CN106990100B

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Info

Publication number: CN106990100B
Application number: CN201710198269.9A
Authority: CN
Inventors: 黄�俊; 路春桃; 赵平锋
Original assignee: Wuhan Hygeianey Bioscience Co ltd
Current assignee: Wuhan Hygeianey Bioscience Co ltd
Priority date: 2017-03-29
Filing date: 2017-03-29
Publication date: 2020-06-23
Anticipated expiration: 2037-03-29
Also published as: CN106990100A

Abstract

The invention discloses a stable HRP enzymatic chemiluminescence substrate solution, which comprises luminol or luminol derivatives or luminol sodium salt in a specific proportion, p-iodophenol as a reinforcing agent, phenolphthalein as a synergistic reinforcing agent, a luminescent agent solution prepared from a buffer solution, and an oxidant solution prepared from carbamide peroxide, disodium ethylene diamine tetraacetate, Tween-20 and the buffer solution in a specific proportion, wherein the luminescent agent solution and the oxidant solution are mixed before use. The HRP enzymatic chemiluminescence substrate liquid is simple in preparation, p-iodophenol is used as a reinforcing agent, phenolphthalein is used as a synergistic reinforcing agent, and tween-20 is used as a stabilizing agent, so that the stability of the luminescence liquid is further improved, and the stable HRP enzymatic chemiluminescence substrate liquid which is high in signal-to-noise ratio, high in sensitivity, long in platform period and purplish red is obtained.

Description

Stable HRP enzymatic chemiluminescence substrate solution

Technical Field

The invention relates to the field of immunoassay, in particular to a chemiluminescent substrate solution taking HRP as a detection system for enzymatic reaction.

Background

The chemiluminiscence immunoassay (CLIA) is a novel labeled immunoassay technology for detecting trace antigens or antibodies, which is established by combining luminescence analysis and immunoreaction, and has the specificity of immunoreaction and high sensitivity of chemiluminescence reaction. It overcomes the problems of radioactive hazard and pollution caused by using radioactive isotope in Radioimmunoassay (RIA); the defects of complex required instruments and large background interference in Fluorescence Immunoassay (FIA) are overcome. The kit is favored by people due to the unique advantages of high sensitivity, low instrument price, simple and convenient use, safety, no radioactive pollution and the like, and becomes an important direction of label immunization. Among them, chemiluminescence immunoassay using Acridinium Ester (AE), alkaline phosphatase (ALP) and horseradish peroxidase (HRP) as labels is the main development trend.

Luminol (Luminol), Isoluminol (Isoluminol) and its derivatives are the first chemiluminescent substances used in chemiluminescence methods, the chemical name of Luminol is 3-aminophthalic acid hydrazide, the characteristics of luminescence of which were first reported by Albrecht, which produces α -hydroxyperoxide intermediate by oxidation, the decomposition of which releases light energy, the property of luminescence is related to the pH of the reaction system, the oxidation step of Luminol compounds is related to the solvent composition, in protic solvents, enzymes are required to catalyze various oxygen derivatives to oxidize Luminol, in aprotic solvents, chemiluminescence only requires oxygen and a strong base, while strong alkalinity produces a certain inhibition effect on the activity of enzymes, and thus the direct determination of biomacromolecules using Luminol-hydroperoxide-horseradish peroxidase has low sensitivity.

The existing luminescent substrate commonly used in the market has poor stability and high background; the mixture of the luminescent agent solution and the oxidant solution is extremely unstable; the luminous liquid is colorless, sample adding is not facilitated, plate-type chemiluminescence manual operation is influenced, repeatability of an experiment is influenced, use cost is high, application of chemiluminescence immunoassay is severely limited, and a formula of the luminous liquid is researched aiming at the problems.

Disclosure of Invention

The invention aims to provide a stable HRP enzymatic chemiluminescence substrate solution to solve the problems of poor stability, high background, inaccurate result, high cost and the like of the conventional chemiluminescence substrate.

The invention adopts the following technical scheme:

a stable HRP enzymatic chemiluminescence substrate solution comprises a luminophore solution and an oxidant solution, wherein the luminophore solution contains luminol or luminol derivative or luminol sodium salt with a final concentration of 0.5-10 mmol/L, p-iodophenol with a final concentration of 0.5-10 mmol/L, phenolphthalein with a final concentration of 0.01-5 mmol/L, and a buffer solution with a pH value of 8.5-10.5;

the oxidant solution is a citric acid buffer solution which contains carbamide peroxide with the final concentration of 0.5-10 mmol/L, disodium ethylene diamine tetraacetate with the mass concentration of 0.01-0.1%, Tween-20 with the volume ratio of 0.05-0.5% and the pH value of 4.5-6.5;

when in use, the luminous agent solution and the oxidant solution are mixed for use.

Preferably, the buffer solution of the luminescent agent solution is 0.1-0.3 mol/L boric acid-borax buffer solution, and the final concentration of the citrate buffer solution in the oxidant solution is 0.1-0.3 mol/L.

Preferably, the HRP enzymatic chemiluminescent substrate solution comprises luminol with a final concentration of 1mmol/L, p-iodophenol with a final concentration of 1-2 mmol/L, phenolphthalein with a final concentration of 0.1-0.2 mmol/L, and boric acid-borax buffer solution with a pH of 9.0-10.0.

Preferably, the HRP enzymatic chemiluminescent substrate solution comprises carbamide peroxide with a final concentration of 1mmol/L, disodium ethylene diamine tetraacetate with a mass concentration of 0.04%, Tween-20 with a volume ratio of 0.1-0.25%, and 0.2mol/L citric acid buffer solution, and the pH value of the buffer solution is 5.0-6.0.

Preferably, the HRP enzymatic chemiluminescent substrate solution further contains ProClin 300 with a mass concentration of 0.01-0.1%.

The HRP enzymatic chemiluminescence substrate solution is preferably prepared by using 0.05% of ProClin 300 by mass.

The HRP enzymatic chemiluminescent substrate solution is preferably prepared by mixing the luminescent agent solution and the oxidant solution in a volume ratio of 1:1 before use.

The HRP enzymatic chemiluminescence substrate solution provided by the invention is simple in preparation, and the stability of the luminescence solution is further improved by adopting p-iodophenol as a reinforcing agent, phenolphthalein as a synergistic reinforcing agent and tween-20 as a stabilizing agent, so that the stable HRP enzymatic chemiluminescence substrate solution with high signal-to-noise ratio, high sensitivity, long platform period and purplish red color is obtained. The blank value of the HRP enzymatic chemiluminescent substrate solution is not increased after 1 day after mixing, the signal value is stable, and after performance test, the HRP enzymatic chemiluminescent substrate solution has higher reaction stability, the reaction can quickly reach the maximum luminous intensity, and a long-time platform period is provided later, compared with the luminescent substrate solution on the market, the HRP enzymatic chemiluminescent substrate solution has high luminous intensity value and high sensitivity. The invention is applied to HRP enzymatic reaction, has the characteristics of short reaction time and long detection platform period, can be stably stored for 2 years at 4-8 ℃, and can be widely applied to various kits taking HRP as a detection target.

The chemiluminescent substrate liquid has low cost and good stability, the color of the mixed luminescent liquid is purple red, the addition of the luminescent liquid is easy to be distinguished by naked eyes compared with a white chemiluminescent plate, the chemiluminescent substrate liquid is particularly suitable for semi-automatic manual operation of chemiluminescence, the operation errors caused by the manual operation are reduced, in addition, the phenolphthalein solution can change the color due to the pH change, the luminescent liquid can identify whether a reagent grows and goes bad according to the color, and the unnecessary cost increase and the false detection rate of a sample caused by using a failed reagent are effectively avoided.

Drawings

FIG. 1 is a graph comparing the luminescence time versus the percentage of the luminescence value of two luminescent liquids for each time period with respect to the initial luminescence value in a preferred embodiment of the present invention.

FIG. 2 is a graph showing the comparison of the luminescence time and the signal-to-noise ratio of two luminescent liquids in each time period in a preferred embodiment of the present invention.

FIG. 3 is a calibration graph of the substrate solution used for the detection of the concentration of interferon-gamma (LnX) -luminescence (LnY) in a preferred embodiment of the present invention.

Fig. 4 is a graph showing the results of plotting the light emission time against the light emission value for each period in example 4 of the present invention.

Fig. 5 is a graph plotting the results of the graphs according to the percentage of the light emitting time-the light emitting value of two light emitting liquids at each time period with respect to the initial light emitting value in example 4 of the present invention.

Detailed Description

According to the invention, phenolphthalein is used as a synergistic enhancer, and the phenolphthalein contains two phenolic hydroxyl groups; tween-20 is used as a stabilizer, and Tween-20 can ensure the slow release of the oxidizing agent.

The present invention will be described in detail with reference to the accompanying drawings and specific embodiments, it should be understood that the detailed description is only for the purpose of illustrating and explaining the technical solutions of the present invention, and is not intended to limit the present invention. The experimental procedures in the following examples, unless otherwise specified, were carried out according to conventional experimental procedures; the test reagents were commercially available unless otherwise specified.

Example 1

A stable HRP enzymatic chemiluminescence substrate solution is prepared by respectively preparing 1000mL of a luminophore solution and an oxidant solution, wherein the luminophore solution has the following specific formula:

the specific formula of the oxidant solution is as follows:

in the above luminescent agent solution, other conventional buffer systems such as phosphate buffer and the like can be used as the buffer solution.

Before use, the luminous agent solution and the oxidant solution are mixed according to the volume ratio of 1:1 and then used. The blank value is not increased after the chemiluminescence substrate solution is mixed for 1 day, and the signal value is stable.

In order to ensure the stability of the reagent after the reagent is stored for a long time, ProClin 300 with the mass concentration of 0.01-0.1% can be added into an oxidant solution, and the mass concentration effectively ensures the shelf life of the solution and does not influence the reaction effect of the reagent.

Example 2

In this example, a chemiluminescent substrate solution and an oxidant solution are prepared based on theembodiment 1, and the specific formula is as follows:

solution of luminescent agent in chemiluminescent substrate solution:

luminol 1mmol/L

Phenolphthalein 0.1mmol/L

P-iodophenol 1.5mmol/L

0.2mol/L boric acid-borax buffer solution, and adjusting the pH value to 9.6 by using hydrochloric acid.

Oxidant solution in chemiluminescent substrate solution:

0.2mol/L citric acid buffer, and adjusting pH to 5.0 with hydrochloric acid

After the prepared luminescent agent solution and the prepared oxidant solution are mixed according to the volume ratio of 1:1, the mixture is used as a luminescent substrate solution to be compared with an ultrasensitive luminescent substrate solution produced by imported ThermoFisher Pierce sold in the market, and the signal to noise ratio (the ratio of a luminescent value to a background value) and the stability of a luminescent value (the percentage of the luminescent value in each time period relative to an initial luminescent value) in HRP enzyme solution of 0.5 mu g/mL are measured.

The method comprises the following specific steps: adding 50 mu L of HRP enzyme solution with the concentration of 0.5 mu g/mL into a luminescent plate, reserving a hole without adding the enzyme solution as a blank hole, respectively adding commercially available luminescence liquid and the luminescence substrate liquid prepared in the invention into the holes, and placing a chemiluminescence detector for reading once every 1 minute.

Drawing a graph according to the percentage of the luminous values of the two luminous liquids in each time period relative to the initial luminous value, wherein the drawn result graph is shown in figure 1;

the signal-to-noise ratios of the two luminescent liquids in the time periods of luminescence are plotted, and the results are shown in FIG. 2.

As can be seen from the figure, the signal-to-noise ratio of the luminescent liquid in the embodiment is obviously superior to that of the imported contrast luminescent liquid, and the plateau stability is slightly superior to that of the imported contrast luminescent liquid.

Example 3

The chemiluminescent substrate solution in example 2 was used in a double antibody sandwich method to detect gamma-interferon, wherein the antibody was the antibody produced by the Biotech limited of Gili, Wuhan Hai.

The experimental procedure was as follows:

1. sample application

(1) Diluting a gamma-interferon calibrator reference product into 6 dilution concentrations of 12.5pg/mL, 25pg/mL, 50pg/mL, 100pg/mL, 200pg/mL and 400pg/mL by using a calibrator diluent, sequentially adding 50 mu L of each dilution concentration into a plate hole, and making two holes in parallel;blank control 1 hole, blank control add 50 μ L of calibrator diluent;

(2) adding 50 mu L of enzyme-labeled reagent into each hole;

2. and (3) incubation: gently mixing, and incubating in a constant temperature incubator at 37 deg.C for 1 hr;

3. washing, namely throwing off liquid in the holes, adding 300 mu L of 1 × washing liquid into each hole for washing, beating to dry, and repeating for 5 times;

4. luminescence: adding 100 mu L of luminescent agent mixed solution into each hole;

5. and (3) determination: reading the luminescence value by a chemiluminescence immunoassay analyzer within 20 minutes;

the linear fitting is carried out by using the gamma-interferon concentration-luminescent value data of the luminescent substrate liquid of the invention: ln (X) -Ln (Y), fitting equation: y 1.540x +3.584, wherein the correlation coefficient R²0.998. The calibration curve of the substrate liquid gamma-interferon concentration (LnX) -luminescence value (LnY) in this example is shown in FIG. 3.

The result shows that the reagent can be used for the detection of the kit and has good linearity.

Example 4

Based on example 2, luminescent agent A1 (without phenolphthalein) and oxidant B1 (without Tween-20) were prepared, and the luminescent agent and the oxidant in example 2 were labeled as luminescent agent A and oxidant B.

Luminescent agent a1 solution:

luminol 1mmol/L

P-iodophenol 1.5mmol/L

Oxidant B1 solution:

carbamide peroxide 1mmol/L

Disodium Ethylenediaminetetraacetate (EDTA) 0.04%

ProClin 300 0.05％

0.2mol/L citric acid buffer, and adjusting pH to 5.0 with hydrochloric acid

Mixing the luminous agent A with the oxidant B, the luminous agent A1 with the oxidant B, and the luminous agent A with the oxidant B1 at a ratio of 1:1 respectively, and respectively measuring the signal to noise ratio (ratio of luminous value to background value) and the stability of the luminous value (percentage of the luminous value to the initial luminous value in each time period) in the HRP enzyme solution of 0.5 mu g/mL.

Drawing according to the luminous time-luminous value of each time period, and drawing a result graph as shown in figure 4; and (3) drawing a graph according to the percentage of the luminous values of the two luminous liquids in each time period relative to the initial luminous value, and drawing a result graph as shown in figure 5.

As can be seen from FIG. 4, the luminescent intensity of the luminescent liquid is weaker without adding phenolphthalein as a synergistic enhancer. As can be seen from FIG. 5, the stability of the luminescence value of the luminescent liquid is obviously improved by adding Tween-20 as a stabilizer, and the synergistic enhancer also has the function of stabilizing the luminescence value.

Claims

1. A stable HRP enzymatic chemiluminescence substrate solution comprises a luminophore solution and an oxidant solution, wherein the luminophore solution contains luminol or luminol derivative or luminol sodium salt with a final concentration of 0.5-10 mmol/L, p-iodophenol with a final concentration of 0.5-10 mmol/L, phenolphthalein with a final concentration of 0.01-5 mmol/L, and a buffer solution with a pH value of 8.5-10.5;

the oxidant solution is a citric acid buffer solution which contains carbamide peroxide with the final concentration of 0.5-10 mmol/L, ethylene diamine tetraacetic acid with the mass concentration of 0.01-0.1%, Tween-20 with the volume ratio of 0.05-0.5%, the pH value of the citric acid buffer solution is 4.5-6.5, and ProClin 300 with the mass concentration of 0.01-0.1%.

2. The HRP enzymatic chemiluminescent substrate solution of claim 1 wherein the buffer solution of the luminophore solution is 0.1mol/L to 0.3mol/L boric acid-borax buffer solution and the final concentration of the citrate buffer solution in the oxidant solution is 0.1mol/L to 0.3 mol/L.

3. The HRP enzymatic chemiluminescent substrate solution of claim 2, wherein the solution of the luminescent agent is a buffer solution containing luminol with a final concentration of 1mmol/L, p-iodophenol with a final concentration of 1-2 mmol/L, phenolphthalein with a final concentration of 0.1-0.2 mmol/L, and boric acid-borax with a pH of 9.0-10.0 with a final concentration of 0.2 mol/L.

4. The HRP enzymatic chemiluminescent substrate solution of any one of claims 1-3 wherein the oxidizing agent solution is a buffer solution containing urea peroxide at a final concentration of 1mmol/L, disodium edetate at 0.04% by mass, Tween-20 at 0.1-0.25% by volume, and 0.2mol/L citric acid at a pH of 5.0-6.0.

5. The HRP enzymatic chemiluminescent substrate solution of claim 1 wherein the mass concentration of the ProClin 300 is 0.05%.

6. The HRP enzymatic chemiluminescent substrate solution of any one of claims 1-3 or 5 wherein in use, the luminophore solution and the oxidant solution are mixed at a volume ratio of 1:1 for use.

7. The HRP enzymatic chemiluminescent substrate solution of claim 4 wherein the solution of the luminescent agent and the solution of the oxidizing agent are mixed at a volume ratio of 1:1 for use.