WO2019015156A1

WO2019015156A1 - Screening method for a stabilizer for a chemiluminescent reaction

Info

Publication number: WO2019015156A1
Application number: PCT/CN2017/107931
Authority: WO
Inventors: 欧赛英; 潘杨斌; 周慧琳
Original assignee: 苏州长光华医生物医学工程有限公司
Priority date: 2017-07-20
Filing date: 2017-10-27
Publication date: 2019-01-24
Also published as: CN109283171A; CN109283171B

Abstract

A screening method for a stabilizer in a chemiluminescent reaction. The method uses a first structure type acridine sulfonamide to conduct hydrogen peroxide testing in order to observe the ability of different stabilizers to consume hydrogen peroxide. The stabilizers comprise the following substances with reducing properties: BSA, reduced amino acids such as glycine, lycine and histidine, citric acid, and TRIS buffers. The method is used to provide a reference for the selection and usage of a stabilizer in a chemiluminescent reaction.

Description

Method for screening chemiluminescence reagent stabilizer

Technical field

The invention relates to a screening method of a chemiluminescence reagent stabilizer, and belongs to the technical field of chemical analysis.

Background technique

Chemiluminescence immunoassay is a novel labeling immunoassay technology that combines a chemiluminescent system with an immune response for the detection of trace antigens or antibodies. It has good selectivity, high sensitivity, strong specificity, no radioactive hazard, and analysis. The advantages of fast speed, simple equipment, etc., have been widely used in the fields of environment, clinical, food, drug testing, etc., and become the research hotspot and development trend of immunoassay methods.

As a marker for chemiluminescent immunoassay reagents, acridine compounds have been widely used in chemiluminescence immunoassays. Acridine compounds have many important advantages over other markers (such as radioactive elements, enzymatic labels), such as relatively high sensitivity, low cost, wide linear range, and relatively simple signal detection equipment.

Although acridine compounds have many advantages in the use of chemiluminescence immunoassays, there are still some problems, especially when the chemiluminescence signal is initiated by an oxidation reaction, and some occasional oxidants such as hydrogen peroxide in the liquid reagent. The presence of (for example, the surfactant in the reagent will automatically generate hydrogen peroxide, some liquids also have a small amount of hydrogen peroxide, and the reagent will also produce hydrogen peroxide during production, transportation and storage), acridine The marker has a destabilizing effect. In addition, components of various stabilizing reagents used in chemiluminescence immunoassay methods, such as immunoglobulins, albumin, etc., contain oxidation-sensitive amino acids such as cysteine, methionine, tryptophan, and tyrosine. Etc., can also be oxidized by hydrogen peroxide to eliminate or change biological activity.

A change in the activity of the above acridine label or protein results in a decrease in the accuracy of the reagent detection result, and therefore it is necessary to remove some accidental oxidant such as hydrogen peroxide when optimizing the reagent component, but existing The technology has no reference to the choice and dosage of hydrogen peroxide consumption reagents. It is usually necessary to add stabilizers in real time to investigate the real-time stability of the reagents to investigate the hydrogen peroxide consumption capacity of the stabilizers, but this takes up to several months or even one. In two years, it is necessary to establish a rapid measurement method to investigate the hydrogen peroxide consumption capacity of the stabilizer, and pre-screen the chemiluminescence reagent stabilizer with suitable hydrogen peroxide consumption ability, which provides a guiding basis for the stability study of the reagent.

Summary of the invention

The technical problem to be solved by the present invention is to provide a screening method for a chemiluminescent reagent stabilizer, which solves the technical problems in the prior art that there is no reference to the selection and dosage of hydrogen peroxide depleting reagents. The chemiluminescence reagent stabilizer detects the hydrogen peroxide consumption ability, and selects a chemiluminescence reagent stabilizer with a suitable hydrogen peroxide consumption capacity according to the detection result, which greatly saves the working intensity and time for screening the chemiluminescence reagent stabilizer.

The technical solution adopted by the present invention to solve the technical problem thereof is:

A screening method for a chemiluminescent reagent stabilizer, comprising the following steps,

Selected several stabilizers;

Take a series of C0-C1 concentration range of hydrogen peroxide standard solution with a volume of V1, and add high-purity water and a chemiluminescence detection reagent with a volume of V2 and a concentration of C2, respectively, and immediately add a volume of V3 and a concentration of C3. The excitation liquid collects the total number of photons in a period of time, and obtains a standard curve by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution;

Take a certain concentration of hydrogen peroxide standard solution in the same volume of C0-C1 concentration range as the standard curve, the number of parts is at least 1 part more than the stabilizer, and add to one of the hydrogen peroxide standard solutions. The high-purity water with the same volume of high-purity water in the process of establishing the standard curve, and the other hydrogen peroxide standard solution are added with the same volume of the high-purity water in the process of establishing the standard curve, respectively, and then separately added to the volume. V2, a chemiluminescence detection reagent with a concentration of C2, and immediately added Collecting the total number of photons in the same period of time as the standard curve is established with the excitation liquid with volume V3 and concentration C3; comparing the total photon number measured after adding a certain stabilizer and high purity water with the established standard curve , respectively, the hydrogen peroxide concentration C' after adding each stabilizer and the hydrogen peroxide concentration C without the stabilizer are obtained, and the calculation rate is calculated according to the formula degradation rate (H2O2)=(CC')/C*100%. a hydrogen peroxide degradation rate of a stabilizer;

A chemiluminescent reagent stabilizer having a suitable hydrogen peroxide consumption ability is selected according to the hydrogen peroxide degradation rate of each stabilizer.

Preferably, V2 · C2 ≥ 1.5 (V1 · C1), and V2 · C2 = (2-5) V3 · C3.

Preferably, the chemiluminescence detecting reagent is an acridine sulfonamide solution, and the structural formula of the acridine sulfonamide is as follows:

Wherein R and R' are one selected from the group consisting of an alkyl group and a substituent thereof, and X and X' are one selected from the group consisting of a sulfonyl group, a halogen, a carboxyl group, and an N-hydroxysuccinimide.

Preferably, the acridine sulfonamide is one of the following structures:

Preferably, the stabilizer is one or more of BSA, a reducing amino acid, and a reducing buffer.

Preferably, the stabilizer is BSA, glycine, glutamic acid, lysine, histidine, PB, citric acid-Na ₂ HPO ₄ , citric acid-TRIS, MES-TRIS, MES-NaOH buffer One or several.

Preferably, the buffer is acidic.

Preferably, the incubation temperature is 37 ° C and the time is from 0.5 h to 240 h.

Preferably, said D is at least 5 seconds.

The beneficial effects of the invention are:

(1) Screening method of the chemiluminescent reagent stabilizer of the present invention First, several kinds of chemiluminescent reagent stabilizers are selected, and the consumption ability of each chemiluminescent reagent stabilizer for hydrogen peroxide is detected, and hydrogen peroxide is selected according to the detection result. A chemiluminescent reagent stabilizer that is suitable for consumption. The method greatly saves the working intensity and time of the screening reagent stabilizer, and the detection is fast and efficient, and provides a reference for the selection and dosage of the chemiluminescent reagent stabilizer.

(2) Screening method of the chemiluminescent reagent stabilizer of the present invention When measuring the consumption rate of hydrogen peroxide, the pH of the solution changes due to the addition of a stabilizer and no stabilizer, and the standard curve is without a stabilizer. In the present invention, the acridine sulfonamide of the present invention is suitable for determining the hydrogen peroxide content of different pH solutions after the addition of the stabilizer, and the measurement result is accurate.

Detailed ways

Example 1

The present embodiment provides a screening method for a chemiluminescent reagent stabilizer, which comprises the steps of selecting amino acids such as glycine, lysine, histidine and glutamic acid as stabilizers for chemiluminescent reagents, and peroxidizing each amino acid. The degradation rate of hydrogen is detected, and according to the detection result, an amino acid having a suitable hydrogen peroxide consumption capacity can be selected, and the steps of detecting the hydrogen peroxide consumption ability of the different amino acids are as follows:

Take 50 μL of 2.5 μM, 10 μM, 40 μM, 100 μM, 250 μM hydrogen peroxide standard solution, add 50 μL of high purity water and 50 μL of 2 M acridine sulfonamide solution, and immediately add 100 μL of hydrogen at a concentration of 0.25 M. The sodium oxide solution was used to collect the total photon number of 5S, and the linear standard curve was obtained by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution, y=10658x+5963.1, R ² =0.9998;

Take 50 μL of a 10 μM hydrogen peroxide standard solution, add 50 μL of 1% amino acid to one part, and add 50 μL of high purity water as a control, and incubate at 37 ° C for 0.5 h, 12 h, 24 h, respectively. After 72h and 240h, 50 μL of 2M acridinesulfonamide solution was added, and 100 μL of 0.25 M sodium hydroxide solution was added immediately, and the total photon number after 5S was collected.

Comparing the total number of photons measured in the detection step with the established linear standard curve, and calculating the hydrogen peroxide concentration C' after adding the stabilizer and the hydrogen peroxide concentration C without the stabilizer, according to the formula degradation rate (H) ₂ O ₂ )=(CC')/C*100%, and the degradation rate of hydrogen peroxide was calculated.

The structural formula of the acridine sulfonamide is as follows:

The degradation rates of various amino acids at different incubation times are shown in the following table:

It can be seen from the above table that the amino acid stabilizers selected in the experiment all have certain hydrogen peroxide consumption ability, among which glycine, lysine and histidine all show strong hydrogen peroxide consumption ability, while glutamic acid pair The degradation ability of hydrogen peroxide is weak. Actually, according to the test results, amino acids with suitable hydrogen peroxide consumption capacity can be selected. For example, when the peroxide consumption capacity is strong, glycine, lysine and histidine can be selected as stabilizers, when peroxide consumption capacity is required. When it is weak, glutamic acid can be selected as a stabilizer.

Example 2

This embodiment provides a screening method for a chemiluminescent reagent stabilizer, comprising the following steps: selecting 0.1 M PB (pH=6.8), 0.1 M citric acid-Na ₂ HPO ₄ (pH=6.8), 0.1 M citric acid- Buffers such as TRIS (pH=6.8), 0.1M MES-TRIS (pH=6.8), 0.1M MES-NaOH (pH=6.8) as chemiluminescent reagent stabilizers, the ability to consume hydrogen peroxide for each buffer The detection is performed, and according to the detection result, a buffer having a suitable hydrogen peroxide consumption capacity can be selected, and the detection steps of the different buffers for the hydrogen peroxide consumption ability are as follows:

100 μL of a 2.5 μM, 10 μM, 40 μM, 100 μM, 250 μM hydrogen peroxide standard solution was added thereto, and 150 μL of high-purity water and 150 μL of a 1 M acridine sulfonamide solution were added thereto, and 50 μL of hydrogen having a concentration of 0.6 M was immediately added. The sodium oxide solution was used to collect the total photon number of 5S, and the linear standard curve was obtained by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution, y=15628x+4543.2, R ² =0.9999;

Take 100 μL of 10 μM hydrogen peroxide standard solution, add 150 μL of 0.1 M buffer to one part, and add 150 μL of high purity water as control. Incubate at 37 ° C for 0.5 h, 12 h, 24 h. After 72h and 240h, 150 μL of a 1 M acridine sulfonamide solution was added, and 50 μL of a 0.6 M sodium hydroxide solution was immediately added to collect the total photon number after 5 S;

The structural formula of the acridine sulfonamide is as follows:

The degradation rates of various buffers at different incubation times are shown in the following table:

Since acridinesulfonamide itself is unstable in an alkaline environment, it is slow to select a luminescent reagent. The flushing is all acidic. It can be seen from the above table that the buffers of PB and MES-NaOH in the buffer selected from the experiment cannot consume hydrogen peroxide, and the citric acid-Na2HPO4 and citric acid-TRIS show strong hydrogen peroxide consumption capacity, while MES -TRIS has a relatively weak ability to degrade hydrogen peroxide. Actually, a buffer suitable for hydrogen peroxide consumption can be selected according to the detection result. For example, when a peroxide consumption capacity is required, citric acid-Na2HPO4 and citric acid-TRIS can be selected as stabilizers, and when peroxide consumption is required. When the ability is weak, MES-TRIS can be selected as a stabilizer.

Example 3

In order to investigate the ability of different concentrations of stabilizers to consume hydrogen peroxide, this example uses BSA as a stabilizer to select different concentrations of BSA to detect the hydrogen peroxide consumption capacity. According to the test results, hydrogen peroxide consumption capacity can be selected. A suitable concentration of stabilizer, the steps of detecting the hydrogen peroxide consumption capacity of the different concentrations of BSA are as follows:

150 μL of a 2.5 μM, 10 μM, 40 μM, 100 μM, 250 μM hydrogen peroxide standard solution was added thereto, and a volume of 100 μL of high-purity water and 100 μL of a 0.5625 M acridine sulfonamide solution were added thereto, and 150 μL of a concentration of 0.1875 M was immediately added. The sodium hydroxide solution was used to collect the total photon number of 5S, and the linear standard curve was obtained by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution, y=6758x+6356.5, R ² =0.9998;

Take 150 μL of a 40 μM hydrogen peroxide standard solution, add 100 μL of BSA in the range of 0.1%-10% to one part, and add 100 μL of high-purity water as a control in the other, and incubate at 37 ° C for 0.5 h. After 12h, 24h, 72h, 240h, 100 μL of 0.5625 M acridine sulfonamide solution was added, and 150 μL of 0.1875 M sodium hydroxide solution was immediately added to collect the total photon number after 5S;

The structural formula of the acridine sulfonamide is as follows:

The degradation rates of different concentrations of BSA under different incubation times are shown in the following table:

BSA is a globulin in bovine serum. Since it has a reducing cysteine residue on the surface and is reducible, it can consume hydrogen peroxide in the solution to protect the stability of the luminescent reagent. As can be seen from the above table, it can be seen from the results that the higher the BSA concentration, the higher the degradation rate of hydrogen peroxide under the same incubation time. Actually, a stabilizer suitable for the consumption of hydrogen peroxide can be selected according to the test result. For example, when the peroxide consumption capacity is required, a higher concentration of the stabilizer can be selected, and when the peroxide consumption capacity is required to be weak, Choose a lower concentration of stabilizer.

Example 4

In order to investigate the ability of stabilizers to consume hydrogen peroxide at different concentrations, this example uses BSA as a stabilizer to select 1% BSA to detect the consumption capacity of 2.5μM-250μM hydrogen peroxide. The concentration of hydrogen peroxide is selected to be a certain concentration and kind of stabilizer having a suitable hydrogen peroxide consumption capacity. The procedure for detecting the consumption capacity of the 1% BSA for 2.5 μM-250 μM hydrogen peroxide is as follows:

Take 50 μL of 2.5 μM, 10 μM, 40 μM, 100 μM, 250 μM hydrogen peroxide standard solution, add 50 μL of high purity water and 50 μL of 2 M acridine sulfonamide solution, and immediately add 100 μL of hydrogen at a concentration of 0.35 M. The sodium oxide solution was used to collect the total photon number of 5S, and the linear standard curve was obtained by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution, y=11028x+5375.4, R ² =0.9998;

Take 50 μL of 2.5 μM, 10 μM, 40 μM, 100 μM, 250 μM hydrogen peroxide standard solution in two portions, add 50 μL of 1% BSA to the same concentration of one part of hydrogen peroxide standard solution, and add 50 μL of high purity water to the other part. As a control, after incubation at 37 ° C for 0.5 h, 12 h, 24 h, 72 h, 240 h, add 50 μL of 2 M acridine sulfonamide solution, and immediately add 100 μL of 0.25 M sodium hydroxide solution to collect separately. The total number of photons after 5S;

The structural formula of the acridine sulfonamide is as follows:

The degradation rates of different concentrations of hydrogen peroxide standard solutions under different incubation times are shown in the following table:

As can be seen from the above table, the consumption of hydrogen peroxide by BSA is a gradual process. The higher the concentration of hydrogen peroxide, the slower the degradation rate of hydrogen peroxide by the stabilizer BSA. Actually, a stabilizer suitable for hydrogen peroxide consumption can be selected according to the detection result. For example, when the hydrogen peroxide content in the chemiluminescence reagent is high, a stabilizer having a relatively high concentration of peroxide consumption can be selected, when chemiluminescence is obtained. When the hydrogen peroxide content of the reagent is low, a stabilizer having a relatively low concentration of peroxide consumption can be selected.

The invention adopts a kind of structure of acridine sulfonamide, and the quantitative analysis method of hydrogen peroxide is used to determine the consumption capacity of the stabilizer for hydrogen peroxide. The pH of the solution changes due to the addition of a stabilizer and no stabilizer, and the standard The curve is made without a stabilizer, and the acridine sulfonamide used in the present invention is suitable for determining the hydrogen peroxide content of different pH solutions after the addition of the stabilizer.

In view of the above-described embodiments of the present invention, various changes and modifications may be made by those skilled in the art without departing from the scope of the invention. The technical scope of the present invention is not limited to the contents of the specification, and the technical scope thereof must be determined according to the scope of the claims.

Claims

A screening method for a chemiluminescent reagent stabilizer, characterized in that it comprises the following steps,

Selected several stabilizers;

Take a series of C0-C1 concentration range of hydrogen peroxide standard solution with a volume of V1, and add high-purity water and a chemiluminescence detection reagent with a volume of V2 and a concentration of C2, respectively, and immediately add a volume of V3 and a concentration of C3. The excitation liquid collects the total number of photons in a period of time, and obtains a standard curve by the correspondence between the total photon number and the concentration of the hydrogen peroxide standard solution;

Take a certain concentration of hydrogen peroxide standard solution in the same volume of C0-C1 concentration range as the standard curve, the number of parts is at least 1 part more than the stabilizer, and add to one of the hydrogen peroxide standard solutions. The high-purity water with the same volume of high-purity water in the process of establishing the standard curve, and the other hydrogen peroxide standard solution are added with the same volume of the high-purity water in the process of establishing the standard curve, respectively, and then separately added to the volume. V2, the chemiluminescence detection reagent with the concentration of C2, and immediately add the excitation liquid with the volume of V3 and the concentration of C3, respectively collect the total photon number in the same time period as the standard curve is established; a certain stabilizer and The total photon number measured after high purity water is compared with the established standard curve, and the hydrogen peroxide concentration C' after adding each stabilizer and the hydrogen peroxide concentration C without stabilizer are obtained respectively, according to the formula degradation rate (H2O2). = (CC') / C * 100%, calculate the degradation rate of hydrogen peroxide under each stabilizer;

A chemiluminescent reagent stabilizer having a suitable hydrogen peroxide consumption ability is selected according to the degradation rate of each stabilizer.
The method for screening a chemiluminescent reagent stabilizer according to claim 1, wherein V2 · C2 ≥ 1.5 (V1 · C1), and V2 · C2 = (2-5) V3 · C3.
The method for screening a chemiluminescent reagent stabilizer according to claim 1, wherein the chemiluminescence detecting reagent is an acridinesulfonamide solution, and the structure of the acridinesulfonamide The formula is as follows:

Wherein R and R' are one selected from the group consisting of an alkyl group and a substituent thereof, and X and X' are one selected from the group consisting of a sulfonyl group, a halogen, a carboxyl group, and an N-hydroxysuccinimide.
The method for screening a chemiluminescent reagent stabilizer according to claim 3, wherein the acridinesulfonamide is one of the following structures:
The method for screening a chemiluminescent reagent stabilizer according to claim 1, wherein the stabilizer is one or more of BSA, a reducing amino acid, and a reducing buffer.
The method for screening a chemiluminescent reagent stabilizer according to claim 5, wherein the stabilizer is BSA, glycine, glutamic acid, lysine, histidine, PB, citric acid-Na 2 HPO 4 One or more of citric acid-TRIS, MES-TRIS, MES-NaOH buffer.
The method for screening a chemiluminescent reagent stabilizer according to claim 6, wherein the buffer is acidic.
The method for screening a chemiluminescent reagent stabilizer according to any one of claims 1 to 7, wherein the incubation temperature is 37 ° C and the time is from 0.5 h to 240 h.
The method for screening a chemiluminescent reagent stabilizer according to any one of claims 1 to 8, characterized in that the total photon number collection time is at least 5 seconds.