CN110132945B

CN110132945B - Chemiluminescence method kit formula for eliminating urea interference

Info

Publication number: CN110132945B
Application number: CN201910495126.3A
Authority: CN
Inventors: 李立和; 杨朕; 张超
Original assignee: Tianjin Baodi Hospital
Current assignee: Tianjin Baodi Hospital
Priority date: 2019-06-10
Filing date: 2019-06-10
Publication date: 2021-09-03
Anticipated expiration: 2039-06-10
Also published as: CN110132945A

Abstract

The invention discloses a formula of a chemiluminescence method kit for eliminating urea interference, belongs to a method for testing materials by using visible light and generating color change through a test reaction result, and belongs to the technical field of immunoassay. The technical scheme of the invention is as follows: the chemiluminescence method sample diluent is Tris-HCL buffer solution containing urease, the dosage of the urease is calculated according to the volume of a serum sample, the dilution volume, the reaction time and an enzyme kinetic equation, so that the urea enzymatic reaction is completely decomposed before the determination reaction.

Description

Chemiluminescence method kit formula for eliminating urea interference

Technical Field

The present invention pertains to a method of assay comprising an enzyme; or a method for testing materials by using visible light and generating color change through the result of test reaction, in particular to a determination method for eliminating chemiluminescence interference by using urease.

Background

Chemiluminescence immunoassay (CLIA) is a technology that combines chemiluminescence assay technology with high sensitivity and high specificity immunoreaction, is used for detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like, and is a latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Clinical laboratories for immunological analysis often include three types, 1, direct chemiluminescence, and acridinium ester (yapei) or ABEI (new industry) as a marker. 2. Enzymatic chemiluminescence, and the marker is alkaline phosphatase (xiamenbo boshi) or horseradish peroxidase (johnson). 3. Electrochemiluminescence, and the marker is terpyridyl ruthenium (Roche). In clinical immunoassay, urea often interferes with antigen-antibody combination to influence a determination result, and experimental research shows that the interference of urea on the determination result can reach 19-31% in 6M urea/5 min, 7M urea/5 min, 8M urea/5 min and 7M urea/10 min, the urea content in serum of healthy people and patients is respectively 3.1-8.0 mmol/L and 8.0-70 mmol/L (such as renal failure patients), and the urea in serum often causes certain interference when a chemiluminescence method is applied for detection.

The combination of antigen and antibody is the combination between antigen epitope and antigen combining point in antibody hypervariable region, and because the two are in complementary relation in chemical structure and space configuration, the antigen determinant and the antibody molecule variable region are in complementary configuration, so that the two molecules have relatively strong affinity. The spatial configuration has different complementary degrees, the bonding force between the antigen and the antibody molecules is different, the complementary degree is high, the affinity is strong, the antigen-antibody combination is actually the combination of the molecular surface, just like the combination of enzyme and a substrate, and is a non-covalent bond combination; the binding is the result of the combined action of electrostatic attraction, van der waals force, hydrogen bonding and hydrophobic bonding, which stabilizes antigen-antibody binding and exhibits reversibility of dissociation. After high-concentration urea is added in an experiment, a large number of urea molecules and an antibody peptide chain form stronger hydrogen bond capability than water molecules, hydrogen atoms in the urea molecules and oxygen atoms in the antibody peptide chain react to form hydrogen bonds, and the hydrogen bonds in antibody protein molecules can be destroyed, so that secondary structures such as spiral, folding and free rotary structures of a hydrogen bond maintenance system in the antibody peptide chain are destroyed, the hydrophobic effect of protein nonpolar genes and water is further reduced, the hydrogen bonds and the hydrophobic effect among antigen and antibodies are greatly reduced, the low-affinity antibody and the antigen are combined with poor complementary degree and are dissociated, and the antibody is eluted by a washing solution; the high affinity antibody is not eluted by the eluent because of its low influence due to its high degree of complementarity to the antigen.

Urease is often used as a tool enzyme in biochemical reagents to measure serum urea nitrogen, urea in serum is hydrolyzed to generate ammonia and carbon dioxide under the action of urease, and NADH is oxidized to generate NAD under the action of glutamate dehydrogenase under the action of alpha-ketoglutarate and reduced coenzyme I⁺The change in absorbance at a wavelength of 340nm was monitored and the urea content in the serum was calculated. In the chemiluminescence method measurement of the invention, when the serum is added into the diluent containing urease, the urea in the serum is hydrolyzed to generate ammonia and carbon dioxide under the action of the urease, and the positive reaction is easy to be carried out in the acidic buffer solution, so that the urea in the reaction solution does not interfere the combination of antigen and antibody in the chemiluminescence method, thereby not influencing the reaction result. Particularly, in patients before and after dialysis, the test results showed large variation due to the change of urea in serum.

In the chemiluminescence method reagent, a serum diluent is phosphate buffer solution or normal saline in hepatitis series detection, a phosphate buffer solution containing calf serum is commonly used in the serum diluent in free thyroxine and free triiodothyronine detection, and in the invention, Tris-HCL buffer solution containing urease is used as a sample diluent for measuring the treponema pallidum antibody as an example, so that the interference is obviously reduced. The dosage of the urease in the diluent is related to the dilution ratio and the dilution action time of the sample, and the dosage of the urease is calculated according to an enzyme kinetics method.

The reaction formula is as follows:

the invention content is as follows:

in order to solve the problem that the endogenous urea in serum interferes with antigen-antibody reaction to influence the measurement result in the prior art, the invention provides the chemiluminescence measurement method which is economic, convenient and feasible, has higher accuracy and can eliminate the influence of the endogenous urea.

The technical scheme is as follows: Tris-HCL containing urease is used as a buffer solution for sample dilution, the invention takes the angstrom bioassay treponema pallidum antibody as an example to prepare the sample dilution, and the Japanese east ocean spinning urease is used, and the Mie constant of the Japanese east ocean spinning urease is 1.05 multiplied by 10^-2And M. The specific parameters are as follows: the volume of the serum sample is 10 mu L, the volume of the diluent is 300 mu L, the dilution action time is 5 minutes, the tool enzyme dosage is calculated according to an enzyme kinetic equation, and 41.66mL of urease solution is dissolved in 1L of Tris-HCL buffer solution.

Compared with the prior art, the invention has the following advantages: the chemiluminescence method sample diluent prepared by the invention is not interfered by endogenous urea in serum, particularly for renal failure patients, and the chemiluminescence method is influenced for determination due to nonspecific interference caused by high-concentration urea.

The specific implementation mode is as follows:

the present invention will be described in further detail by the following examples of the determination of antibodies to treponema pallidum using the FIG. A2000PLUS chemiluminescence analyzer.

Example 1

The composition of the reagent is as follows:

a. the treponema pallidum antibody reagent is a reagent matched with Zhengzhou Antu bioengineering GmbH, the detection is carried out by adopting a double-antigen sandwich method principle, magnetic particles are coated by treponema pallidum antigen, horseradish peroxidase marks the treponema pallidum antigen to prepare an enzyme conjugate, an antigen-antibody-antigen-enzyme complex is formed through immune reaction, the complex catalyzes a luminescent substrate to emit photons, and the luminescent intensity is in direct proportion to the content of the treponema pallidum antibody.

b. The sample diluent is a diluent prepared by the invention, each liter of Tris-HCl buffer solution contains 150mmol (pH6.0) of Tris dissolved in 41.66mL of urease solution and 200 mul of Proclin-300 preservative, and the final concentration of the urease is more than or equal to 1000U/L.

Example 2

The composition of the reagent is as follows:

a. the treponema pallidum antibody reagent is a matched reagent of Zhengzhou AnTu bioengineering GmbH.

b. The sample diluent is a reagent matched with Zhengzhou Antu bioengineering GmbH.

Example 3

1. Detecting an object: 66 renal failure patients, 50 non-syphilitic patients, 28 men, and an average age of 38.5 years; women 22 cases, mean age 34.5 years. 16 cases of syphilis infected patients, of which 9 men had an average age of 44.5 years; female 7 cases, average age 36.5 years.

2. The method comprises the following steps: the antibodies to treponema pallidum of renal failure patients were measured in example 1 and example 2, respectively, and the change in luminescence values before and after dialysis was compared between patients.

3. The instrument comprises the following steps: chemiluminescence detector for AutoLumo A2000PLUS

4. Results

4.1 comparison of luminescence values before and after dialysis of patients with renal failure not infected with syphilis, as determined in example 1 and example 2, respectively, is shown in Table 1:

TABLE 1 comparison of the luminescence values (RLU) of patients with renal failure due to non-syphilis infection before and after dialysis measured by the two methods

4.2 comparison of luminescence values before and after dialysis of patients with syphilis-infected renal failure measured using example 1 and example 2, respectively, are shown in Table 2:

TABLE 2 comparison of the luminescence values (RLU) of syphilis-infected renal failure patients before and after dialysis measured by the two methods

Statistical analysis of tables 1 and 2 reveals: the result of luminescence values of the patients with renal failure in the non-syphilis infection group is obviously different by adopting the example 2, wherein t is 11.98, and P is 0.0338; the result of luminescence values is highly significant difference before and after the determination of the renal failure patients in the syphilis infection group, wherein t is 79.89, and P is 0.0091. The luminous value results of the patients with renal failure in the non-syphilis infection group are not significantly different by adopting the example 1, wherein t is 1.04, and P is 0.5338; the results of the luminescence values before and after the renal failure of the syphilis infection group are determined to have no significant difference, t is 2.05, and P is 0.3776.

The statistical analysis shows that the interference of endogenous urea in serum can be effectively reduced by adopting the dilution liquid containing urease and adopting the chemiluminescence method, and particularly, the measurement result of patients with high uremia has consistency and reliability, thereby having popularization and application prospects and application values.

Claims

1. A reagent kit formula for eliminating urea interference by a chemiluminescence method is characterized in that a sample diluent contains urease, urea in serum can be decomposed by the urease to eliminate interference of the urea on the antigen-antibody affinity, the sample diluent is Tris-HCL buffer solution containing the urease, and the enzyme dosage is calculated according to the volume of a serum sample, the volume of the diluent, the reaction time and an enzyme kinetic equation, so that urea enzymatic reaction can degrade the urea before the determination reaction, and the urea does not interfere with the chemiluminescence method for determination.

2. The formula of the chemiluminescence method kit for eliminating urea interference according to claim 1, wherein 41.66mL of urease solution and 200. mu.l of proclin-300 preservative are dissolved in each liter of sample diluent, and the final concentration of urease is more than or equal to 1000U/L.

3. The chemiluminescence method kit formula for eliminating urea interference according to claim 1, wherein the concentration of Tris-HCl buffer solution in sample dilution is150 mmol/L.