CN112710609B

CN112710609B - Anti-chyle interference uric acid determination kit

Info

Publication number: CN112710609B
Application number: CN202011535494.5A
Authority: CN
Inventors: 岳彩琴
Original assignee: BIOSINO BIO-TECHNOLOGY AND SCIENCE Inc
Current assignee: BIOSINO BIO-TECHNOLOGY AND SCIENCE Inc
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2022-03-04
Anticipated expiration: 2040-12-23
Also published as: CN112710609A

Abstract

The invention provides an anti-chylomicron interference uric acid determination kit, which comprises an anti-chylomicron interference reagent and reagents R1 and R2 for uric acid determination; wherein the anti-chylomicron interference reagent is contained in a reagent R1 or R2, and the concentration of the anti-chylomicron interference reagent in R1 or R2 is 0.01-10%. The invention takes the basic components of the uric acid determination kit as the basis, and 0.01 to 10 percent of one or more surfactants are added into the detection reagent, thereby eliminating the interference of chyle in a lipemia sample on the uric acid determination and improving the accuracy of the uric acid detection of the lipemia sample; the reagent has simple components and is easy to produce and prepare; low cost and easy commercialization.

Description

Anti-chyle interference uric acid determination kit

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to an anti-chyle interference uric acid determination kit.

Background

Uric acid is the end product of purine metabolism and is excreted by the kidneys with urine. Purine anabolism disorder, energy metabolism disorder and uric acid excretion disorder of kidney can cause uric acid concentration to increase or decrease. The uric acid determination has important significance for diagnosing gout, kidney diseases, metabolic diseases, liver diseases and the like.

Triglyceride (TG) in blood is one of the major components of lipoprotein, and dyslipidaemia is caused by disturbances of lipoprotein metabolism. The increase of the contents of Cholesterol (TC), Triglyceride (TG) and lipoprotein in a lipemia sample can cause the turbidity of the sample to increase or the sample to be chyle; hyperlipidemia is also often accompanied by hyperuricemia. Currently, the clinical determination of uric acid and triglyceride is mostly carried out on a full-automatic biochemical analyzer by adopting Trinder's reaction principle according to the Lamber-Beer law. Because chylomicrons in the lipemia sample have turbidities of different degrees and have certain interference on a uric acid detection result, effective measures are taken to reduce the interference of chylomicrons in the lipemia sample on uric acid determination, and the method is helpful for judging whether the hyperlipidemia is accompanied by uric acid increase in clinic, thereby providing a basis for diagnosis and treatment of clinical diseases.

The basic principle of the prior art for measuring uric acid is as follows: the uric acid is catalyzed by uricase to generate allantoin and hydrogen peroxide (H)₂O₂)，H₂O₂4-aminoantipyrine (4-AAP) and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS) generate a colored quinonimine compound under the catalysis of Peroxidase (POD), and the generated amount is in direct proportion to the concentration of uric acid. And detecting the change of the absorbance at the wavelength of 546nm (540 nm-560 nm) by using a biochemical analyzer to obtain the concentration of the uric acid in the sample to be detected.

In the prior art, ascorbic acid oxidase is added into a formula to eliminate the interference of ascorbic acid (VC) on reaction; the stability problem of the reagent is further solved by adding the stabilizing agent into the formula, but for the lipemia sample, chylomicron in the sample is aggregated, so that the turbidity of the reagent is increased to cause the abnormity of reaction absorbance, and the accuracy of the detection result of the lipemia sample is influenced.

Disclosure of Invention

The invention aims to provide an anti-chyle interference uric acid determination kit.

In order to achieve the purpose of the invention, the invention provides an anti-chylomicron interference uric acid determination kit, which comprises an anti-chylomicron interference reagent and reagents R1 and R2 for uric acid determination; wherein, the anti-chylomicron interference reagent is contained in the reagent R1 (preferably contained in the reagent R1), and the concentration of the anti-chylomicron interference reagent in the reagent R1 or R2 is 0.01-10%.

The anti-chyle interference agent is at least one selected from polyoxyethylene fatty alcohol nonionic surfactants, polyoxyethylene-propylene oxide fatty alcohol nonionic surfactants, polyethylene glycol alkyl ether nonionic surfactants, mixed fatty acid glyceride nonionic surfactants and the like.

The reagent R1 is: 10-100mM phosphate buffer solution with pH6.8-8.0, 1800-2400U/L peroxidase, 700-800U/L ascorbate oxidase and 0.2-0.5mM 4-aminoantipyrine.

The reagent R2 is: 10-100mM phosphate buffer pH6.8-8.0, 500-700U/L uricase and 1.0-2.0mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt or 3, 5-dichloro-2-hydroxybenzenesulfonic acid.

The polyoxyethylene fatty alcohol nonionic surfactant is a polymer containing 8-10 EO (Ethylene Oxide), and is preferably ZUSOLAT 1008/85 and/or OXETAL 800/85.

The polyoxyethylene-propylene oxide fatty alcohol nonionic surfactant is a polymer containing 8-10 EO-PO (propylene oxide), and preferably at least one of PROPETIAL 100, PROPETIAL 200, PROPETIAL 105, PROPETIAL 120, PROPETIAL 130, PROPETIAL 140, PROPETIAL 150, PROPETIAL 160 and the like.

The polyethylene glycol alkyl ether nonionic surfactant is preferably polyethylene glycol monoalkyl ether (Genapol X-080) and/or ethylphenyl polyethylene glycol (Nonidet P40).

The mixed fatty acid glyceride type nonionic surfactant is an ethoxylated triglyceride compound, preferably RT7 and/or RT163 and the like.

Preferably, the anti-chylomicron interference agent is contained in agent R1.

Further, the kit also contains 280-320 mu M uric acid calibrator.

The detection method of the kit comprises the following steps: the method adopts a full-automatic biochemical analyzer with double reagent functions to measure, and the detection wavelength is as follows: 546nm (540 nm-560 nm); temperature: 37 ℃; optical path of cuvette: 1 cm.

The method comprises the following specific operation steps:

the invention takes the basic components of the uric acid determination kit as the basis, and 0.01 to 10 percent of one or more nonionic surfactants are added into the detection reagent, so that the interference of chyle in a lipemia sample on the uric acid determination is eliminated, and the accuracy of the uric acid detection of the lipemia sample is improved; the reagent has simple components and is easy to produce and prepare; low cost and easy commercialization.

By using the surfactant and the buffer solution in the detection reagent, the interference of chyle on the measurement of uric acid in the lipemic sample is eliminated, which is probably because in a specific buffer solution, the nonionic surfactant has solubilization effect on the insoluble suspended particles in the lipemic sample, and the solubility is increased so as to reduce the turbidity caused by the insoluble suspended particles: when the triglyceride in the sample is less than or equal to 11.29mmol/L, the chyle (Intralipid) is less than or equal to 1000mg/dL, and the uric acid determination result is not obviously interfered.

Drawings

FIG. 1 is a graph showing the reaction curves of the

test groups

1, 2, 3 and example 1 for eliminating chyle samples on Hitachi 7180 full-automatic biochemical analyzer when the concentration of chyle in the sample is 1000mg/dL in the preferred embodiment of the present invention.

FIG. 2 is a graph showing the reaction curves of the elimination of chyle samples in each of

test groups

1, 2, and 3 and example 1 on Hitachi 7180 full-automatic biochemical analyzer when the triglyceride concentration in the sample is 11.29mmol/L in the preferred embodiment of the present invention.

Detailed Description

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

Peroxidase (horseradish peroxidase, POD), ascorbate oxidase, and Uricase (Uricase) used in the following examples were purchased from Toyo Boseki Biotech, Inc. and Roche, respectively; oxetal 800/85, PROPETAL 120 and RT163 were purchased from German Stelma chemical,

X-080 was purchased from Sigma.

EXAMPLE 1 anti-chylomicron interference uric acid determination reagent

The kit comprises three components of a reagent R1, a reagent R2 and a calibrator (297 mu M uric acid), and the compositions of the reagent R1 and the reagent R2 are shown in Table 1.

Table 1 example 1 reagent composition

EXAMPLE 2 anti-chylomicron interference uric acid determination reagent

The kit comprises three components of a reagent R1, a reagent R2 and a calibrator (297 mu M uric acid), and the compositions of the reagent R1 and the reagent R2 are shown in Table 2.

Table 2 example 2 reagent composition

Example 3 accuracy of anti-chylomicron interference uric acid reagent assay

Clinically approved uric acid detection reagent is taken as a control group 1; the reagent without the surfactant was control 2; triton X-100, a nonionic surfactant, was added as control 3, and the composition of the reagents is shown in Table 3.

Table 3 comparative example 3 reagent composition

Based on the reagent compositions of the control groups 1 to 3 and examples 1 to 2, the uric acid concentrations in the lipemia samples were measured, and the deviations of the measurement results of the control group 2, the control group 3, the example 1, and the example 2 from the control group 1 were calculated, respectively, to evaluate the accuracy. The test results are shown in Table 4.

Table 4 example 3 test results

The results show that when the concentration of triglyceride and chyle is 11.29mmol/L and 1000mg/dL respectively, the deviation of the measurement results of the example 1 and the example 2 from the control group 1 is within +/-5%, and the deviation of the control group 2 and the control group 3 from the control group 1 is more than +/-10%, which indicates that the accuracy of the measurement of the lipemia interference sample of the example 1 and the example 2 can reach the level of the control group 1, and the clinical requirement can be met.

Example 4 stability of anti-chylomicron interference uric acid assay reagent

Decap stability and potency stability tests were performed with examples 1 and 2 using the preferred reagents provided in CN104198686A (see detailed description) as control 3.

Test methods and results: the reagent is respectively uncapped at the temperature of 2-8 ℃ for 40 days and hermetically stored at the temperature of 2-8 ℃ for 24 months, the concentrations of uric acid in the level 1 and the level 2 of the biochemical composite quality control product of Zhongsheng Bei accuse biotechnology, Inc. are measured, the deviation of the measurement result and the target value is calculated, and the test result is shown in a table 5.

Table 5 example 4 test results

The results show that the deviation between the target values of the example 1 and the example 2 and the control group 3 is within +/-10% when the cover is opened at 2-8 ℃ for 40 days and the sample is hermetically stored at 2-8 ℃ for 24 months, and the deviation between the example 1 and the example 2 is smaller than that of the control group 3, which indicates that the cover opening stability and the actual effect stability of the example 1 and the example 2 are better than those of the control group 3.

EXAMPLE 5 selection of the amount of anti-chylomicron Interferon

Adding surfactants with different concentrations and proportions according to the table 6 to prepare a reagent R1 of

test groups

1, 2 and 3 and a reagent R1 of example 1; reagent R2 was prepared according to the composition of reagent R2 in Table 1.

On Hitachi 7180 full-automatic biochemical analyzer,

test groups

1, 2 and 3 tested the hypertriglyceridemia and chyle samples simultaneously with example 1. As shown in fig. 1 and 2, the abscissa is the time point (17.6 seconds per point) at which the instrument reads the absorbance value, and the ordinate is the absorbance value generated by the reaction of the sample with the reagent; adding the sample and the reagent R1 at the point of 0, uniformly mixing, starting to reduce the absorbance value, starting the reaction for eliminating chyle, wherein the reduction speed of the absorbance value represents the speed of eliminating the turbidity of chyle; prior to 16 points, absorbance values due to chylomicron turbidity were reduced to baseline levels (sample was purified water), thereby eliminating chylomicron interference; the reaction for uric acid determination was initiated by adding the reagent R2 at 16 o' clock (about 5 minutes).

As can be seen from the graph, when the concentration of triglyceride in the sample was 11.29mmol/L, and the concentration of chyle was 1000mg/dL, the

test groups

1, 2, 3 and example 1, the absorbance value due to the chyle turbidity was reduced to the baseline level before 16 o' clock, thereby eliminating chyle interference; the rate of decrease of absorbance values is related to the amount and ratio of surfactant, and the amount and ratio in example 1 works best.

TABLE 6 ingredient Table of reagent R1 in example 5

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

Claims

1. The kit for determining uric acid by resisting chylomicron interference is characterized by comprising an anti-chylomicron interference reagent and reagents R1 and R2 for determining uric acid; wherein the anti-chylomicron interference reagent is contained in a reagent R1 or R2, and the concentration of the anti-chylomicron interference reagent in a reagent R1 or R2 is 1-10%;

the anti-chylomicron interference agent is at least one selected from polyoxyethylene fatty alcohol nonionic surfactants, polyoxyethylene-propylene oxide fatty alcohol nonionic surfactants, polyethylene glycol alkyl ether nonionic surfactants and mixed fatty glyceride nonionic surfactants;

the reagent R1 is: 10-100mM phosphate buffer solution with pH value of 6.8-8.0, 1800-2400U/L peroxidase, 700-800U/L ascorbate oxidase and 0.2-0.5mM 4-aminoantipyrine;

the reagent R2 is: 10-100mM phosphate buffer pH6.8-8.0, 500-700U/L uricase and 1.0-2.0mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt or 3, 5-dichloro-2-hydroxybenzenesulfonic acid;

the polyoxyethylene fatty alcohol nonionic surfactant is ZUSOLAT 1008/85 and/or OXETAL 800/85;

the polyoxyethylene-propylene oxide fatty alcohol nonionic surfactant is selected from at least one of PROPETAL 100, PROPETAL 200, PROPETAL 105, PROPETAL 120, PROPETAL 130, PROPETAL 140, PROPETAL 150 and PROPETAL 160;

the polyethylene glycol alkyl ether nonionic surfactant is GenapolX-080 and/or Nonidet P40;

the mixed fatty acid glyceride type nonionic surfactant is RT7 and/or RT 163.

2. The kit of claim 1, wherein the anti-chylomicron interference agent is contained in reagent R1.

3. The kit according to claim 1 or 2, wherein the kit further comprises 280-320 μ M uric acid calibrator.