阅读说明：本技术 一种用于检测同型半胱氨酸的试剂盒 (Kit for detecting homocysteine ) 是由 王小龙 赵伟亨 于 2020-05-18 设计创作，主要内容包括：本发明实施例涉及医学检测技术领域,特别是涉及一种用于检测同型半胱氨酸的试剂盒,包括：壳体、试剂以及信息卡。信息卡存储有吸光度值与浓度值的标准曲线,信息卡存储有校正因子,校正因子用于当待检测血液样本为全血时,校正血液样本中的同型半胱氨酸的浓度。试剂包括处理液、第一反应液和第二反应液；处理液用于将血液样本中的蛋白结合型同型半胱氨酸转换为游离型同型半胱氨酸；第一反应液与第二反应液用于与游离型同型半胱氨酸反应。使用用于检测同型半胱氨酸的试剂盒,分析仪根据信息卡中存储的吸光度值与浓度值的标准曲线可直接获得同型半胱氨酸的浓度,非常方便。此外,可通过校正因子校正全血样本的同型半胱氨酸的浓度。(The embodiment of the invention relates to the technical field of medical detection, in particular to a kit for detecting homocysteine, which comprises: a housing, a reagent, and an information card. The information card stores a standard curve of the absorbance value and the concentration value, the information card stores a correction factor, and the correction factor is used for correcting the concentration of homocysteine in the blood sample when the blood sample to be detected is whole blood. The reagent comprises a treatment solution, a first reaction solution and a second reaction solution; the treatment fluid is used for converting protein-bound homocysteine in a blood sample into free homocysteine; the first reaction solution and the second reaction solution are used for reacting with free homocysteine. By using the kit for detecting homocysteine, the analyzer can directly obtain the concentration of homocysteine according to the standard curve of the absorbance value and the concentration value stored in the information card, and the kit is very convenient. In addition, the concentration of homocysteine in a whole blood sample may be corrected by a correction factor.)

1. A kit for detecting homocysteine is characterized by comprising a shell, a reagent and an information card;

the blood sample detection device comprises a shell, an information card and a correction factor, wherein the shell is provided with an accommodating space, a reagent is accommodated in the accommodating space, the information card is arranged in the shell and stores a standard curve of an absorbance value and a concentration value, and the information card stores the correction factor which is used for correcting the concentration of homocysteine in the blood sample when the blood sample to be detected is whole blood;

the reagent comprises: a treatment liquid, a first reaction liquid and a second reaction liquid;

the treatment fluid is used for converting protein-bound homocysteine in the blood sample into free homocysteine;

the first reaction solution comprises reduced nicotinamide adenine dinucleotide phosphate, dithiothreitol, alpha-ketoglutarate, modified HCY methyltransferase, succinate dehydrogenase and sodium acetate, wherein the content of the reduced nicotinamide adenine dinucleotide phosphate is 0.2-0.3%, the content of the dithiothreitol is 0.1-0.12%, the content of the alpha-ketoglutarate is 0.6-0.8%, the concentration of the modified HCY methyltransferase is 3.0KU/L-6.0KU/L, the concentration of the succinate dehydrogenase is 5.0KU/L-8.0KU/L, and the content of the sodium acetate is 0.1-0.15%;

the second reaction solution comprises modified S-adenosylhomocysteine hydrolase, adenosine deaminase, methionine adenosyltransferase, bovine serum albumin and triton X-100, wherein the concentration of the modified S-adenosylhomocysteine hydrolase is 3.0KU/L-6.0KU/L, the concentration of the adenosine deaminase is 4.0KU/L-8.0KU/L, the concentration of the methionine adenosyltransferase is 4.0KU/L-8.0KU/L, the content of the bovine serum albumin is 0.5-1, and the content of the triton X-100 is 0.1-0.5%.

2. The kit according to claim 1, wherein the treatment solution comprises PEG6000, sodium ethylene diamine tetracetate, sodium dodecyl sulfate and proclin300, wherein the content of the PEG6000 is 0.1% -0.5%, the content of the sodium ethylene diamine tetracetate is 0.01% -0.05%, the content of the sodium dodecyl sulfate is 0.01% -0.2%, and the content of the proclin300 is 0.1% -0.3%.

3. The kit of claim 2, wherein the process fluid further comprises a first Tris buffer, the first Tris buffer having a concentration of 50mmol/L, and the first Tris buffer having a PH of 7.4 at a temperature of 25 ℃.

4. The kit of claim 1, wherein the first reaction solution further comprises a second Tris buffer, the second Tris buffer having a concentration of 100mmol/L, and the second Tris buffer having a PH of 9.2 at a temperature of 25 ℃.

5. The kit of claim 1, wherein the second reaction solution further comprises a third Tris buffer, wherein the third Tris buffer has a concentration of 80mmol/L, and wherein the third Tris buffer has a PH of 7.6 at a temperature of 25 ℃.

6. A homocysteine detection method applied to an analyzer, wherein the detection method uses the kit for detecting homocysteine according to any one of claims 1-5, and the method comprises the following steps:

diluting the blood sample with the treatment fluid to obtain a first mixture;

adding the first reaction liquid into the first mixture to obtain a second mixture;

adding the second reaction solution into the second mixture to obtain a third mixture;

testing the second mixture for a first absorbance value and the third mixture for a second absorbance value at a predetermined wavelength;

calculating an absorbance difference value according to the first absorbance value and the second absorbance value;

and obtaining the concentration of homocysteine in the blood sample according to the absorbance difference and the standard curve of the absorbance value and the concentration value stored in the information card.

7. The method of claim 6, wherein when the blood sample is whole blood, the step of calculating an absorbance difference based on the first absorbance value and the second absorbance value further comprises:

and calculating the difference value of the second absorbance value, the first absorbance value and the correction factor, wherein the difference value of the second absorbance value, the first absorbance value and the correction factor is the absorbance difference value.

8. The method of claim 6, wherein the step of calculating an absorbance difference based on the first absorbance value and the second absorbance value when the blood sample is serum further comprises:

and calculating the difference value between the second absorbance value and the first absorbance value, wherein the difference value between the second absorbance value and the first absorbance value is the absorbance difference value.

9. The detection method according to claim 6, wherein prior to said testing at a predetermined wavelength a first absorbance value of said second mixture and a second absorbance value of said third mixture, said detection method further comprises:

and testing a third absorbance value of the physiological saline at a preset wavelength, and zeroing the third absorbance value.

10. A test system comprising an analyzer and a kit for detecting homocysteine according to any of claims 1-5.

Technical Field

The embodiment of the invention relates to the technical field of medical detection, in particular to a kit for detecting homocysteine.

Background

Homocysteine (HCY), an important risk factor for atherosclerotic vascular disease, is an intermediate formed by methionine metabolism. The increase of the concentration of Homocysteine (HCY) in blood (the concentration is more than or equal to 12umol/L) is a risk factor of coronary heart disease, stroke, peripheral vascular atherosclerosis and arteriovenous embolism, and the increase of Homocysteine (HCY) in blood increases the morbidity and mortality of cardiovascular diseases, so that the determination of the concentration of Homocysteine (HCY) in blood is very important in clinical significance.

The current methods for measuring Homocysteine (HCY) mainly comprise the following methods: a circulating enzyme method technology; high Performance Liquid Chromatography (HPLC), Enzyme Immunoassay (EIA), ion chromatography, capillary electrophoresis, and fluorescence polarization immunoassay. Currently, in the global market, the enzymatic recycling method is mainly divided into two technologies, namely a hydrolase enzymatic recycling method and a cystathionine enzymatic recycling method. Wherein

However, in the process of implementing the embodiment of the present invention, the inventors of the present invention found that: cystathionine cycler method mainly determines the content of Homocysteine (HCY) by measuring cystathionine generated by converting Homocysteine (HCY) into beta-cystathionine, but cannot eliminate the interference of cystathionine carried by patients; the hydrolase cycling enzyme method has high specificity and strong anti-interference capability, but the current test reagent has low sensitivity and complex operation.

Disclosure of Invention

In view of the above, embodiments of the present invention provide a kit for detecting homocysteine, which overcomes or at least partially solves the above problems.

According to an aspect of the embodiments of the present invention, there is provided a kit for detecting homocysteine, comprising a housing, a reagent and an information card; the blood sample detection device comprises a shell, an information card and a correction factor, wherein the shell is provided with an accommodating space, a reagent is accommodated in the accommodating space, the information card is arranged in the shell and stores a standard curve of an absorbance value and a concentration value, and the information card stores the correction factor which is used for correcting the concentration of homocysteine in the blood sample when the blood sample to be detected is whole blood; the reagent comprises: a treatment liquid, a first reaction liquid and a second reaction liquid; the treatment fluid is used for converting protein-bound homocysteine in the blood sample into free homocysteine; the first reaction solution comprises reduced nicotinamide adenine dinucleotide phosphate, dithiothreitol, alpha-ketoglutarate, modified HCY methyltransferase, succinate dehydrogenase and sodium acetate, wherein the content of the reduced nicotinamide adenine dinucleotide phosphate is 0.2-0.3%, the content of the dithiothreitol is 0.1-0.12%, the content of the alpha-ketoglutarate is 0.6-0.8%, the concentration of the modified HCY methyltransferase is 3.0KU/L-6.0KU/L, the concentration of the succinate dehydrogenase is 5.0KU/L-8.0KU/L, and the content of the sodium acetate is 0.1-0.15%; the second reaction solution comprises modified S-adenosylhomocysteine hydrolase, adenosine deaminase, methionine adenosyltransferase, bovine serum albumin and triton X-100, wherein the concentration of the modified S-adenosylhomocysteine hydrolase is 3.0KU/L-6.0KU/L, the concentration of the adenosine deaminase is 4.0KU/L-8.0KU/L, the concentration of the methionine adenosyltransferase is 4.0KU/L-8.0KU/L, the content of the bovine serum albumin is 0.5-1, and the content of the triton X-100 is 0.1-0.5%.

In an optional mode, the treatment solution comprises PEG6000, sodium ethylene diamine tetracetate, sodium dodecyl sulfate and proclin300, wherein the content of PEG6000 is 0.1% -0.5%, the content of sodium ethylene diamine tetracetate is 0.01% -0.05%, the content of sodium dodecyl sulfate is 0.01% -0.2%, and the content of proclin300 is 0.1% -0.3%.

In an alternative embodiment, the process fluid further comprises a first Tris buffer, the concentration of the first Tris buffer is 50mmol/L, and the pH of the first Tris buffer is 7.4 at a temperature of 25 ℃.

In an alternative embodiment, the first reaction solution further comprises a second Tris buffer, wherein the concentration of the second Tris buffer is 100mmol/L, and the pH of the second Tris buffer is 9.2 at a temperature of 25 ℃.

In an alternative embodiment, the second reaction solution further comprises a third Tris buffer, wherein the concentration of the third Tris buffer is 80mmol/L, and the pH of the third Tris buffer is 7.6 at a temperature of 25 ℃.

According to an aspect of the embodiments of the present invention, there is provided a method for detecting homocysteine, which is applied to an analyzer, the method using the above kit for detecting homocysteine, comprising: diluting the blood sample with the treatment fluid to obtain a first mixture; adding the first reaction liquid into the first mixture to obtain a second mixture; adding the second reaction solution into the second mixture to obtain a third mixture; testing the second mixture for a first absorbance value and the third mixture for a second absorbance value at a predetermined wavelength; calculating an absorbance difference value according to the first absorbance value and the second absorbance value; and obtaining the concentration of homocysteine in the blood sample according to the absorbance difference and the standard curve of the absorbance value and the concentration value stored in the information card.

In an alternative mode, when the blood sample is whole blood, the step of calculating an absorbance difference from the first absorbance value and the second absorbance value further includes: and calculating the difference value of the second absorbance value, the first absorbance value and the correction factor, wherein the difference value of the second absorbance value, the first absorbance value and the correction factor is the absorbance difference value.

In an alternative mode, when the blood sample is serum, the step of calculating an absorbance difference from the first absorbance value and the second absorbance value further comprises: and calculating the difference value between the second absorbance value and the first absorbance value, wherein the difference value between the second absorbance value and the first absorbance value is the absorbance difference value.

In an alternative manner, before the testing the first absorbance value of the second mixture and the second absorbance value of the third mixture at the preset wavelength, the detection method further includes: and testing a third absorbance value of the physiological saline at a preset wavelength, and zeroing the third absorbance value.

According to an aspect of the embodiments of the present invention, there is provided a detection system, comprising an analyzer and the above-mentioned kit for detecting homocysteine.

The embodiment of the invention has the beneficial effects that: different from the existing kit for detecting homocysteine, the kit provided by the embodiment of the invention comprises: a housing, a reagent, and an information card. The information card stores a standard curve of an absorbance value and a concentration value, and stores a correction factor, wherein the correction factor is used for correcting the concentration of homocysteine in a blood sample when the blood sample to be detected is whole blood. The reagent comprises a treatment solution, a first reaction solution and a second reaction solution; the treatment fluid is used for converting protein-bound homocysteine in the blood sample into free homocysteine; the first reaction solution comprises reduced nicotinamide adenine dinucleotide phosphate, dithiothreitol, alpha-ketoglutarate, modified HCY methyltransferase, succinate dehydrogenase and sodium acetate, wherein the content of the reduced nicotinamide adenine dinucleotide phosphate is 0.2-0.3%, the content of the dithiothreitol is 0.1-0.12%, the content of the alpha-ketoglutarate is 0.6-0.8%, the concentration of the modified HCY methyltransferase is 3.0KU/L-6.0KU/L, the concentration of the succinate dehydrogenase is 5.0KU/L-8.0KU/L, and the content of the sodium acetate is 0.1-0.15%; the second reaction solution comprises modified S-adenosylhomocysteine hydrolase, adenosine deaminase, methionine adenosyltransferase, bovine serum albumin and triton X-100, wherein the concentration of the modified S-adenosylhomocysteine hydrolase is 3.0KU/L-6.0KU/L, the concentration of the adenosine deaminase is 4.0KU/L-8.0KU/L, the concentration of the methionine adenosyltransferase is 4.0KU/L-8.0KU/L, the content of the bovine serum albumin is 0.5-1, and the content of the triton X-100 is 0.1-0.5%. The kit for detecting homocysteine is used for detecting the concentration of homocysteine on an analyzer, and the analyzer can directly obtain the concentration of homocysteine according to a standard curve of the absorbance value and the concentration value stored in an information card, so that the kit is very convenient. In addition, because the information card stores the correction factor, the kit for detecting homocysteine is suitable for detecting the concentration of homocysteine in a whole blood sample.

Drawings

One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the figures in which like reference numerals refer to similar elements and which are not to scale unless otherwise specified.

FIG. 1 is a schematic diagram of a kit for detecting homocysteine according to an embodiment of the present invention;

FIG. 2 is a graph of absorbance values versus concentration values provided by an embodiment of the present invention;

FIG. 3 is a scattergram of an assessment reagent and a contrast reagent provided in an embodiment of the present invention;

FIG. 4 is an absolute bias diagram of the assessment reagent and the contrast reagent provided by the embodiment of the present invention;

FIG. 5 is a graph showing the relative bias of the assessment reagent and the contrast reagent provided in the examples of the present invention;

FIG. 6 is a schematic flow chart of a method for detecting homocysteine according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The application of the kit for detecting homocysteine has the following action principle: the protein-bound homocysteine in the blood sample is converted into free homocysteine, free homocysteine converted by the protein-bound homocysteine and original free homocysteine in blood react with covalent substrates to generate adenosine, the adenosine is immediately hydrolyzed into ammonia and hypoxanthine, the ammonia enables reduced nicotinamide adenine dinucleotide phosphate (NADH) to be converted into NAD + under the action of enzyme, and the concentration of homocysteine in the blood sample is in direct proportion to the change of the reduced nicotinamide adenine dinucleotide phosphate (NADH).

Referring to fig. 1, a kit 100 includes a housing 101, a reagent (not shown), and an information card 102. The housing 101 is provided with an accommodating space 1011, the reagent is accommodated in the accommodating space 1011, and the information card 102 is provided in the housing 101.

For the above reagents, the reagents include: the reaction system comprises a treatment liquid, a first reaction liquid and a second reaction liquid. The treatment solution, the first reaction solution and the second reaction solution are placed separately.

The treatment fluid is used for converting the protein-bound homocysteine in the blood sample into free homocysteine. The homocysteine circulates in vivo, most of the homocysteine exists in an oxidized form combined with plasma protein, namely, protein-combined homocysteine is formed by being combined with albumin in plasma through disulfide bonds, and only a small amount of homocysteine exists in a free form, namely, free homocysteine, so that the blood sample can be subjected to further operation by diluting and pre-reacting the blood sample with the treatment solution before being tested. In some embodiments, the treatment fluid comprises PEG6000, sodium edetate, sodium lauryl sulfate, proclin300, and a first Tris buffer, wherein the PEG6000 is present in an amount of 0.1% to 0.5%, the sodium edetate is present in an amount of 0.01% to 0.05%, the sodium lauryl sulfate is present in an amount of 0.01% to 0.2%, the proclin300 is present in an amount of 0.1% to 0.3%, the first Tris buffer is present at a concentration of 50mmol/L, and the first Tris buffer has a PH of 7.4 at a temperature of 25 ℃.

The first reaction solution comprises reduced nicotinamide adenine dinucleotide phosphate, dithiothreitol, alpha-ketoglutarate, modified HCY methyltransferase, succinate dehydrogenase, sodium acetate and a second Tris buffer solution, wherein the reduced nicotinamide adenine dinucleotide phosphate is 0.2-0.3%, the dithiothreitol is 0.1-0.12%, the alpha-ketoglutarate is 0.6-0.8%, the modified HCY methyltransferase is 3.0KU/L-6.0KU/L, the succinate dehydrogenase is 5.0KU/L-8.0KU/L, the sodium acetate is 0.1-0.15%, the second Tris buffer solution is 100mmol/L, and the second Tris buffer solution has a pH of 9.2 at a Tris temperature of 25 ℃.

The second reaction solution comprises modified S-adenosylhomocysteine hydrolase, adenosine deaminase, methionine adenosyltransferase, bovine serum albumin, triton X-100 and a third Tris buffer solution, wherein the concentration of the modified S-adenosylhomocysteine hydrolase is 3.0KU/L-6.0KU/L, the concentration of the adenosine deaminase is 4.0KU/L-8.0KU/L, the concentration of the methionine adenosyltransferase is 4.0KU/L-8.0KU/L, the content of the bovine serum albumin is 0.5-1, the content of the triton X-100 is 0.1-0.5%, the concentration of the third Tris buffer solution is 80mmol/L, and the pH of the third Tris buffer solution at the temperature of 25 ℃ is 7.6.

For the information card 102, a calibration curve of absorbance values and concentration values and a calibration factor are stored in the information card 102.

The standard curve of the absorbance value versus concentration value is shown in fig. 2. The standard curve of the absorbance value and the concentration value is obtained by fitting the absorbance values of a plurality of standard solutions with known homocysteine concentrations on an analyzer. According to the standard curve of the absorbance value and the concentration value, the concentration x of homocysteine and the absorbance value y satisfy the linear equation of 0.0054x +0.0148, R²0.9921. The information card 102 is contacted with an information card sensing area on the analyzer, after the card swiping is successful, the information card sensing area can identify standard curve information of the absorbance value and the concentration value stored in the information card 102, and the absorbance value of the blood sample to be detected can be detected by the analyzer and can be directly converted into the concentration of homocysteine.

The correction factor is used for correcting the concentration of homocysteine in a blood sample when the blood sample to be detected is whole blood. The correction factors were obtained by testing whole serum samples and serum samples for a number of known homocysteine concentrations.

For example, whole serum samples were tested for absorbance at a known concentration of homocysteine at 37 ℃ and a wavelength of 340nm, and serum samples were taken after centrifugation. And subtracting the absorbance value of the serum sample of the homocysteine from the absorbance value of the whole serum sample of the homocysteine by the average value A to obtain a difference C, and then calculating an average value D to obtain the correction factor. Wherein the concentrations of the whole serum sample of homocysteine and the serum sample of homocysteine are respectively No. 1, No. 2, No. 12, No. 3, No. 22, No. 4, No. 32 and No. 5, No. 42 umol/L. The absorbance measurements for whole serum samples of homocysteine are reported in table 1. The absorbance values of the serum samples of homocysteine were measured and reported in Table 2, and the mean A, the mean B, the difference C and the mean D in Table 3.

TABLE 1 Absorbance values of Whole serum samples of homocysteine

Number of tests	Number 1	Number 2	No. 3	Number 4	Number 5
						1	0.7443	0.7651	0.8048	0.9014	0.9528
2	0.7439	0.7651	0.8052	0.9015	0.9526
						3	0.7441	0.7649	0.8039	0.9017	0.9534
4	0.7445	0.7652	0.8041	0.9021	0.9537
						5	0.7438	0.7638	0.8046	0.9019	0.9529
6	0.7439	0.7640	0.8045	0.9023	0.9538
						7	0.7440	0.7644	0.8051	0.9025	0.9531
8	0.7446	0.7649	0.8049	0.9019	0.9532
						9	0.7441	0.7643	0.8053	0.9020	0.9541
10	0.7442	0.7638	0.8049	0.9024	0.9573
						Average value A	0.74414	0.76455	0.80473	0.90197	0.95369

TABLE 2 absorbance values of serum samples of homocysteine

TABLE 3 correction factor

It is understood that, in some embodiments, the standard solution with known concentration of homocysteine has different concentration intervals, the calibration factors of the concentration intervals are respectively detected, and the concentration intervals and the corresponding calibration factors are recorded into the information card 102, so as to improve the accuracy of detecting the concentration of homocysteine.

The performance of the kit for detecting homocysteine was evaluated using an analyzer as follows:

(1) repeatability test

Blood samples at concentrations of (10.0. + -. 2.0) umol/L were tested repeatedly and the Coefficient of Variation (CV) calculated as required by the national industry Standard YY/T1158-2015. Blood samples at a concentration of (20.0. + -. 4.0) umol/L were repeatedly tested, and the Coefficient of Variation (CV) was calculated, and the results of the measurements are reported in Table 4.

TABLE 4 repeatability tests

As can be seen from Table 4, the Coefficient of Variation (CV) obtained by repeating the test (10.0. + -. 2.0) on the umol/L sample was not more than 5%. The kit for detecting homocysteine has good repeatability, and the obtained Coefficient of Variation (CV) of the result is not more than 3% when the sample of umol/L (20.0 +/-4.0) is repeatedly tested.

(2) Blank evaluation test

Under the conditions that the temperature is 37 ℃ and the wavelength is 340nm, physiological saline is used as a blank sample, three kits for detecting homocysteine with different batches are used for testing, the detection is carried out for 20 times continuously, the detection results are recorded in a table 5, wherein the three kits for detecting homocysteine with different batches are marked as batch number 1, batch number 2 and batch number 3.

TABLE 5 blank evaluation test

As can be seen from Table 5, the absorbance values of the blank samples were all less than 0.05, and were in accordance with the industry standards.

(3) Assay sensitivity test

The absorbance change rate (. DELTA.A/t) of 10.0umol/L samples was measured at a temperature of 37 ℃ and a wavelength of 340nm using three different lots of the kit for homocysteine detection, which were designated as lot 1, lot 2 and lot 3, and the measurement results are shown in Table 6.

TABLE 6 sensitivity of analysis

Number of detections	Batch number 1	Batch number 2	Batch No. 3
				1	0.0635	0.0609	0.0608
2	0.0621	0.0615	0.0599
				3	0.0615	0.0617	0.0621
4	0.0624	0.0613	0.0637
				5	0.0614	0.0621	0.0633
6	0.0633	0.0635	0.0632
				7	0.0625	0.0641	0.0625
8	0.0610	0.0632	0.0624
				9	0.0613	0.0620	0.0633
10	0.0615	0.0619	0.0629
				Mean value of	0.06205	0.06222	0.06241

As can be seen from Table 6, the absorbance change rates of 10.0umol/L samples tested by three different lots of the kit for detecting homocysteine were all greater than 0.01. The analysis sensitivity of the kit for detecting homocysteine meets the test requirements.

(4) Interference experiment

1. Bilirubin

Selecting one part of mixed serum, dividing the mixed serum into three parts, adding interferents into the three parts to be used as interference samples, and not adding interferents into the other part to be used as a basic sample, and preparing an interferent sample series according to the following method:

base sample X1: 0.9mL of serum sample + 0.1mL of distilled water;

interference sample X2: 0.9mL of serum sample + 0.1mL of 20mg/dL bilirubin solution;

interference sample X3: 0.9mL of serum sample + 0.1mL of 35mg/dL bilirubin solution.

The assay was repeated 5 times for each sample, the experimental results are reported in table 7, and the assay interferent concentrations were calculated, and the interference values for each group are the assay mean for each group of interfering samples-the assay mean for the basal sample.

TABLE 7 bilirubin interference test

As can be seen from Table 7, bilirubin had substantially no effect on the test results when bilirubin was less than or equal to 20 mg/dL.

2. Triglyceride (fat turbidity interference)

The light interference effect of chylomicron on reagent reaction in a serum sample is evaluated by simulating chylomicron in serum by using fat milk, and the content of the chylomicron (the chylomicron mainly consists of triglyceride) in the sample is measured according to the content of triglyceride in triglyceride standard solution (fat milk).

Selecting one part of a mixed serum sample, dividing the mixed serum sample into three parts, adding interferents into the three parts to be used as interference samples, and not adding interferents into the other part to be used as a basic sample, and preparing an interferent sample series according to the following method:

base sample X4: 0.9mL of serum sample + 0.1mL of distilled water;

interference sample X5: 0.9mL of serum sample + 0.1mL of 500mg/dL triglyceride solution;

interference sample X6: 0.9mL of serum sample + 0.1mL of 750mg/dL triglyceride solution.

Each sample was repeated 5 times, the experimental results are recorded in table 8, and the analyte interferent concentrations were calculated, and the interference values for each group are the mean of the interference samples for each group-the mean of the basal samples.

TABLE 8 triglyceride interference experiments

As can be seen from Table 8, when the triglyceride was 500mg/dL or less, the triglyceride had substantially no effect on the test results.

3. Hemoglobin

Selecting one part of a mixed serum sample, dividing the mixed serum sample into three parts, adding interferents into the three parts to be used as interference samples, and not adding interferents into the other part to be used as a basic sample, and preparing an interferent sample series according to the following method:

base sample X7: 0.9mL of serum sample + 0.1mL of distilled water;

interference sample X8: 0.9mL of serum sample + 0.1mL of 500mg/dL hemoglobin solution;

interference sample X9: 0.9mL of serum sample +750mg/dL of hemoglobin solution 0.1 mL.

The assay was repeated 5 times for each sample, the results are reported in table 9, and the assay interferent concentrations were calculated, and the interference values for each group are the assay mean for each group of interfering samples-the assay mean for the basal sample.

TABLE 9 hemoglobin interference test

As can be seen from Table 9, when hemoglobin was less than or equal to 500mg/dL, hemoglobin had substantially no effect on the test results.

(5) Clinical analysis

The kit for detecting homocysteine in the embodiment of the invention is used as an examination reagent and compared with the detection results of a Jianli kit and a complete Beckman AU680 full-automatic biochemical analyzer as a contrast reagent. 116 serum samples (54 samples with abnormal values and 59 samples with normal values) were tested separately.

The results show that: the positive coincidence rate is 96.29 percent, the negative coincidence rate is 100 percent, the total coincidence rate is 98.27 percent, and the coincidence rate is more than 95 percent; the correlation coefficient r was 0.9962. The kits of two different manufacturers have higher coincidence rate, consistency and relevance and can be regarded as equivalent clinically.

The test results of the two kits were plotted in a scatter plot, as shown in FIG. 3. The correlation coefficient r value of the two kits is 0.9962(t is 17.3, p is approximately equal to 0.643 > 0.05), and is better than the index 0.975, which indicates that the two kits have good correlation.

The mean (Md) and Standard Deviation (SD) of the difference between the test results of the two kits were calculated to calculate the 95% consistency limit (Md + -2 SD), the absolute bias of the two kits is shown in FIG. 4, and as can be seen from FIG. 4, 111 scatter points are within the Md + -2 SD range, i.e., 95.68% (≧ 95%) scatter points are within the Md + -2 SD range, indicating that the two kits have good consistency.

The mean (Md) and Standard Deviation (SD) of the ratio of the test results of the two kits were calculated to calculate the 95% consistency limit (Md + -2 SD), the relative bias of the two kits is shown in FIG. 5, as can be seen from FIG. 5, 111 scatter points are within the Md + -2 SD range, and 95.68% (≧ 95%) scatter points are within the Md + -2 SD range, indicating that the two kits have good consistency.

In an embodiment of the present invention, a kit for detecting homocysteine comprises a housing, a reagent and an information card. The information card stores a standard curve of an absorbance value and a concentration value, and stores a correction factor, wherein the correction factor is used for correcting the concentration of homocysteine in a blood sample when the blood sample to be detected is whole blood. The reagent comprises a treatment solution, a first reaction solution and a second reaction solution; the treatment fluid is used for converting protein-bound homocysteine in the blood sample into free homocysteine; the first reaction solution comprises reduced nicotinamide adenine dinucleotide phosphate, dithiothreitol, alpha-ketoglutarate, modified HCY methyltransferase, succinate dehydrogenase and sodium acetate, wherein the content of the reduced nicotinamide adenine dinucleotide phosphate is 0.2-0.3%, the content of the dithiothreitol is 0.1-0.12%, the content of the alpha-ketoglutarate is 0.6-0.8%, the concentration of the modified HCY methyltransferase is 3.0KU/L-6.0KU/L, the concentration of the succinate dehydrogenase is 5.0KU/L-8.0KU/L, and the content of the sodium acetate is 0.1-0.15%; the second reaction solution comprises modified S-adenosylhomocysteine hydrolase, adenosine deaminase, methionine adenosyltransferase, bovine serum albumin and triton X-100, wherein the concentration of the modified S-adenosylhomocysteine hydrolase is 3.0KU/L-6.0KU/L, the concentration of the adenosine deaminase is 4.0KU/L-8.0KU/L, the concentration of the methionine adenosyltransferase is 4.0KU/L-8.0KU/L, the content of the bovine serum albumin is 0.5-1, and the content of the triton X-100 is 0.1-0.5%. The kit for detecting homocysteine is used for detecting the concentration of homocysteine on an analyzer, and the analyzer can directly obtain the concentration of homocysteine according to a standard curve of the absorbance value and the concentration value stored in an information card, so that the kit is very convenient. In addition, because the information card stores the correction factor, the kit for detecting homocysteine is suitable for detecting the concentration of homocysteine in a whole blood sample.

The embodiment of the present invention further provides a homocysteine detection method, which is applied to an analyzer, wherein the detection method uses the kit for detecting homocysteine, please refer to fig. 6, and the detection method comprises the following steps:

step S101, diluting the blood sample with the treatment fluid to obtain a first mixture.

The ratio of the treatment fluid to the blood sample is 30: 1-60: 1.

step S102, adding the first reaction solution to the first mixture to obtain a second mixture.

Step S103, adding the second reaction solution to the second mixture to obtain a third mixture.

The ratio of the first reaction solution to the second reaction solution is 7:1-10:1

Step S104, testing a first absorbance value of the second mixture and a second absorbance value of the third mixture at a preset wavelength.

The preset wavelength is 340 nm. It will be appreciated that the predetermined wavelength may be other, such as 600 nm. The preset wavelengths are different, and the standard curves of the absorbance value and the concentration value are different.

It is understood that, in some embodiments, to improve the accuracy of the test, before step S104, the detection method further includes: and testing a third absorbance value of the physiological saline at a preset wavelength, and zeroing the third absorbance value.

And step S105, calculating an absorbance difference value according to the first absorbance value and the second absorbance value.

When the blood sample is whole blood, step S105 specifically includes: and calculating the difference value of the second absorbance value, the first absorbance value and the correction factor, wherein the difference value of the second absorbance value, the first absorbance value and the correction factor is the absorbance difference value.

When the blood sample is serum, step S105 specifically includes: and calculating the difference value between the second absorbance value and the first absorbance value, wherein the difference value between the second absorbance value and the first absorbance value is the absorbance difference value.

And step S106, obtaining the concentration of homocysteine in the blood sample according to the absorbance difference and the standard curve of the absorbance value and the concentration value stored in the information card.

Referring again to fig. 2, the concentration x of homocysteine in a blood sample and the absorbance value y satisfy the linear equation of 0.0054x +0.0148, R²0.9921. In the present application, the absorbance difference corresponds to the absorbance value, for example, if the absorbance difference is 0.2, the concentration of homocysteine is 34.3 umol/L.

In an embodiment of the present invention, the method for detecting homocysteine comprises diluting a blood sample with said treatment fluid to obtain a first mixture; adding the first reaction liquid into the first mixture to obtain a second mixture; adding the second reaction solution into the second mixture to obtain a third mixture; testing the second mixture for a first absorbance value and the third mixture for a second absorbance value at a predetermined wavelength; calculating an absorbance difference value according to the first absorbance value and the second absorbance value; and obtaining the concentration of homocysteine in the blood sample according to the absorbance difference and the standard curve of the absorbance value and the concentration value stored in the information card. The detection method is suitable for detecting the concentration of homocysteine in a whole blood sample

The embodiment of the invention also provides a detection system, which comprises an analyzer and the kit for detecting homocysteine. The analyzer is suitable for detecting the concentration of the homocysteine by using the kit for detecting homocysteine.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.