 3(μmol/L)	3.99	8.98	16.87
Mean value of measurement (μmol/L)	4.13	8.95	16.81
				Indicating value (μmol/L)	3.98	8.85	17.70
Relative deviation of	3.85％	1.17％	-5.01％

The results in Table 3 show that three horizontal deviations of the HCY reagent for measuring the SRM 1955 are less than 10 percent, which indicates that the kit has better detection accuracy.

Example 5 in-line precision testing of homocysteine assay kit

Repeated testing of high concentration quality control Using the kit prepared in example 1The samples of the sample (20. mu. mol/L) and the low concentration quality control sample (10. mu. mol/L) were each measured 10 times, and the mean value of the measured values was calculated

And standard deviation(s). According to the formula:

and calculating the Coefficient of Variation (CV) and judging the precision of the kit.

TABLE 4 HCY precision measurement results

The results in table 4 show that the coefficient of variation CV of HCY reagent for measuring high-concentration quality control products and low-concentration quality control products is less than 3%, which indicates that the detection precision of the kit is better.

Example 6 serum sample testing

The kit prepared in example 1 was subjected to a sample alignment test with the HCY reagent of DIAZYME, and the correlation coefficient r was determined in Table 5²0.9926, the correlation was good, indicating that the kit can be used for clinical diagnosis.

TABLE 5 alignment of HCY clinical samples

Example 7 component optimization experiment for detection reagent R1

Preparing HCY detection reagent according to the method of example 1, optimizing the content of partial components (Tris buffer solution, Triton X-100, mannitol and tert-butyl hydroquinone) in the reagent R1 to obtain 8 groups of formulas (table 6), forming the HCY reagent by R1 and R2 prepared by the 8 groups of formulas, uncapping and placing in Hitachi 7180 biochemical analyzer, operating according to the kit specification, detecting the same sample, observing the stability conditions of reagents with different formulas in the biochemical analyzer uncapping for 1 day, 3 days, 5 days, 7 days, 10 days and 14 days, and the result is shown in table 7. The data was analyzed to yield the optimal protectant composition.

TABLE 6 optimization experiment of partial composition of reagent R1

Component numbering	Tris(mM)	Triton X-100(g/L)	Mannitol (g/L)	Tert-butyl Hydroquinone (mM)
					1	50	0.5	1	2
2	50	0.5	5	1
					3	50	1	1	2
4	50	1	5	1
					5	100	0.5	1	2
6	100	0.5	5	1
					7	100	1	1	2
8	100	1	5	1

Table 7 reagent decap stability results

The results in table 7 show that, under the condition of uncapping storage of HCY reagents prepared by different component formulas, uncapping reagents at different times have smaller measured values of the same sample and good stability compared with reagents on the same preparation day, wherein the measured values of component 4 in 7 days have relatively small changes and are used as the optimal formula.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (1)

1. A stable homocysteine circulating enzyme method detection kit is characterized in that the detection kit consists of reagents R1, R2 and HCY calibrator solution;

the formula of the reagent R1 is as follows: 50mM Tris buffer solution with pH8.5, Triton X-1001 g/L, mannitol 5g/L, tert-butyl hydroquinone 1mM, disodium ethylene diamine tetraacetate 0.5mM, Tris (2-carbonyl ethyl) phosphate 2mM, S-adenosylmethionine 1mM, alpha-ketoglutarate 5mM, NADH 0.4mM, NaN₃ 1g/L；

The formula of the reagent R2 is as follows: 50mM HEPES buffer solution with pH of 9.0, Triton X-1001 g/L, mannitol 5g/L, S-adenosylhomocysteine hydrolase 3KU/L, Hcy methyltransferase 5KU/L, adenosine deaminase 2KU/L, glutamate dehydrogenase 10KU/L, NaN₃ 1g/L；

HCY calibrator solutions were used at concentrations of 2. mu.M, 8. mu.M, 15. mu.M, 30. mu.M, and 60. mu.M, respectively.

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