CN109799352B - Chemiluminescent reagent and application thereof in immunoassay - Google Patents
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Abstract
The invention discloses a chemiluminescent reagent and application thereof in immunoassay. The chemiluminescence reagent provided by the invention contains chlorogenic acid and a chemiluminescence substrate, wherein the chemiluminescence substrate is luminol or luminol soluble salt. The invention proves that the sensitivity of the chemiluminescent reagent containing chlorogenic acid for detecting horseradish peroxidase is improved by more than 100 times compared with the chemiluminescent reagent containing 4-iodophenol through the chemiluminescence analysis of standard horseradish peroxidase catalysis luminol and hydrogen peroxide. The chemiluminescence reagent containing chlorogenic acid is used for a protein immunoblotting experiment, and the detection sensitivity of target protein can be remarkably improved. The invention can be used for detecting trace protein in protein immunoblotting.
Description
Technical Field
The invention relates to a chemiluminescence reagent in biology and immunology and application thereof in immunoassay.
Background
In the biological detection technology, immunological detection is very important, and the characteristic of high specificity is that the antigen-antibody reaction is utilized to detect the corresponding antigen-antibody concentration. However, the classical precipitation method and agglutination method, which are characterized by direct reaction of antibody and antigen, have low sensitivity and poor accuracy in detecting antigen and antibody. Researchers then use peroxidase to label the second antibody or streptavidin, and convert the antigen or antibody signal to be detected in the immunoreaction into enzyme activity convenient for detection. The sensitivity and accuracy of the detection is greatly improved by the amplification and good linearity of the enzymatic reaction. The horseradish peroxidase is a peroxidase with high stability and strong catalytic capability. It is often used to label a second antibody or streptavidin for converting the antigen or antibody signal to be detected in an immune reaction into a detectable enzyme activity.
The enzyme activity can be quantitatively determined by the redox reaction catalyzed by horseradish peroxidase, and commonly used horseradish peroxidase substrates are chromogenic substrates diaminobenzidine and tetramethylbenzidine. The horseradish peroxidase catalyzes the oxydating diaminobenzidine to generate a brown insoluble product, and is suitable for western blot detection and immunohistochemical detection; the horseradish peroxidase catalyzes tetramethyl benzidine to generate a blue product, the blue product is changed into a stable yellow substance under an acidic condition, the product generation amount and the corresponding enzyme activity can be accurately determined through absorbance, and the method is suitable for enzyme-linked immunosorbent assay. However, the chromogenic substrates of horseradish peroxidase still have the serious defects of low detection sensitivity, narrow quantitative linear range and the like. Researchers have found that the use of chemiluminescent substrates can significantly improve the sensitivity and linear range of horseradish peroxidase detection, and various chemiluminescent substrates are gradually being developed. In particular, a redox substrate based on luminol and hydrogen peroxide, and a phenol chemiluminescence enhancer such as 4-iodophenol (Pure and Applied Chemistry,1987,59(5),651-654) are added, so that the enzymatic activity of horseradish peroxidase can be detected well by a chemiluminescence reaction.
In western blotting experiments, many proteins to be detected are trace proteins. The sensitivity of the enhanced chemiluminescence solution sold in the market at present still cannot meet the requirement of a protein immunoblotting experiment.
Disclosure of Invention
The invention aims to solve the technical problem of how to enhance the chemiluminescence intensity in the reaction process of horseradish peroxidase catalyzed luminol and hydrogen peroxide.
In order to solve the above technical problems, the present invention provides a chemiluminescent reagent.
The chemiluminescence reagent provided by the invention is a chemiluminescence composition, the chemiluminescence reagent contains chlorogenic acid and a chemiluminescence substrate, and the chemiluminescence substrate is luminol or luminol soluble salt. The chemiluminescent reagent of the present invention may also be referred to as a chlorogenic acid containing chemiluminescent reagent.
In the above chemiluminescent reagent, the luminol soluble salt may be a luminol sodium salt.
In the above chemiluminescent reagent, the chemiluminescent reagent may be comprised of chlorogenic acid and the chemiluminescent substrate. In the chemiluminescence reagent, the chlorogenic acid and the chemiluminescence substrate can be packaged separately or mixed together. In the chemiluminescence reagent, the ratio of the chlorogenic acid to the chemiluminescence substrate can be 0.1-20.0mmol of chlorogenic acid: 2.5mmol of the chemiluminescent substrate, 0.5-2.0mmol of chlorogenic acid: 2.5mmol of the chemiluminescent substrate, or 1.0-2.0mmol of chlorogenic acid: 2.5mmol of the chemiluminescent substrate.
In the above-mentioned chemiluminescent reagent, the chemiluminescent reagent may further contain other substances required for carrying out a chemiluminescent reaction, such as tris (hydroxymethyl) aminomethane and/or hydrochloric acid and/or water.
In the above chemiluminescent reagent, the chemiluminescent reagent may be composed of chlorogenic acid, the chemiluminescent substrate and at least one of the following: tris (hydroxymethyl) aminomethane, hydrochloric acid and water.
In the above chemiluminescent reagent, the chemiluminescent reagent may further comprise hydrogen peroxide. The chemiluminescent reagent may consist of chlorogenic acid, the chemiluminescent substrate and hydrogen peroxide. In the chemiluminescence reagent, hydrogen peroxide is packaged separately, and chlorogenic acid and the chemiluminescence substrate can be packaged separately or mixed together. In the chemiluminescence reagent, the ratio of chlorogenic acid, the chemiluminescence substrate and hydrogen peroxide can be 0.1-20.0mmol of chlorogenic acid: 2.5mmol of the chemiluminescent substrate: 6mmol hydrogen peroxide, 0.5-2.0mmol chlorogenic acid: 2.5mmol of the chemiluminescent substrate: 6mmol hydrogen peroxide, or 1.0-2.0mmol chlorogenic acid: 2.5mmol of the chemiluminescent substrate: 6mmol of hydrogen peroxide.
In the above-mentioned chemiluminescent reagent, the chemiluminescent reagent may further contain other substances required for carrying out a chemiluminescent reaction, such as tris (hydroxymethyl) aminomethane and/or hydrochloric acid and/or water.
In the above chemiluminescent reagent, the chemiluminescent reagent may be composed of chlorogenic acid, the chemiluminescent substrate, hydrogen peroxide and at least one of the following: tris (hydroxymethyl) aminomethane, hydrochloric acid and water.
In the above chemiluminescent reagent, the chemiluminescent reagent may be a reagent for chemiluminescent immunoassay.
In practical application, the chemiluminescence reagent can be composed of a solution A and a solution B, wherein the solution A can be an aqueous solution with the pH of 8.6 and composed of a solute and a solvent, the solvent is water, and the solute and the concentration thereof are respectively 0.1M trihydroxymethyl aminomethane, 0.03M hydrochloric acid, 2.5mM luminol sodium salt and 0.1-20.0mM chlorogenic acid. The solution B can be aqueous solution with pH of 8.6 composed of solute and solvent, wherein the solute and its concentration are 0.1M tris, 0.03M hydrochloric acid and 6mM hydrogen peroxide, respectively, and the solvent is water. The solution A and the solution B are mixed in equal volume for use.
In the chemiluminescence reagents, compared with the chemiluminescence reagent containing 4-iodophenol, the chemiluminescence reagent (containing chlorogenic acid) disclosed by the invention has the advantage that the chemiluminescence intensity of horseradish peroxidase in the reaction process of catalyzing luminol and hydrogen peroxide is increased by 114 times. Compared with the chemiluminescence reagent containing 4-iodophenol in composition, the chemiluminescence reagent of the invention (the chemiluminescence reagent containing chlorogenic acid) has the same components and contents except that equimolar 4-iodophenol is replaced by equimolar chlorogenic acid.
Any of the following applications also fall within the scope of the present invention:
use of U0, chlorogenic acid or said chemiluminescent reagent in chemiluminescent detection or chemiluminescent immunoassay (chemiluminescent immunoassay, such as western blot detection);
u01, application of chlorogenic acid in preparing chemiluminescence reagents or application of chlorogenic acid as a chemiluminescence enhancer;
u1, chlorogenic acid and the application of the chemiluminescence substrate in preparing a chemiluminescence reagent;
u2, chlorogenic acid, the chemiluminescent substrate and hydrogen peroxide in preparing a chemiluminescent reagent;
use of U3, chlorogenic acid, said chemiluminescent substrate and at least one of the following in the preparation of a chemiluminescent reagent: tris and/or hydrochloric acid and/or water;
u4, chlorogenic acid, the chemiluminescent substrate, hydrogen peroxide and at least one of the following substances in the preparation of a chemiluminescent reagent: tris and/or hydrochloric acid and/or water;
u5, chlorogenic acid and hydrogen peroxide in preparing chemiluminescence reagent;
u6, chlorogenic acid and tris (hydroxymethyl) aminomethane are applied to the preparation of a chemiluminescent reagent;
u7, chlorogenic acid and hydrochloric acid in preparing chemiluminescence reagent.
In the above application, the chemiluminescent reagent may be the above chemiluminescent reagent. The chemiluminescence enhancing agent can be a reagent for enhancing the chemiluminescence intensity in the reaction process of horseradish peroxidase catalyzed luminol and hydrogen peroxide.
In the application, compared with 4-iodophenol, chlorogenic acid enhances the chemiluminescence intensity of horseradish peroxidase in the reaction process of luminol and hydrogen peroxide by 114 times.
According to the invention, a large amount of luminol chemiluminescence enhancement effect screening is carried out, so that chlorogenic acid has a strong chemiluminescence enhancement effect, and the detection sensitivity of a protein immunoblotting experiment can be greatly improved. The invention proves that the sensitivity of the chemiluminescent reagent containing chlorogenic acid for detecting horseradish peroxidase is improved by more than 100 times compared with the chemiluminescent reagent containing 4-iodophenol through the chemiluminescence analysis of standard horseradish peroxidase catalysis luminol and hydrogen peroxide. The chemiluminescence reagent containing chlorogenic acid is used for a protein immunoblotting experiment, and the detection sensitivity of target protein can be remarkably improved. The invention can be used for detecting trace protein in protein immunoblotting.
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FIG. 1 shows the results of Western blotting using 4-iodophenol-containing enhanced chemiluminescent solutions and chlorogenic acid-containing enhanced chemiluminescent solutions.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Reagent: tris-hydroxymethyl (cat # T110600) available from Shanghai Allan Biotech Co., Ltd; luminol sodium salt (cat # V900354), chlorogenic acid (cat # C3878), hydrogen peroxide (cat # 88597), and 4-iodophenol (cat # I10201) were purchased from Sigma-Aldrich, USA; other chemicals were purchased from Beijing chemical industries. DMEM cell culture medium and fetal bovine serum, purchased from Corning, USA. Protein electrophoresis related reagents purchased from Bai Rui Da Biotech, Inc. of Beijing.
Consumable material: white 96-well plates (cat # T110600) and common centrifuge and pipette tips were purchased from Corning, USA. Polyvinylidene fluoride (PVDF) membranes, available from merck, germany; cell culture flasks, centrifuge tubes, and pipette tips were purchased from Corning, usa.
The instrument comprises the following steps: enspire Multimode Plate Reader, available from platinum-Acer, Germany. Protein electrophoresis related equipment, available from berle, usa; ImageQuant LAS 4000 chemiluminescent imager, available from GE, USA.
Cell: RAW264.7 cells, purchased from the institute of basic medicine, institute of medical science, china, cell center.
Example 1 chlorogenic acid enhancement of chemiluminescence intensity during reaction of luminol and Hydrogen peroxide catalyzed by horseradish peroxidase
This example provides 9 chemiluminescent reagents, each of which consists of solution A and solution B. The 9 chemiluminescent reagents were identical in solution B, but different in solution A. The solution B of the 9 chemiluminescent reagents was an aqueous solution of pH 8.6 consisting of 0.1M Tris, 0.03M HCl and 6mM hydroperoxide in the concentrations of solute and solvent, water.
The 9 kinds of chemiluminescence reagents are respectively 0mM chlorogenic acid chemiluminescence reagent, 0.1mM chlorogenic acid chemiluminescence reagent, 0.2mM chlorogenic acid chemiluminescence reagent, 0.5mM chlorogenic acid chemiluminescence reagent, 1.0mM chlorogenic acid chemiluminescence reagent, 2.0mM chlorogenic acid chemiluminescence reagent, 5.0mM chlorogenic acid chemiluminescence reagent, 10.0mM chlorogenic acid chemiluminescence reagent and 20.0mM chlorogenic acid chemiluminescence reagent.
The solution A of 0mM chlorogenic acid chemiluminescence reagent was 0mM chlorogenic acid solution (control). The solution A of the 0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with the pH of 8.6, which consists of a solute and a solvent, wherein the solute and the concentration thereof are respectively 0.1M trihydroxymethyl aminomethane, 0.03M hydrochloric acid and 2.5mM luminol sodium salt, and the solvent is water.
The solution A of 0.1mM chlorogenic acid chemiluminescence reagent is aqueous solution with pH of 8.6 composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 0.1mM chlorogenic acid chemiluminescence reagent was 0.1 mM.
The solution A of 0.2mM chlorogenic acid chemiluminescence reagent is aqueous solution with pH of 8.6 composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 0.2mM chlorogenic acid chemiluminescence reagent was 0.2 mM.
The solution A of 0.5mM chlorogenic acid chemiluminescence reagent is aqueous solution with pH of 8.6 composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 0.5mM chlorogenic acid chemiluminescence reagent was 0.5 mM.
A solution A of 1.0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with pH of 8.6, which is composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 1.0mM chlorogenic acid chemiluminescence reagent was 1.0 mM.
A solution A of 2.0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with pH of 8.6, which is composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 2.0mM chlorogenic acid chemiluminescence reagent was 2.0 mM.
A solution A of 5.0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with pH of 8.6, which is composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 5.0mM chlorogenic acid chemiluminescence reagent was 5.0 mM.
A solution A of 10.0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with pH of 8.6, which is composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 10.0mM chlorogenic acid chemiluminescence reagent was 10.0 mM.
A solution A of 20.0mM chlorogenic acid chemiluminescence reagent is an aqueous solution with pH of 8.6, which is composed of solute and solvent, wherein the solute is chlorogenic acid, and the solvent is the above 0mM chlorogenic acid solution. Chlorogenic acid content in solution A of 20.0mM chlorogenic acid chemiluminescence reagent was 20.0 mM.
The chemiluminescence intensity of the chemiluminescence reaction of the 9 chemiluminescence reagents catalyzed by horseradish peroxidase is respectively tested according to the following chemiluminescence analysis method: the solution A and the solution B were mixed in equal volumes, and then pipetted into a white 96-well plate at 100. mu.l/well, followed by addition of 0.001U/ml horseradish peroxidase solution at 10. mu.l/well, after thorough mixing, the reaction was carried out at room temperature for 5 minutes, and the chemiluminescence intensity (Relative light unit, RLU) was measured with a microplate reader.
The results of the chemiluminescence analysis show that the chemiluminescence intensity of the horseradish peroxidase catalyzing the chemiluminescence reaction of 0.1mM chlorogenic acid chemiluminescence reagent, 0.2mM chlorogenic acid chemiluminescence reagent, 0.5mM chlorogenic acid chemiluminescence reagent, 1.0mM chlorogenic acid chemiluminescence reagent, 2.0mM chlorogenic acid chemiluminescence reagent, 5.0mM chlorogenic acid chemiluminescence reagent, 10.0mM chlorogenic acid chemiluminescence reagent and 20.0mM chlorogenic acid chemiluminescence reagent is respectively 3660 times (183/0.05 ═ 3660) of the chemiluminescence intensity of the horseradish peroxidase catalyzing the chemiluminescence reaction of 0mM chlorogenic acid chemiluminescence reagent, 7120 times (356/0.05 ═ 7120), 9760 times (488/0.05 ═ 9760), 10760 times (538/0.05 ═ 10760), 10240 times (512/0.05 ═ 10240), 8560 times (428/0.05 ═ 8560), 7060 times (353/0.05 ═ 7060), and 4460 times (223/0.05 ═ 4460). The chlorogenic acid added into the chemiluminescence liquid can obviously improve the chemiluminescence intensity of the reaction of horseradish peroxidase catalyzed luminol and hydrogen peroxide, so that the chemiluminescence intensity is improved by 3660-10760 times. The experiment was repeated 3 times, each time treating 3 wells.
TABLE 1 chemiluminescence intensity of the 9 chemiluminescent reagents catalyzed by horseradish peroxidase
Note: RLU values are expressed as mean. + -. standard deviation, and were statistically analyzed using One way ANOVA and Dunnett's post hoc test. P <0.05vs 0mM chlorogenic acid chemiluminescent reagent; # P <0.05vs 1.0mM chlorogenic acid chemiluminescent reagent.
Example 2 comparison of the enhancement of luminol chemiluminescence by chlorogenic acid and 4-iodophenol Using horseradish peroxidase standards
This example provides 3 chemiluminescent reagents, a control chemiluminescent solution, an enhanced chemiluminescent solution containing 4-iodophenol, and an enhanced chemiluminescent solution containing chlorogenic acid. The 3 kinds of chemiluminescent reagents consist of liquid A and liquid B. The solutions B of the 3 chemiluminescent reagents were the same except that the solution A was different. The solutions B of the 3 chemiluminescent reagents were aqueous solutions of pH 8.6 composed of solute and solvent, the solute and its concentration were 0.1M Tris, 0.03M HCl and 6mM hydroperoxide, respectively, and the solvent was water.
The solution A of the control chemiluminescent solution was an aqueous solution of pH 8.6 composed of a solute and a solvent, the solute and its concentration were 0.1M Tris, 0.03M HCl and 2.5mM luminol sodium salt, respectively, and the solvent was water.
The solution A of the enhanced chemiluminescent solution containing 4-iodophenol was an aqueous solution of pH 8.6 consisting of a solute and a solvent, the solute was 4-iodophenol, the solvent was the solution A of the control chemiluminescent solution, and the content of 4-iodophenol in the solution A of the enhanced chemiluminescent solution containing 4-iodophenol was 1 mM. (note: 1-5mM 4-iodophenol solution can exert the maximum chemiluminescence enhancement effect on luminol, and 4-iodophenol with the concentration of 1mM is selected to prepare the enhanced chemiluminescence solution in order to compare the effect with chlorogenic acid).
The A solution of enhanced chemiluminescence solution containing chlorogenic acid is aqueous solution with pH of 8.6 composed of solute and solvent, the solute is chlorogenic acid, and the solvent is the A solution of the above control chemiluminescence solution. The chlorogenic acid content in the A solution of the enhanced chemiluminescence liquid containing chlorogenic acid is 1 mM.
The chemiluminescence intensity of the chemiluminescence reaction of the 3 chemiluminescence reagents catalyzed by horseradish peroxidase is respectively tested according to the following chemiluminescence analysis method: the solution A and the solution B were mixed in equal volumes, and then pipetted into a white 96-well plate at 100. mu.l/well, followed by addition of 0.001U/ml horseradish peroxidase solution at 10. mu.l/well, after thorough mixing, the reaction was carried out at room temperature for 5 minutes, and the chemiluminescence intensity (Relative light unit, RLU) was measured with a microplate reader. The experiment was repeated 3 times, each time treating 3 wells.
The results of the chemiluminescence analysis showed that the chemiluminescence intensities of the enhanced chemiluminescent solution containing 4-iodophenol and the enhanced chemiluminescent solution containing chlorogenic acid catalyzed by horseradish peroxidase were 1205 times (4.82/0.04 ═ 1205) and 13800 times (552/0.04 ═ 13800) of the chemiluminescence intensity of the chemiluminescent reaction of the control chemiluminescent solution catalyzed by horseradish peroxidase, respectively, and the chemiluminescence intensity of the enhanced chemiluminescent solution containing chlorogenic acid catalyzed by horseradish peroxidase was 115 times (552/4.82 ═ 115) of the chemiluminescence intensity of the chemiluminescent reaction of the enhanced chemiluminescent solution containing 4-iodophenol catalyzed by horseradish peroxidase (table 2). The results show that the addition of 4-iodophenol and chlorogenic acid to the chemiluminescent solution can improve the chemiluminescence intensity of the reaction of the horseradish peroxidase catalyzed luminol and hydrogen peroxide, and the chemiluminescence enhancement effect of chlorogenic acid is improved by more than 100 times compared with 4-iodophenol (Table 2).
TABLE 2 chemiluminescence intensity of the chemiluminescent reaction catalyzed by horseradish peroxidase with the 2 chemiluminescent reagents
| Chemiluminescent reagent | RLU(×106) |
| Contrast chemiluminescence liquid | 0.04±0.01* |
| Enhanced chemiluminescence liquid containing 4-iodophenol | 4.82±0.09 |
| Enhanced chemiluminescence liquid containing chlorogenic acid | 552±15* |
Note: RLU values are expressed as mean. + -. standard deviation and were statistically analyzed using Student's t test. P <0.05vs enhanced chemiluminescence solution containing 4-iodophenol.
Example 3 comparison of chlorogenic acid and 4-iodophenol on luminol chemiluminescence enhancement Using Western blotting
1 million RAW264.7 cells in logarithmic growth phase were collected, lysed with RIPA medium strength lysate for 30 minutes at 4 ℃ and centrifuged at 10000g for 10 minutes to collect protein supernatant, which was then added to loading buffer and incubated in 98 ℃ metal bath for 10 minutes to prepare denatured protein. The samples were loaded (5 parallel wells) onto 10% polyacrylamide gels for protein electrophoresis, after which the proteins were electroporated onto PVDF membranes. The membrane was blocked with 5% skimmed milk powder TBS solution for 1 hour, incubated with rat anti-mouse β -actin antibody overnight at 4 ℃, washed with TBST solution, incubated with horseradish peroxidase-labeled goat anti-rat IgG for 2 hours at room temperature, washed with TBST solution, and exposed to the enhanced chemiluminescence solutions containing 4-iodophenol and chlorogenic acid of example 2, respectively. Wherein, before exposure, the A liquid and the B liquid of the two enhanced chemiluminescence liquids are mixed in equal volume, then the mixture is absorbed onto a PVDF membrane, an automatic exposure mode is selected, an exposure picture is stored, and the exposure time is recorded.
The result of western blot analysis shows that the enhanced chemiluminescence liquid containing chlorogenic acid in example 2 can detect the target protein (beta-actin) more sensitively than the enhanced chemiluminescence liquid containing 4-iodophenol in example 2, and has a thicker exposure band and shorter exposure time (fig. 1). The upper panel in FIG. 1 shows the result of Western blotting detection using the 4-iodophenol-containing enhanced chemiluminescent solution of example 2 exposed for 5 seconds, and the lower panel in FIG. 1 shows the result of Western blotting detection using the chlorogenic acid-containing enhanced chemiluminescent solution of example 2 exposed for 0.5 seconds.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (1)
1. The chemiluminescence immunoassay is applied to a chemiluminescence immunoassay reagent, wherein the chemiluminescence reagent consists of a solution A and a solution B, the solution A is an aqueous solution which consists of a solute and a solvent and has the pH value of 8.6, the solvent is water, and the solute and the concentration thereof are respectively 0.1M trihydroxymethyl aminomethane, 0.03M hydrochloric acid and 2.5mM luminol sodium salt, and 0.1-20.0mM chlorogenic acid; the solution B is an aqueous solution with the pH value of 8.6 and consists of a solute and a solvent, wherein the solute and the concentration thereof are respectively 0.1M trihydroxymethyl aminomethane, 0.03M hydrochloric acid and 6mM hydrogen peroxide, and the solvent is water; the chemiluminescence immunoassay comprises the steps of mixing the solution A and the solution B in equal volume, sucking the mixture into a white 96-well plate with 100 mu l/well, adding 0.001U/ml horseradish peroxidase solution with 10 mu l/well, reacting at room temperature for 5 minutes after fully mixing, and detecting chemiluminescence intensity by an enzyme-linked immunosorbent assay.
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