Alanine aminotransferase detection kit against heparin interference and application thereof

Abstract

The present application relates to the technical field of detection reagent, in particular to a heparin interference resistant alanine aminotransferase detection kit and application thereof.Compared with traditional polycations, mucopolysaccharidase is more specific to heparin sulfate, and can quickly form a complex with heparin to achieve the purpose of removing heparin interference.The present application adds mucopolysaccharidase in alanine aminotransferase (ALT) reagent R1, and when the reagent is used to detect heparin samples, the mucopolysaccharidase in R1 can quickly bind with heparin, so that after reagent R2 is added, the absorbance can steadily decrease, and under the specified parameters, the normal sample detection value can be obtained according to the calibration curve and the change value of absorbance, thereby solving the phenomenon that the heparin sample appears 0 value or even negative value.The stability of the reagent is not affected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection reagent, in particular to an alanine aminotransferase detection reagent kit against heparin interference and application thereof. BACKGROUND

[0002] Alanine aminotransferase (ALT) is an enzyme involved in human protein metabolism, which accelerates the conversion of protein amino acids in the body. It exists widely in various tissues, organs, muscles and bones of the human body, and is most in the cytoplasm of liver cells. The clinical significance of alanine aminotransferase elevation is in the diagnosis and analysis of acute hepatitis B, chronic hepatitis, HBV carriers, severe hepatitis, liver cirrhosis, liver cancer and a series of viral hepatitis. The elevation of ALT only indicates that the liver may be damaged. In addition to hepatitis, many other diseases can cause ALT to rise. In acute hepatitis, chronic hepatitis and liver cirrhosis activity, the permeability of liver cell membrane changes, and ALT overflows from the cells to the blood, which makes the blood test result high.

[0003] At present, the rate method is generally used to detect ALT in the clinic. However, this method has poor reagent stability, and the test heparin sample may appear 0 value or even negative value. This is because heparin will cause the absorbance to decrease sharply after the addition of ALT reagent R1, so that the absorbance at the subsequent reading point is basically flat, not showing a downward trend, resulting in 0 value or even negative value, so that the accurate measurement value cannot be obtained. SUMMARY

[0004] In order to solve the above problems, the present application provides an alanine aminotransferase detection reagent kit against heparin interference and application thereof. The reagent kit prepared by using mucopolysaccharidase can solve the phenomenon of 0 value or even negative value of heparin sample.

[0005] In order to achieve the above purpose, the present application provides the following technical scheme:

[0006] The present application provides the application of mucopolysaccharidase in anti-heparin interference, and the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the item number is LMM03936.

[0007] The present application also provides an alanine aminotransferase detection reagent kit against heparin interference, which comprises reagent R1 and reagent R2 packaged independently, the reagent R1 comprises buffer 50-200mmol / L, L-alanine 100-400mmol / L, lactate dehydrogenase 0.2-2ku / L, surfactant 1-10g / L, mucopolysaccharidase 0.1-2ku / L, preservative 0.1-2g / L and NADH 0.1-1mmol / L; the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the item number is LMM03936.

[0008] The reagent R2 comprises a buffer 50-200 mmol / L, a preservative 0.1-2 g / L and alpha-ketoglutarate 100-400 mmol / L.

[0009] Preferably, the reagent R1 comprises a buffer 50-150 mmol / L, L-alanine 150-350 mmol / L, lactate dehydrogenase 1-1.5 ku / L, a surfactant 1-3 g / L, a mucopolysaccharidase 0.2-2 ku / L, a preservative 0.3-2 g / L and NADH 0.4-1 mmol / L.

[0010] The reagent R2 comprises a buffer 50-150 mmol / L, a preservative 0.3-2 g / L and alpha-ketoglutarate 150-400 mmol / L.

[0011] Preferably, the buffer in the reagent R1 comprises one or more of TAPS, TRIS, HEPES, MOPSO and MOPS buffer, and the pH value of the buffer is 7.5-8.0.

[0012] Preferably, the surfactant comprises one or more of TX-100, AEO-9, TWEEN20 and B66.

[0013] Preferably, the preservative in the reagent R1 comprises one or more of sodium azide, chloramphenicol and erythromycin.

[0014] Preferably, the buffer in the reagent R2 comprises one or more of TAPS, TRIS, HEPES, MOPSO and MOPS buffer, and the pH value of the buffer is 9-9.5.

[0015] Preferably, the preservative in the reagent R2 comprises one or more of sodium azide, chloramphenicol and erythromycin.

[0016] The application further provides the use of the above alanine aminotransferase detection kit in the detection of alanine aminotransferase.

[0017] Preferably, the sample to be detected comprises a sample containing heparin.

[0018] Beneficial effects:

[0019] The application provides the use of a mucopolysaccharidase in anti-heparin interference, and the mucopolysaccharidase is purchased from Nanjing Luomaimei Company and has a product number of LMM03936.

[0020] Compared with traditional polycations, the mucopolysaccharidase is more specific to heparin sulfate and can quickly form a complex with heparin, so as to remove the interference of heparin. The present application adds mucopolysaccharidase in alanine aminotransferase (ALT) reagent R1, when the reagent is used to detect a heparin sample, the mucopolysaccharidase in R1 can quickly bind with heparin, so that after the addition of reagent R2, the absorbance can stably decrease, under the specified parameters, according to the calibration curve and the change value of absorbance, the normal sample detection value is obtained, so as to solve the phenomenon that the heparin sample appears 0 value or even negative value. And the stability of the reagent is not affected. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0022] Figure 1 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0023] Figure 2 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0024] Figure 3 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0025] Figure 4 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0026] Figure 5 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0027] Figure 6 The correlation graph of the reagent kit prepared for example 1 and the control reagent kit when detecting ordinary samples (not containing heparin samples);

[0028] Figure 7 The linear graph of the reagent kit prepared for example 1 and the control reagent kit;

[0029] Figure 8 The linear graph of the reagent kit prepared for example 1 and the control reagent kit;

[0030] Figure 9 The linear graph of the reagent kit prepared for example 1 and the control reagent kit;

[0031] Figure 10 The linear graph of the reagent kit prepared for example 1 and the control reagent kit; DETAILED DESCRIPTION

[0032] The application provides application of a mucopolysaccharidase in anti-heparin interference, the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the article number is LMM03936.

[0033] The mucopolysaccharidase can hydrolyze the beta-1, 4-bond between N-acetyl-beta-D-glucosamine and D-glucuronic acid in mucopolysaccharide to generate a tetrasaccharide residue without releasing monosaccharide, and the enzyme reaction is: mucopolysaccharide + H2O = oligosaccharide. Since heparin also contains the above-mentioned groups, under the action of the mucopolysaccharidase, heparin is hydrolyzed from polysaccharide into oligosaccharide, so that the heparin interference can be avoided, the detection result is accurate, and the problem of 0 value or even negative value can be avoided; in particular, the problem that the detection result of alanine aminotransferase in a sample containing heparin appears 0 value or even negative value in the prior art can be solved.

[0034] The application also provides an alanine aminotransferase detection kit for anti-heparin interference, comprising reagent R1 and reagent R2 packaged independently, wherein the reagent R1 comprises buffer 50-200 mmol / L, L-alanine 100-400 mmol / L, lactate dehydrogenase 0.2-2 ku / L, surfactant 1-10 g / L, mucopolysaccharidase 0.1-2 ku / L, preservative 0.1-2 g / L and NADH 0.1-1 mmol / L.

[0035] The reagent R2 comprises buffer 50-200 mmol / L, preservative 0.1-2 g / L and alpha-ketoglutaric acid 100-400 mmol / L.

[0036] Unless otherwise specified, the application does not have special requirements for the source of each component in the kit, and commercially available products known to those skilled in the art can be used.

[0037] In the application, the volume ratio of the reagent R1 to the reagent R2 is preferably 4:1.

[0038] The reagent R1 comprises buffer 50-200 mmol / L, preferably 50-150 mmol / L, and more preferably 100 mmol / L. In the application, the buffer preferably comprises one or more of TAPS, TRIS, HEPES, MOPSO and MOPS buffer, and more preferably TRIS buffer; the pH value of the buffer is preferably 7.5-8.0, and more preferably 7.6. The application adjusts the pH of the reagent by the buffer, thereby improving the stability of the reagent, and further improving the accuracy of the detection result.

[0039] The reagent R1 comprises L-alanine 100-400 mmol / L, preferably 150-350 mmol / L, and more preferably 150 mmol / L. The L-alanine is a reaction substrate.

[0040] The reagent R1 of the present application comprises lactate dehydrogenase 0.2~2ku / L, preferably 1~1.5ku / L, and more preferably 1ku / L. The lactate dehydrogenase of the present application is to convert NADH into NAD + The catalyst of the reaction.

[0041] The reagent R1 of the present application comprises surfactant 1~10g / L, preferably 1~3g / L, and more preferably 3g / L. In the present application, the surfactant preferably comprises one or more of TX-100, AEO-9, TWEEN20 and B66, and more preferably TX-100. The surfactant of the present application has the effect of improving the sensitivity of the detection reaction.

[0042] The reagent R1 of the present application comprises mucopolysaccharidase 0.1~2ku / L, preferably 0.2~2ku / L, and more preferably 0.2ku / L. The mucopolysaccharidase of the present application is more specific to heparin sulfate than traditional polycations, and can quickly form a complex with heparin. Under the action of mucopolysaccharidase, heparin is hydrolyzed from polysaccharide into oligosaccharide, thereby achieving the purpose of removing heparin interference. The mucopolysaccharidase of the present application is preferably purchased from Nanjing Luomaimei Company, and the item number is LMM03936.

[0043] The reagent R1 of the present application comprises preservative 0.1~2g / L, preferably 0.3~2g / L, and more preferably 0.3g / L. In the present application, the preservative preferably comprises one or more of sodium azide, chloramphenicol and erythromycin, and more preferably erythromycin. The preservative of the present application has the effect of inhibiting the growth of miscellaneous bacteria and prolonging the effective period of the kit.

[0044] The reagent R1 of the present application comprises NADH 0.1~1mmol / L, preferably 0.4~1mmol / L, and more preferably 0.8mmol / L. The NADH of the present application has a specific absorption at 340nm, and the ALT activity concentration is calculated by continuously monitoring the absorbance decrease rate at 340nm.

[0045] The reagent R2 of the present application comprises buffer 50~200mmol / L, preferably 50~150mmol / L, and more preferably 100mmol / L. In the present application, the buffer preferably comprises one or more of TAPS, TRIS, HEPES, MOPSO, GOODS and MOPS buffer, and more preferably TRIS buffer; the pH value of the buffer is preferably 9~9.5, and more preferably 9.2. The present application adjusts the pH of the reagent by buffer, thereby improving the stability of the reagent, and further improving the accuracy of the detection result.

[0046] The reagent R2 of the present application comprises a preservative 0.1-2 g / L, preferably 0.3-2 g / L, and more preferably 0.3 g / L. In the present application, the preservative preferably comprises one or more of sodium azide, chloramphenicol and erythromycin, and more preferably erythromycin. The preservative of the present application has the effect of inhibiting the growth of miscellaneous bacteria and prolonging the effective period of the kit.

[0047] The reagent R2 of the present application comprises α-ketoglutaric acid 100-400 mmol / L, preferably 1-3 mmol / L, and more preferably 3 mmol / L.

[0048] The present application preferably further provides a preparation method of the above-mentioned kit, comprising the following steps:

[0049] The buffer, surfactant, preservative and L-alanine are mixed, and hydrochloric acid or sodium hydroxide is used to adjust the pH to the specified pH ± 0.10, and then the mucopolysaccharidase, NADH and LDH are sequentially added, and finally hydrochloric acid or sodium hydroxide is used to adjust the pH to the specified pH ± 0.05, and then the volume is adjusted to obtain the reagent R1.

[0050] The buffer, preservative and α-ketoglutaric acid are mixed, and hydrochloric acid or sodium hydroxide is used to adjust the pH to the specified pH ± 0.05, and then the volume is adjusted to obtain the reagent R2.

[0051] The present application can avoid the influence of a large amount of hydrochloric acid or NAOH on the enzyme activity by adjusting the pH value first, then adding the mucopolysaccharidase, NADH and LDH, and finally adjusting the pH value again.

[0052] The present application further provides the use of the above-mentioned kit in detecting alanine aminotransferase. The kit of the present application for detecting alanine aminotransferase can be for the purpose of non-diagnosis, in order to obtain an intermediate value of alanine aminotransferase.

[0053] In the present application, the sample to be detected whether containing alanine aminotransferase can preferably be a sample containing heparin.

[0054] The present application effectively removes the interference of heparin by adding a suitable concentration of mucopolysaccharidase in the reagent and combining with a specific buffer and a suitable concentration of other components, without affecting the stability of the reagent and the accuracy of the detection result, solving the problem that the alanine aminotransferase kit is not resistant to heparin. And the stability of the reagent is basically not affected, and the reagent is stable at least for 18 months under the condition of 2-8℃.

[0055] In order to further illustrate the present application, the anti-heparin interference alanine aminotransferase detection kit and its application provided by the present application are described in detail in conjunction with the following examples, but they should not be understood as limiting the scope of protection of the present application.

[0056] Example 1

[0057] A heparin interference resistant alanine aminotransferase detection kit, consisting of reagent R1 and reagent R2 packaged independently, wherein the volume ratio of reagent R1 and reagent R2 is 4:1;

[0058] The reagent R1 consists of the following components:

[0059] TRIS buffer with pH value of 7.6 100 mmol / L, TX-100 3 g / L, mucopolysaccharidase 0.2 ku / L, erythromycin 0.3 g / L, L-alanine 150 mmol / L, NADH 0.8 mmol / L, lactate dehydrogenase (LDH) 1 ku / L; the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the article number is LMM03936;

[0060] The reagent R2 consists of the following components:

[0061] TRIS buffer with pH value of 9.2 50 mmol / L, erythromycin 0.3 g / L, α-ketoglutaric acid 150 mmol / L.

[0062] The reagent R1 is prepared by the following method:

[0063] Take 800 mL of purified water, add TRIS, dissolve thoroughly, then add TX-100, erythromycin and L-alanine in sequence, adjust the pH to 7.6 with hydrochloric acid and sodium hydroxide, then add mucopolysaccharidase, NADH and LDH in sequence, finally add purified water to 1000 mL, and adjust the pH to 7.6 with hydrochloric acid and sodium hydroxide.

[0064] The reagent R2 is prepared by the following method:

[0065] Take 200 mL of purified water, add TRIS, dissolve thoroughly, then add erythromycin and α-ketoglutaric acid in sequence, finally add purified water to 250 mL, and adjust the pH to 9.2 with hydrochloric acid and sodium hydroxide.

[0066] Example 2

[0067] A heparin interference resistant alanine aminotransferase detection kit, consisting of reagent R1 and reagent R2 packaged independently, wherein the volume ratio of reagent R1 and reagent R2 is 4:1;

[0068] The reagent R1 consists of the following components:

[0069] TRIS buffer solution 50 mmol / L with pH value of 7.8, AEO-9 1 g / L, mucopolysaccharidase 0.5 ku / L, sodium azide 0.3 g / L, L-alanine 180 mmol / L, NADH 1 mmol / L, lactate dehydrogenase (LDH) 1 ku / L;

[0070] The reagent R2 is prepared by the following method:

[0071] TRIS buffer solution 50 mmol / L with pH value of 9.5, sodium azide 0.3 g / L, alpha-ketoglutaric acid 180 mmol / L.

[0072] The reagent R1 is prepared by the following method:

[0073] Take 800 mL of purified water, add TRIS, dissolve thoroughly, then add AEO-9, sodium azide and L-alanine in sequence, adjust pH to 7.8 with hydrochloric acid and sodium hydroxide, then add mucopolysaccharidase, NADH and LDH in sequence, and finally add purified water to make up to 1000 mL, and adjust pH to 7.8 with hydrochloric acid and sodium hydroxide.

[0074] The reagent R2 is prepared by the following method:

[0075] Take 200 mL of purified water, add TRIS, dissolve thoroughly, then add sodium azide and alpha-ketoglutaric acid in sequence, and finally add purified water to make up to 250 mL, and adjust pH to 9.5 with hydrochloric acid and sodium hydroxide.

[0076] Example 3

[0077] An alanine aminotransferase detection kit resistant to heparin interference, which is composed of reagent R1 and reagent R2 packaged independently, wherein the volume ratio of reagent R1 to reagent R2 is 4:1;

[0078] The reagent R1 is prepared by the following method:

[0079] TAPS buffer solution 100 mmol / L with pH value of 7.8, TX-100 1 g / L, mucopolysaccharidase 1 ku / L, sodium azide 0.5 g / L, L-alanine 200 mmol / L, NADH 1 mmol / L, lactate dehydrogenase (LDH) 1 ku / L; the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the item number is LMM03936;

[0080] The reagent R2 is prepared by the following method:

[0081] TAPS buffer solution 100 mmol / L with pH value of 9.5, sodium azide 0.5 g / L, alpha-ketoglutaric acid 250 mmol / L.

[0082] The reagent R1 is prepared by the following method:

[0083] Take 800 mL of purified water, add TAPS, dissolve thoroughly, then add TX-100, sodium azide and L-alanine in turn, adjust the pH to 7.8 with hydrochloric acid and sodium hydroxide, then add mucopolysaccharidase, NADH and LDH in turn, and finally add purified water to make up to 1000 mL, and adjust the pH to 7.8 with hydrochloric acid and sodium hydroxide.

[0084] The reagent R2 is prepared by the following method:

[0085] Take 200 mL of purified water, add TAPS, dissolve thoroughly, then add sodium azide and α-ketoglutaric acid in turn, and finally add purified water to make up to 250 mL, and adjust the pH to 9.5 with hydrochloric acid and sodium hydroxide.

[0086] Example 4

[0087] An anti-heparin interference alanine aminotransferase detection kit, consisting of reagent R1 and reagent R2 packaged separately, wherein the volume ratio of reagent R1 to reagent R2 is 4:1;

[0088] The reagent R1 is composed of the following components:

[0089] TAPS buffer with pH value of 7.6 100 mmol / L, MOPS buffer with pH value of 7.6 50 mmol / L, TX-100 1 g / L, B-66 1 g / L, mucopolysaccharidase 1 ku / L, chloramphenicol 2 g / L, L-alanine 300 mmol / L, NADH 0.4 mmol / L, lactate dehydrogenase (LDH) 1.5 ku / L; the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the item number is LMM03936;

[0090] The reagent R2 is composed of the following components:

[0091] TAPS buffer with pH value of 9.2 100 mmol / L, MOPS buffer with pH value of 9.2 50 mmol / L, chloramphenicol 2 g / L, α-ketoglutaric acid 350 mmol / L.

[0092] The reagent R1 is prepared by the following method:

[0093] Take 800 mL of purified water, add TAPS and MOPS, dissolve thoroughly, then add TX-100, B-66, chloramphenicol and L-alanine in turn, adjust the pH to 7.6 with hydrochloric acid and sodium hydroxide, then add mucopolysaccharidase, NADH and LDH in turn, and finally add purified water to 1000 mL, and adjust the pH to 7.6 with hydrochloric acid and sodium hydroxide.

[0094] The reagent R2 is prepared by the following method:

[0095] Take 200 mL of purified water, add TAPS and MOPS, dissolve thoroughly, then add chloramphenicol and alpha-ketoglutaric acid, and finally add purified water to 250 mL, and adjust the pH to 9.2 with hydrochloric acid and sodium hydroxide.

[0096] Example 5

[0097] An alanine aminotransferase detection kit resistant to heparin interference, consisting of reagent R1 and reagent R2 packaged separately, wherein the volume ratio of reagent R1 to reagent R2 is 4:1;

[0098] The reagent R1 consists of:

[0099] HEPES buffer with a pH of 7.5 25 mmol / L, MOPS buffer with a pH of 7.5 25 mmol / L, TX-100 2 g / L, AEO-9 2 g / L, mucopolysaccharidase 2 ku / L, chloramphenicol 1.5 g / L, L-alanine 350 mmol / L, NADH 0.4 mmol / L, lactate dehydrogenase (LDH) 1.5 ku / L; the mucopolysaccharidase is purchased from Nanjing Luomaimei Company, and the item number is LMM03936;

[0100] The reagent R2 consists of:

[0101] HEPES buffer with a pH of 9.3 25 mmol / L, MOPS buffer with a pH of 9.3 25 mmol / L, chloramphenicol 1.5 g / L, alpha-ketoglutaric acid 400 mmol / L.

[0102] The reagent R1 is prepared by the following method:

[0103] Take 800 mL of purified water, add HEPES and MOPS, dissolve thoroughly, then add TX-100, AEO-9, chloramphenicol and L-alanine in turn, adjust the pH to 7.5 with hydrochloric acid and sodium hydroxide, then add mucopolysaccharidase, NADH and LDH in turn, and finally add purified water to 1000 mL, and adjust the pH to 7.5 with hydrochloric acid and sodium hydroxide.

[0104] The reagent R2 is prepared by the following method:

[0105] Take 200 mL of purified water, add HEPES and MOPS, dissolve thoroughly, then add chloramphenicol and α-ketoglutaric acid in turn, and finally add purified water to 250 mL, and adjust the pH to 9.3 with hydrochloric acid and sodium hydroxide.

[0106] Comparative Example 1

[0107] A kit similar to Example 1, the only difference is that no mucopolysaccharidase is added.

[0108] Comparative Example 2

[0109] A kit similar to Example 2, the only difference is that no mucopolysaccharidase is added.

[0110] Comparative Example 3

[0111] A kit similar to Example 3, the only difference is that no mucopolysaccharidase is added.

[0112] Comparative Example 4

[0113] A kit similar to Example 4, the only difference is that no mucopolysaccharidase is added.

[0114] Comparative Example 5

[0115] A kit similar to Example 5, the only difference is that no mucopolysaccharidase is added.

[0116] Application Example 1

[0117] Sample correlation: 50 fresh serum samples were collected from the hospital, numbered 1~50, and compared with Example 1, no mucopolysaccharidase (Comparative Example 1) and commercially available alanine aminotransferase reagent kit (as a control kit, purchased from Beijing Jiujiang Technology Co., Ltd., with the product number GS0001G). The content of ALT in samples 1~50 was detected. The instrument used was the automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, secondary wavelength 380 nm, sample volume 16 μl, reagent R1 volume 200 μl, reagent R2 volume 50 μl. The results are shown in Table 1, Figure 1 and Figure 2 .

[0118] Table 1: Sample correlation test results (unit: U / L)

[0119]

[0120] From the above results, the correlation of Example 1 with the control kit sample is 0.9972, which is better than the comparison example, indicating that the correlation of the serum sample tested by the present application is good.

[0121] Application Example 2

[0122] Heparin homologous sample detection: Determine the sample obtained from the same patient using different blood collection tubes (serum and heparin tubes) to determine whether heparin will affect the sample value. From the hospital, 20 groups of samples of the same patient using different blood collection tubes (serum and heparin tubes) were obtained, and the serum tubes were numbered as "serum 1-serum 20", and the heparin tubes were numbered as "heparin homologous 1-heparin homologous 20". The content of ALT in the above samples was detected by using Example 1, Example 2, the glycosaminoglycanase-free comparative example (Comparative Example 2) and the alanine aminotransferase kit circulating in the market (as a control kit, purchased from Beijing Jiujiang Technology Co., Ltd., with the product number GS0001G). The instrument used was a full-automatic biochemical analyzer TBA-2000FR (manufacturer: Toshiba, Japan), and the detection parameters were set as follows: main wavelength 340 nm, secondary wavelength 380 nm, sample volume 16 μl, reagent R1 volume 200 μl, and reagent R2 volume 50 μl. The results are shown in Table 2 and Figures 3-6 .

[0123] Table 2: Content of ALT in different samples (unit: U / L)

[0124]

[0125] From the above results, the correlation of the kit of the present application is better than that of the control kit, and Example 1 performs best. Comparative Example 2 has 5 cases of 0 values, the control kit has 3 cases of 0 values and 1 case of negative value, while the kits of the present application, Examples 1 and 2, have no 0 values and negative values. It is shown that the glycosaminoglycanase has a positive effect on solving the problem of 0 values of heparin samples.

[0126] Application Example 3

[0127] Precision experiment: two patient serum samples were taken from the hospital, one patient serum alanine aminotransferase content is high, the other patient serum alanine aminotransferase content is low, two patient samples are recorded as high and low value samples, and the high and low value serum samples are tested by example 1, example 3, no mucopolysaccharidase comparative example (comparative example 3) and the alanine aminotransferase kit circulating in the market (as a control kit, purchased from Beijing Jiujiang Technology Co., Ltd., with the goods number GS0001G), each repeated determination 20 times, 20 determination values were obtained respectively, the mean value (X) and standard deviation (S) and coefficient of variation (CV) of the determination results were calculated, and the precision. The instrument used is automatic biochemical analyzer TBA-2000FR (manufacturer: Japan Toshiba), and the detection parameters are set as follows: main wavelength 340nm, secondary wavelength 380nm, sample volume 16μl, reagent R1 volume 200μl, reagent R2 volume 50μl. The results are shown in Table 3.

[0128] Table 3 Precision experiment results (unit: U / L)

[0129]

[0130]

[0131] From Table 3, the results of the kit and the control kit of the application can meet the line standard variation coefficient <5%, and the kit prepared by example 1 performs best.

[0132] Application example 4

[0133] The standard solution was prepared by adding ALT (source leaf biological product No. S25364) to purified water, and the standard solution with a concentration of 1000U / L was diluted with purified water to 6 different concentrations of test samples (concentrations: 1000U / L, 500U / L, 250U / L, 125U / L, 63U / L, 32U / L). Example 1, example 4, no mucopolysaccharidase comparative example (comparative example 4) and the circulating control kit on the market were tested (the control kit and the detection method are the same as application example 1), each concentration of test sample was repeated for 3 times, the mean value (yi) of the detection results was calculated, the linear regression equation was calculated with the dilution concentration (xi) as the independent variable and the detection result mean value (yi) as the dependent variable, and the linear regression correlation coefficient (r) was calculated. The results are shown in Table 4 and Figures 7-10 .

[0134] Table 4 Linear results of different groups (unit: U / L)

[0135]

[0136] From the above results, the kit, the comparative example and the control kit can meet the linear requirements of the line standard; it can be seen that the addition of mucopolysaccharidase does not affect the detection results while resisting the interference of heparin.

[0137] Application Example 5

[0138] Stability: Examples 1, 5 and the comparative example without mucopolysaccharidase (Comparative Example 5), and the alanine aminotransferase reagent kit circulating on the market (as a control kit, purchased from Beijing Jiujiang Technology Co., Ltd., with the product number GS0001G) were respectively taken out of the instrument with the cap opened and placed in a constant temperature incubator at 37℃ for acceleration. The 4℃ reagent was calibrated with the Langdo multi-component biochemical calibrator (target value 148 U / L), and then the Langdo multi-component biochemical quality control level 2 and level 3 (target values 39 U / L and 138 U / L, respectively) were taken out and tested under the 4℃ calibration channel every 1-2 times a week, and the detection method was the same as in the application example. The determination results are shown in Table 5.

[0139] Table 5 Stability results of different groups (unit: U / L)

[0140]

[0141] From Table 5, it can be seen that the opening of the bottle and the accelerated stability of the reagent kit of the application can be maintained for one month, while the opening of the bottle and the accelerated stability of the control reagent for one month are out of control, which shows that the reagent kit of the application is better than the control reagent and the comparative example in stability, and the reagent kit prepared in Example 1 has better stability.

[0142] In summary, the reagent kit of the application can solve the problem of easy 0 value of heparin samples, and the precision, linearity and stability are higher than the industry standard and the commercially available control reagent kit, and have high clinical use value.

[0143] Although the application has been disclosed as above with preferred embodiments, it is not intended to limit the application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, therefore the protection scope of the application should be defined by the claims.

Claims

1. An alanine aminotransferase test kit against heparin interference, consisting of reagent Rl and reagent R2 packaged separately, characterized in that, The reagent R1 is composed of the following components with the following concentrations: buffer 50-150 mmol / L, L-alanine 150-350 mmol / L, lactate dehydrogenase 1-1.5 ku / L, surfactant 1-3 g / L, mucopolysaccharidase 0.2-2 ku / L, preservative 0.3-2 g / L, and NADH 0.4-1 mmol / L; the buffer in the reagent R1 includes one or more of TAPS, TRIS, HEPES, MOPSO, and MOPS buffer; the pH value of the buffer is 7.5-8.0; the surfactant includes one or more of TX-100, AEO-9, TWEEN 20, and B66; the preservative in the reagent R1 includes one or more of sodium azide, chloramphenicol, and erythromycin; The reagent R2 is composed of the following components with the following concentrations: buffer 50-150 mmol / L, preservative 0.3-2 g / L, and α-ketoglutaric acid 150-400 mmol / L; the buffer in the reagent R2 includes one or more of TAPS, TRIS, HEPES, MOPSO, and MOPS buffer, and the pH value of the buffer is 9-9.5; the preservative in the reagent R2 includes one or more of sodium azide, chloramphenicol, and erythromycin.

2. Use of the alanine aminotransferase detection kit against heparin interference according to claim 1 in detecting alanine aminotransferase, wherein the direct purpose of the detection is non-diagnostic and non-therapeutic.

3. The use of the anti-heparin interfering alanine aminotransferase detection kit according to claim 2 for detecting alanine aminotransferase, characterized in that, The sample to be detected includes a sample containing heparin.

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