A kind of assay kit of leucine aminopeptidase

Abstract

The present invention provides a leucine aminopeptidase assay kit, including R1 reagent and R2 reagent, R1 reagent including: 10-100mmol / L zwitterionic buffer, 1-10mmol / L metal chelator, 1-10g / L nonionic surfactant, 10-100g / L NaCl and 0.1-1g / L preservative; R2 reagent including: 5-50mmol / L citrate buffer, 0.5-5mmol / L metal chelator, 8-40 mmol / L L-leucyl-P-nitroaniline and 0.1-1g / L preservative. The present invention improves the stability and selectivity of leucine aminopeptidase, the kit shows good repeatability and a wide linear range, shows excellent anti-interference ability, and can meet the clinical demand for blood and urine co-testing.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic kits, and in particular relates to a leucine aminopeptidase assay kit. Background Art

[0002] LAP is widely distributed in various tissues and organs of the human body, and is highly active in the liver, gallbladder, pancreas, kidney, small intestine and uterus. LAP covers different types according to their sources and molecular structures. The two most common types in the human body are leucine aminopeptidase (LAP) and placental leucine aminopeptidase (P-LAP). Different types of LAP have the same ion-binding active center and similar properties.

[0003] The role of LAP in the body is closely related to the physiological functions of various tissues and organs. Its increased concentration is mainly manifested in impaired liver and kidney function, as well as in the middle and late stages of normal pregnancy. In the liver, LAP is mainly distributed in the bile duct area. Various factors that hinder bile circulation, such as biliary infection, destruction of liver lobule structure, and tumor compression, can stimulate the increase of LAP synthesis; in the kidney, LAP is mainly located in the lysosomes of renal tubular epithelial cells. Renal parenchymal damage caused by various reasons, especially renal tubular damage, shows obvious increase in urinary enzyme secretion in the subclinical stage; during pregnancy, especially in the middle and late stages of pregnancy, a large increase in P-LAP can play a positive role. P-LAP can degrade oxytocin and angiotensinogen II and reduce the number of oxytocin receptors on the cell membrane surface, which can relax the blood vessels of the pregnant uterus, regulate uterine blood flow, and reduce the sensitivity of the pregnant uterus to oxytocin, thereby maintaining the stability of the internal environment of the mother and fetus during pregnancy.

[0004] The leucine aminopeptidase assay kits currently on the market have the following defects: 1) The linear range is narrow, resulting in inaccurate high values, especially in late pregnancy when some pregnant women show LAP activity >400U / L. If accompanied by abnormal liver function, the value will be higher, which can easily lead to clinical misjudgment; 2) The normal value of LAP in urine is usually less than 10U / L. The poor repeatability of the reagent will cause unacceptable bias in urine samples near the reference interval, and urine samples are not suitable. 3) Jaundice is common in liver disease. When liver function is impaired, LAP assay kits with poor anti-interference performance, especially poor anti-bilirubin interference performance, will not measure the true concentration of LAP, affecting the judgment of the cause.

[0005] Patent document CN108913752A discloses a leucine aminopeptidase detection kit for detecting leucine aminopeptidase in blood by a continuous monitoring method. The kit only includes reagent R. By selecting BR buffer into reagent R, adding appropriate amounts of polyethylene glycol 20,000, N-β-hydroxyethylethylenediaminetriacetic acid, sodium chloride, L-leucyl-2-naphthylamine hydrochloride and Brom-nitro-dioxan (BND), the stability of the kit is improved, but the maximum linearity can only reach 240U / L, which cannot meet clinical needs.

[0006] Patent document CN111154834A discloses a leucine aminopeptidase detection kit and a preparation method thereof, comprising R1 reagent and R2 reagent, wherein the R1 reagent comprises 100-200 mmol / L of tris(hydroxymethyl)methane buffer, 200-260 mmol / L of glycine buffer, 1.0-5.0 g / L of polycarboxyethylene glycol amino, 30-50 mmol / L of formamide, 1.5-2 g / L of IgG, 20-30 mmol / L of sorbic acid, 50-70 mmol / L of horseradish peroxidase, and the remainder is deionized water; the R2 reagent comprises 15 mmol / L of L-leucine p-nitroaniline, 150-180 mmol / L of glycine buffer, 1-5 mmol / L of piperazine-N,N-di(2-ethanesulfonic acid), and the remainder is deionized water; the kit has strong anti-interference ability and high stability. However, it needs to go through the process of standing, adding dropwise while stirring, removing the supernatant, and the temperature must be strictly controlled in different processes. The preparation process is complicated, time-consuming, laborious and difficult to obtain.

[0007] Patent document CN103266164A discloses a leucine aminopeptidase detection reagent, including a diluent and a reaction reagent, wherein the diluent is a buffer, a surfactant, a preservative, a bilirubin-removing interfering agent and vitamin C oxidase; the reaction reagent is a buffer, sodium chloride, L-leucine-4-nitroaniline, a preservative 0.1-1g / L and a lyophilization protective agent 10-30g / L. However, the stability of the vitamin C oxidase contained in its components is very poor. In addition, the document discloses a POCT technology, which has the characteristics of instant detection, but is not suitable for large-scale rapid detection scenarios and has certain application limitations. Summary of the invention

[0008] The present invention provides a leucine aminopeptidase assay kit to overcome the above-mentioned deficiencies in the prior art.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] A leucine aminopeptidase assay kit of the present invention is characterized by comprising an R1 reagent and an R2 reagent;

[0011] The R1 reagent comprises the following components: 10-100 mmol / L zwitterionic buffer, 1-10 mmol / L metal chelating agent, 1-10 g / L nonionic surfactant, 10-100 g / L NaCl and 0.1-1 g / L preservative;

[0012] The R2 reagent comprises the following components: 5-50 mmol / L citrate buffer, 0.5-5 mmol / L metal chelator, 8-40 mmol / L L-leucyl-P-nitroaniline and 0.1-1 g / L preservative;

[0013] The volume ratio of the R1 reagent to the R2 reagent is 3:1 to 4:1.

[0014] Furthermore, the zwitterionic buffer is a Tris (hydroxymethylaminomethane) buffer, preferably with a pH of 7.0 to 8.0; preferably, the buffer pair of the zwitterionic buffer is a buffer pair of Tris (hydroxymethylaminomethane) and an amino acid and its derivatives; more preferably, the amino acid and its derivatives are glycine or diGly peptide.

[0015] Furthermore, the nonionic surfactant is one or more of polyethylene glycol type nonionic surfactants or polyol type nonionic surfactants, and more preferably one or more of nonylphenol polyoxyethylene ether (NP-9), octylphenol polyoxyethylene ether (OP-9), sorbitan monolaurate (Span-20), and polyoxyethylene sorbitan monolaurate (Tween-20).

[0016] Furthermore, the pH of the citrate buffer is 4.0-5.0; the metal chelator is ethylenediaminetetraacetic acid (EDTA); and the preservative is at least one of sodium azide and proclin300.

[0017] The present invention has the following beneficial effects:

[0018] The present invention improves the stability and selectivity of the detected object, leucine aminopeptidase, in the sample by adjusting a stable buffer and its pH value, and adding a specific and appropriate amount of polyethylene glycol type nonionic surfactant or polyol type nonionic surfactant. The kit of the present invention has excellent anti-interference ability, good repeatability, a wide linear range, high sensitivity and stability, and exhibits excellent anti-interference ability, can meet the clinical demand for simultaneous blood and urine testing, and has good market application prospects. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to specific embodiments. Those skilled in the art should recognize that the embodiments are only used to illustrate the present invention, and are not intended to limit the present invention, and that any changes or modifications to the embodiments within the scope of the present invention are within the scope of the claims of the present invention.

[0020] All the raw materials used in this implementation are commercially available products.

[0021] Example 1

[0022] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0023] The components of R2 reagent include: citric acid-sodium citrate buffer with a pH of 4.5 and a concentration of 20 mmol / L, 1 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline and 0.5 g / L Proclin300;

[0024] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0025] Example 2

[0026] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGly buffer with a pH of 7.5 and a concentration of 100 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0027] The components of R2 reagent include: pH 4.5, concentration of 20mmol / L citric acid-sodium citrate buffer, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0028] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0029] Example 3

[0030] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGly buffer with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 7 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0031] The R2 reagent components include: pH 4.5, concentration of 20mmol / Lol / L citric acid-sodium citrate buffer, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0032] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0033] Example 4

[0034] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L Span-20, 30 g / L NaCl and 0.5 g / L Proclin300;

[0035] The components of R2 reagent include: pH 4.5, concentration of 20mmol / L citric acid-sodium citrate buffer, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0036] The volume ratio of R1 reagent to R2 reagent is 4:1.

[0037] Example 5

[0038] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L Tween-20, 30 g / L NaCl and 0.5 g / L Proclin300;

[0039] The components of R2 reagent include: pH 4.5, concentration of 20mmol / L citric acid-sodium citrate buffer, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0040] The volume ratio of R1 reagent to R2 reagent is 4:1.

[0041] Example 6

[0042] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 2 g / L OP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0043] The components of R2 reagent include: pH 4.5, concentration of 20mmol / L citric acid-sodium citrate buffer, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0044] The volume ratio of R1 reagent to R2 reagent is 4:1.

[0045] Example 7

[0046] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0047] The R2 reagent components include: pH 4.5, 50 mmol / L citric acid-sodium citrate buffer, 1 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline, and 0.5 g / L Proclin300;

[0048] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0049] Example 8

[0050] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0051] The components of R2 reagent include: pH 5.0, citric acid-sodium citrate buffer with a concentration of 20mmol / L, 1mmol / L EDTA, 15mmol / L L-leucyl-p-nitroaniline and 0.5g / L Proclin300;

[0052] The volume ratio of the R1 reagent to the R2 reagent is 3.4:1.

[0053] Example 9

[0054] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0055] The R2 reagent components include: pH 4.5, citric acid-sodium citrate buffer with a concentration of 20 mmol / L, 5 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline, and 0.5 g / L Proclin300;

[0056] The volume ratio of the R1 reagent to the R2 reagent is 3.8:1.

[0057] Example 10

[0058] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0059] The components of R2 reagent include: citric acid-sodium citrate buffer with a pH of 4.5 and a concentration of 20 mmol / L, 1 mmol / L EDTA, 40 mmol / L L-leucyl-p-nitroaniline and 0.5 g / L Proclin300;

[0060] The volume ratio of the R1 reagent to the R2 reagent is 3.5:1.

[0061] Embodiment 11

[0062] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0063] The components of R2 reagent include: citric acid-sodium citrate buffer with a pH of 4.5 and a concentration of 20 mmol / L, 1 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline and 0.5 g / L Proclin300;

[0064] The volume ratio of the R1 reagent to the R2 reagent is 3:1.

[0065] Comparative Example 1

[0066] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGly buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 30 g / L NaCl and 0.5 g / L Proclin300;

[0067] The components of R2 reagent include: citric acid-sodium citrate buffer with a pH of 4.5 and a concentration of 20 mmol / L, 1 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline and 0.5 g / L Proclin300;

[0068] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0069] Comparative Example 2

[0070] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0071] The R2 reagent components include: pH 4.5, citric acid-sodium citrate buffer with a concentration of 20 mmol / L, 15 mmol / L L-leucyl-p-nitroaniline, and 0.5 g / L Proclin300;

[0072] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0073] Comparative Example 3

[0074] A leucine aminopeptidase assay kit comprises R1 reagent and R2 reagent, wherein the R1 reagent comprises: Tris-diGlycine buffer solution with a pH of 7.5 and a concentration of 50 mmol / L, 3 mmol / L EDTA, 5 g / L NP-9, 30 g / L NaCl and 0.5 g / L Proclin300;

[0075] The R2 reagent components include: 100 mmol / L 4-morpholineethanesulfonic acid (MES) buffer at pH 5.5, 1 mmol / L EDTA, 15 mmol / L L-leucyl-p-nitroaniline, and 0.5 g / L Proclin300;

[0076] The volume ratio of the R1 reagent to the R2 reagent is 4:1.

[0077] Experimental Example 1

[0078] For Examples 1, 2, 3, 4, 5, 6 and Comparative Examples 1, 2, 3, 5 samples with low LAP concentration (about 3 U / L, 5 U / L, 10 U / L, 15 U / L, 20 U / L, respectively) were tested, and each sample was tested 10 times. The mean, SD and CV were calculated, and the experimental data and analysis results were recorded in Table 2. The low value repeatability of the LAP assay kit was investigated.

[0079] Experimental Example 2

[0080] For Examples 1, 2, 3, 4, 5, 6 and Comparative Examples 1, 2, 3, a high-value sample with a LAP concentration of about 400 U / L was taken and diluted 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 times with normal saline, and the relative deviation between the reduced concentration and the theoretical concentration was calculated, and the relative deviation analysis results were recorded in Table 3. The ability of the LAP assay kit to determine high-value samples was investigated.

[0081] Experimental Example 3

[0082] For Examples 1, 2, 3, 4, 5, 6 and Comparative Examples 1, 2, 3, total bilirubin (TB), direct bilirubin (DB), triglycerides (TG), ascorbic acid (VC), hemoglobin (HB), and heparin interference samples were obtained by adding; the amount of each interfering substance added is shown in Table 1, and interference samples containing different concentrations were prepared, and the deviation between the interference sample and the matrix sample was calculated and determined, and the analysis results were recorded in Table 4. The interference ability of the LAP assay kit against total bilirubin (TB), direct bilirubin (DB), triglycerides (TG), ascorbic acid (VC), hemoglobin (HB), and heparin was investigated.

[0083] Table 1 Addition amount of interference

[0084]

[0085] Table 2 Repeatability experiment of low value samples

[0086]

[0087]

[0088] Table 3 High value sample dilution recovery experiment

[0089]

[0090] Table 4 Interference experiment

[0091]

[0092]

[0093]

[0094]

[0095] It can be seen that the repeatability of low values ​​determined in Examples 1 to 6 is good, and the recovery rate of high-value samples is better than that of Comparative Examples 1 to 3. In terms of anti-TG interference ability, Examples 1 to 6 and Comparative Examples 2 and 3 are better, and Comparative Example 1 is worse. In terms of anti-HB interference ability, Examples 1 to 6 and Comparative Example 1 are better, and Comparative Examples 2 and 3 are worse. In terms of anti-HB interference ability, Examples 1 to 6 and Comparative Examples 1 to 3 are both good, and in terms of anti-TB and DB interference ability, Examples 1 to 6 are better than Comparative Examples 1 to 3. This is because the metal chelator, surfactant, and appropriate buffer pH and volume ratio of R1 and R2 make the detected object show good stability and selectivity in the reaction system.

Claims

1. A kit for determining leucine aminopeptidase, characterized in that: The invention comprises R1 reagent and R2 reagent; the R1 reagent comprises the following components: 50-100 mmol / L zwitterionic buffer, 3 mmol / L metal chelating agent, 5-7 g / L nonionic surfactant, 30 g / L NaCl and 0.1-1 g / L preservative proclin300; the zwitterionic buffer is tris(hydroxymethylaminomethane) buffer with a pH of 7.5, and the buffer pair of the zwitterionic buffer is tris(hydroxymethylaminomethane) and diglycine buffer pair; The R2 reagent comprises the following components: 20-50 mmol / L citrate buffer, 1-5 mmol / L metal chelator, 15-40 mmol / L L-leucyl-P-nitroaniline and 0.1-1 g / L preservative proclin300; the pH of the citrate buffer is 4.5; The metal chelating agent is ethylenediaminetetraacetic acid; the volume ratio of the R1 reagent to the R2 reagent is 3:1 to 4:1; The nonionic surfactant is one or more of nonylphenol polyoxyethylene ether, octylphenol polyoxyethylene ether, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate.

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