Chemiluminescence immunoassay kit and application thereof in immunoassay

Abstract

This invention belongs to the field of in vitro detection technology, specifically relating to a chemiluminescent immunoassay kit and its application in immunoassay. The chemiluminescent immunoassay kit provided by this invention uses a first target protein recognition molecule and a second target protein recognition molecule to capture proteins. Horseradish peroxidase on the second target protein recognition molecule undergoes a polymerization reaction with a chemiluminescent reagent and norepinephrine, forming a product enriched with the chemiluminescent reagent. Under the action of an alkaline pH adjuster and hydrogen peroxide, a chemiluminescent signal is directly generated. This eliminates the need for covalent modification of the protein recognition molecule with the chemiluminescent reagent, reducing the loss of biomolecule activity, improving the utilization rate of the protein recognition molecule, facilitating the control of batch-to-batch variations of the chemiluminescent reagent, and effectively improving detection sensitivity. The results of the examples show that the chemiluminescent immunoassay kit provided by this invention improves the detection sensitivity of pTau217 to approximately 0.25 pg / mL.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of in vitro detection, and particularly relates to a chemiluminescence immunoassay kit and application thereof in immunoassay. BACKGROUND

[0002] Chemiluminescence immunoassay is the most important detection technology in the current medical examination and diagnosis field. Chemiluminescence is mainly divided into direct chemiluminescence and enzyme-catalyzed chemiluminescence. In direct chemiluminescence, chemiluminescence reagents do not need the catalysis of enzymes in the process of luminescence immunoassay and directly participate in the luminescence reaction. They have unique groups for generating luminescence in chemical structure and can directly label biological macromolecules such as antigens or antibodies. The direct chemiluminescence has fast speed and good reagent stability, but the sensitivity is slightly lower than that of enzyme-catalyzed chemiluminescence. Representative luminescence reagents include acridinium ester, trispyridine ruthenium and isoluminol. Enzyme-catalyzed chemiluminescence uses the catalysis of labeled enzymes to make enzyme-catalyzed reaction luminescence reagents (substrates) luminescence. The enzyme-catalyzed chemiluminescence has high sensitivity. However, the speed is slow and the enzyme activity is easily affected by the outside world. Representative enzyme-catalyzed reaction luminescence reagents include luminol and AMPPD.

[0003] With the development of full automation level of chemiluminescence detection equipment, precision of detection elements and production and preservation process of reagents, the detection sensitivity of chemiluminescence technology has been significantly improved. Under the premise of the existing antibody affinity level, the chemiluminescence technology has approached the limit of its detection capacity. However, medical examination increasingly puts forward higher sensitivity requirements. The existing chemiluminescence technology cannot meet the requirements of medical examination on sensitivity. How to effectively improve the detection sensitivity and accuracy is a technical problem to be solved. SUMMARY

[0004] The purpose of the present application is to provide a chemiluminescence immunoassay kit and application thereof in immunoassay, so as to improve the sensitivity of chemiluminescence detection.

[0005] In order to achieve the above purpose, the present application provides a chemiluminescence immunoassay kit, which comprises norepinephrine or a soluble salt thereof, hydrogen peroxide, an alkaline pH adjuster, a chemiluminescence reagent, a first target protein recognition molecule and a second target protein recognition molecule.

[0006] The second target protein recognition molecule is labeled with horseradish peroxidase.

[0007] Preferably, the first target protein recognition molecule is labeled with magnetic microparticles.

[0008] Preferably, the first target protein recognition molecule and the second target protein recognition molecule each comprise one or more of an antibody, an antigen and a nucleic acid.

[0009] Preferably, the chemiluminescent reagent comprises acridinium ester or isoluminol.

[0010] Preferably, the basic pH regulator comprises sodium hydroxide.

[0011] Preferably, the target protein comprises, but is not limited to, phosphorylated Tau-217 protein or glial fibrillary acidic protein.

[0012] Preferably, the chemiluminescent immunoassay kit further comprises Tris and DMSO.

[0013] The present application also provides the use of the chemiluminescent immunoassay kit in the preparation of a product for assisting clinical diagnosis.

[0014] The present application also provides a method for detecting protein for non-diagnostic purposes, which utilizes the chemiluminescent immunoassay kit described in the above technical solution, comprising the following steps:

[0015] Mixing the sample to be detected and the first target protein recognition molecule, performing first incubation to obtain a first incubation product;

[0016] Mixing the first incubation product and the second target protein recognition molecule, performing second incubation to obtain a second incubation product;

[0017] Mixing the second incubation product, norepinephrine or a soluble salt solution thereof, and hydrogen peroxide solution, performing third incubation to obtain a third incubation product;

[0018] Mixing the third incubation product and the chemiluminescent reagent, performing fourth incubation to obtain a fourth incubation product;

[0019] Mixing the fourth incubation product, hydrogen peroxide, and the basic pH regulator, reading the relative luminescence value by using a full-automatic chemiluminescence instrument, automatically fitting the calibration curve and calculating to obtain the concentration of the target protein.

[0020] Preferably, the temperature of the first incubation is 37℃, and the time is 5-15 min;

[0021] The temperature of the second incubation is 37℃, and the time is 5-15 min

[0022] The temperature of the third incubation is 37℃, and the time is 5-15 min;

[0023] The temperature of the fourth incubation is 37℃, and the time is 5-15 min;

[0024] The concentration of the hydrogen peroxide solution is 1M; the concentration of norepinephrine in the norepinephrine or soluble salt solution is 5mg / mL; and the volume ratio of the hydrogen peroxide solution and the norepinephrine or soluble salt solution is 50:1.

[0025] The working concentration of the chemiluminescent reagent is 1nM.

[0026] Beneficial effects:

[0027] The chemiluminescent immunoassay kit provided by the application comprises norepinephrine or a soluble salt thereof, hydrogen peroxide, an alkaline pH regulator, a chemiluminescent reagent, a first target protein recognition molecule and a second target protein recognition molecule; the second target protein recognition molecule is labeled with horseradish peroxidase, wherein the first target protein recognition molecule and the second target protein recognition molecule capture proteins respectively, the horseradish peroxidase on the second target protein recognition molecule polymerizes with the chemiluminescent reagent and the norepinephrine to form a product rich in chemiluminescent reagent, which directly generates a chemiluminescent signal under the action of the alkaline pH regulator and the hydrogen peroxide. The chemiluminescent immunoassay kit provided by the application does not need to covalently modify the chemiluminescent reagent with the protein recognition molecule, reduces the loss of the activity of biological macromolecules, improves the utilization rate of the protein recognition molecule, is conducive to controlling the batch difference of the chemiluminescent reagent, and can effectively improve the detection sensitivity. The results of the examples show that the chemiluminescent immunoassay kit provided by the application improves the detection sensitivity of pTau217 to close to 0.25pg / mL (coefficient of variation <8%). BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0029] Figure 1 The steps and principle diagram for detecting proteins by using the chemiluminescent immunoassay kit of the application. DETAILED DESCRIPTION

[0030] The chemiluminescent immunoassay kit provided by the application comprises norepinephrine or a soluble salt thereof, hydrogen peroxide, an alkaline pH regulator, a chemiluminescent reagent, a first target protein recognition molecule and a second target protein recognition molecule.

[0031] The second target protein recognition molecule is labeled with horseradish peroxidase.

[0032] In this invention, the first target protein recognition molecule is preferably labeled with magnetic microparticles; the first target protein recognition molecule preferably includes one or more of antibodies, antigens, and nucleic acids, and is more preferably an antibody. This invention utilizes magnetic microparticles to label the first target protein recognition molecule, facilitating separation and washing.

[0033] In this invention, the second target protein recognition molecule preferably comprises one or more of antibodies, antigens, and nucleic acids, and more preferably antibodies. The second target protein recognition molecule of this invention is labeled with horseradish peroxidase. This invention utilizes horseradish peroxidase labeling of the second target protein recognition molecule to catalyze the rapid polymerization of chemiluminescent reagents with norepinephrine, thereby enhancing the chemiluminescent signal.

[0034] This invention uses phosphorylated Tau-217 protein or glial fibrillary acidic protein (GFAP) as examples in the embodiments, but these should not be construed as representing the entire scope of protection of this invention. The phosphorylated Tau-217 protein antibody and GFAP antibody used in the embodiments of this invention were purchased from Medix; horseradish peroxidase, hydrogen peroxide, and norepinephrine or their soluble salts were purchased from Sigma; and magnetic microparticles were purchased from JSR.

[0035] In this invention, the soluble salt of norepinephrine is preferably norepinephrine hydrochloride.

[0036] In this invention, the chemiluminescent reagent preferably includes acridinium ester or isoluminol, and more preferably acridinium ester. In the chemiluminescent immunoassay kit of this invention, the chemiluminescent reagent exists independently, eliminating the need for covalent modification of protein recognition molecules with the chemiluminescent reagent. This reduces the loss of protein recognition molecule activity, improves the utilization rate of protein recognition molecules, facilitates operation, and helps control batch-to-batch variations of the chemiluminescent reagent.

[0037] In this invention, the alkaline pH adjuster preferably comprises sodium hydroxide. The alkaline pH adjuster of this invention can promote the luminescence of acridine esters.

[0038] In this invention, the chemiluminescent immunoassay kit preferably further includes Tris and DMSO; the concentration of Tris is preferably 10 nM, and the pH is preferably 8.5; Tris is preferably used as a solvent for norepinephrine or its soluble salts. The DMSO used in this invention is preferably used as a solvent for acridine esters.

[0039] In view of the advantages of the chemiluminescence immunoassay kit described above, its application in the preparation of auxiliary clinical diagnostic products also falls within the scope of protection of this invention.

[0040] This invention also provides a method for detecting proteins for non-diagnostic purposes, utilizing the chemiluminescent immunoassay kit described above, comprising the following steps:

[0041] The sample to be tested is mixed with the first target protein recognition molecule and incubated for the first time to obtain the first incubation product.

[0042] The first incubation product and the second target protein recognition molecule are mixed and subjected to a second incubation to obtain the second incubation product;

[0043] The second incubation product, norepinephrine or its soluble salt solution, and hydrogen peroxide solution are mixed and subjected to a third incubation to obtain the third incubation product.

[0044] The third incubation product is mixed with a chemiluminescent reagent and subjected to a fourth incubation to obtain a fourth incubation product;

[0045] The fourth incubation product, hydrogen peroxide, and alkaline pH adjuster are mixed, and the relative luminescence value is read by a fully automated chemiluminescence analyzer. The calibration curve is automatically fitted and the concentration of the target protein is calculated.

[0046] In this invention, the sample to be tested and a first target protein recognition molecule are mixed and incubated to obtain a first incubation product. In this invention, the temperature of the first incubation is preferably 37°C; the incubation time is 5–15 min, preferably 10 min.

[0047] After obtaining the first incubation product, the present invention mixes the first incubation product with the second target protein recognition molecule and performs a second incubation to obtain the second incubation product. In the present invention, the temperature of the second incubation is preferably 37°C; the time of the first incubation is 5 to 15 minutes, preferably 10 minutes.

[0048] After obtaining the second incubation product, the present invention mixes the second incubation product, norepinephrine or its soluble salt solution, and hydrogen peroxide solution, and performs a third incubation to obtain the third incubation product. In the present invention, the temperature of the third incubation is preferably 37°C; the incubation time is 5–15 min, preferably 5 min. The concentration of the hydrogen peroxide solution is preferably 1 M; the concentration of norepinephrine in the norepinephrine or its soluble salt solution is preferably 5 mg / mL; the volume ratio of the hydrogen peroxide solution to the norepinephrine or its soluble salt solution is preferably 50:1.

[0049] After obtaining the third incubation product, the present invention mixes the third incubation product with a chemiluminescent reagent and performs a fourth incubation to obtain a fourth incubation product. In the present invention, the temperature of the fourth incubation is preferably 37°C; the incubation time is preferably 5–15 min, more preferably 10 min. The working concentration of the chemiluminescent reagent in the present invention is preferably 1 nM.

[0050] This invention provides a protein recognition molecule that does not require covalent modification of the chemiluminescent reagent, reducing the loss of protein recognition molecule activity and improving the utilization rate of the protein recognition molecule. After the first target protein recognition molecule and the second target protein recognition molecule capture the protein respectively, the horseradish peroxidase on the second target protein recognition molecule undergoes a polymerization reaction with the chemiluminescent reagent and norepinephrine to form a product enriched with the chemiluminescent reagent. Under the action of an alkaline pH adjuster and hydrogen peroxide, a chemiluminescent signal is directly generated, which is easy to operate and helps to control the batch-to-batch variation of the chemiluminescent reagent.

[0051] To further illustrate the present invention, a chemiluminescent immunoassay kit and its application in immunoassay are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] according to Figure 1 The procedure shown is for detecting proteins. The specific procedure is as follows:

[0054] (1) Preparation of norepinephrine solution: Weigh 5 mg of norepinephrine hydrochloride, dissolve it in 3 mL of 10 mM Tris (pH 8.5) solution, and mix well;

[0055] (2) Acridinium ester solution: Weigh acridinium ester and dissolve it in DMSO to make a final concentration of 10 mM, then dilute it to 1 nM with 10 mM Tris (pH 8.5) solution.

[0056] (3) Take 50 μL of magnetic microparticle-labeled phosphorylated Tau protein-217 (pTau217) antibody (5 μg / mL) and incubate with 50 μL of sample at 37℃ for 10 min. After washing and magnetic separation, discard the supernatant.

[0057] (4) Add 50 μL of horseradish peroxidase (HRP) labeled phosphorylated Tau protein-217 (pTau217) antibody (5 μg / mL), incubate at 37℃ for 10 min, and discard the supernatant after washing and magnetic separation.

[0058] (5) Dilute the norepinephrine solution obtained in step (1) to 5 mg / mL with 10 mM Tris (pH 8.5) solution, add 200 μL of diluent and 4 μL of 1 M hydrogen peroxide solution, mix well and incubate at 37 °C for 5 min, and discard the supernatant after washing and magnetic separation.

[0059] (6) Add 50 μL of the 1 nM acridinium ester solution obtained in step (2), incubate at 37 °C for 5 min, and discard the supernatant after washing and magnetic separation;

[0060] (7) Add 100 μL of 0.1 wt.% hydrogen peroxide solution and 100 μL of 0.2 M sodium hydroxide solution, and read the relative luminescence value.

[0061] Example 2

[0062] Same as Example 1, the difference is as follows: When preparing the norepinephrine solution in step (1), weigh 15 mg of norepinephrine hydrochloride, dissolve it in 3 mL of 10 mM Tris (pH 8.5) solution, and mix well;

[0063] After adding the acridinium ester solution in step (6), incubate at 37°C for 10 min.

[0064] Example 3

[0065] Same as Example 1, except that in step (1), when preparing the norepinephrine solution, 25 mg of norepinephrine hydrochloride was weighed and dissolved in 3 mL of 10 mM Tris (pH 8.5) solution and mixed evenly.

[0066] After adding the acridinium ester solution in step (6), incubate at 37°C for 15 min.

[0067] Comparative Example 1

[0068] (1) Take 150 μL of magnetic microparticle-labeled phosphorylated Tau protein-217 (pTau217) antibody and incubate with 50 μL of sample at 37℃ for 10 min. After washing and magnetic separation, discard the supernatant.

[0069] (2) Add 200 μL of acridinium-labeled phosphorylated Tau protein-217 (pTau217) antibody, incubate at 37°C for 10 min, and discard the supernatant after washing and magnetic separation;

[0070] (3) Add 100 μl each of 0.1% hydrogen peroxide solution and 0.2 M sodium hydroxide solution, and read the relative luminescence value.

[0071] Comparative Example 2

[0072] Same as Example 1, except that norepinephrine solution is not added during incubation in step (5).

[0073] Comparative Example 3

[0074] Same as Example 1, except that hydrogen peroxide solution is not added during incubation in step (5).

[0075] Test Example 1

[0076] The methods of Examples 1-3 and Comparative Examples 1-3 were used to detect a series of human pTau217 samples (0, 0.25, 0.5, 1, 2, 4, 8, 16 pg / mL) prepared with bovine serum matrix, and the luminescence intensity was statistically analyzed. The results are shown in Table 1.

[0077] Table 1. Experimental results of luminescence intensity detected by pTau217

[0078]

[0079]

[0080] As shown in Table 1, the luminescence detection signal of Comparative Example 1 (conventional direct luminescence detection of acridine ester with magnetic microparticles) was low, and the sensitivity was approximately 2 pg / mL (coefficient of variation < 8%). Comparative Example 2 did not obtain a gradient detection signal because norepinephrine solution was not added during incubation in step (5). Similarly, in Comparative Example 3, no gradient detection signal was obtained because hydrogen peroxide solution was not added during incubation in step (5). The detection sensitivity of Examples 1-3 was improved compared to Comparative Example 1, and the luminescence detection signal increased with the increase of the incubation time for copolymerization of acridine ester and enzymatically catalyzed norepinephrine. Examples 1-3 effectively improved the luminescence detection signal and increased the sensitivity to approximately 0.25 pg / mL (coefficient of variation < 8%). The experimental results show that adding norepinephrine solution and hydrogen peroxide solution in step (5) is a necessary condition for achieving copolymerization of acridine ester and enzymatically catalyzed norepinephrine.

[0081] Example 4

[0082] Same as Example 1, except that the phosphorylated Tau protein-217 (pTau217) antibody is replaced with GFAP antibody.

[0083] Example 5

[0084] Same as Example 2, except that the phosphorylated Tau protein-217 (pTau217) antibody is replaced with GFAP antibody.

[0085] Example 6

[0086] Same as Example 3, except that the phosphorylated Tau protein-217 (pTau217) antibody is replaced with GFAP antibody.

[0087] Comparative Example 4

[0088] Similar to Comparative Example 1, the only difference is that the phosphorylated Tau protein-217 (pTau217) antibody is replaced with the GFAP antibody.

[0089] Comparative Example 5

[0090] Similar to Comparative Example 2, the only difference is that the phosphorylated Tau protein-217 (pTau217) antibody was replaced with the GFAP antibody.

[0091] Comparative Example 6

[0092] Similar to Comparative Example 3, the only difference is that the phosphorylated Tau protein-217 (pTau217) antibody was replaced with the GFAP antibody.

[0093] Test Example 2

[0094] The methods of Examples 4-6 and Comparative Examples 4-6 were used to detect a series of human GFAP samples (0, 0.05, 0.1, 0.5, 1, 2, 5, 10 ng / mL) prepared with bovine serum matrix, and the luminescence intensity was statistically analyzed. The results are shown in Table 2.

[0095] Table 2. Experimental results of luminescence intensity detected by GFAP.

[0096]

[0097] As shown in Table 2, the detection sensitivity of GFAP using the conventional chemiluminescence method in Comparative Example 4 was 0.1 ng / mL (coefficient of variation < 8%). In Comparative Example 5, no gradient detection signal was obtained because norepinephrine solution was not added during incubation in step (5). Similarly, in Comparative Example 6, no gradient detection signal was obtained because hydrogen peroxide solution was not added during incubation in step (5). The detection sensitivity of Examples 4-6 was improved compared to Comparative Example 4, reaching approximately 0.05 ng / mL (coefficient of variation < 8%).

[0098] As can be seen from the above, the technical solution provided by the present invention can improve the sensitivity of chemical detection of target proteins.

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chemiluminescent immunoassay kit, characterized in that, The chemiluminescent immunoassay kit consists of norepinephrine or its soluble salt, hydrogen peroxide, an alkaline pH adjuster, a chemiluminescent reagent, a first target protein recognition molecule, and a second target protein recognition molecule. The second target protein recognizes a horseradish peroxidase-labeled molecule; The first target protein recognizes molecules labeled with magnetic microparticles; The target protein is phosphorylated Tau-217 protein or glial fibrillary acidic protein; The chemiluminescent reagent is acridine ester.

2. The chemiluminescent immunoassay kit according to claim 1, characterized in that, The first target protein recognition molecule and the second target protein recognition molecule respectively include one or more of antibodies, antigens and nucleic acids.

3. The chemiluminescent immunoassay kit according to claim 1, characterized in that, The alkaline pH adjuster includes sodium hydroxide.

4. The chemiluminescent immunoassay kit according to any one of claims 1 to 3, characterized in that, The chemiluminescent immunoassay kit also includes Tris and DMSO.

5. The use of the chemiluminescent immunoassay kit according to any one of claims 1 to 4 in the preparation of auxiliary clinical diagnostic products.

6. A method for detecting proteins for non-diagnostic purposes, characterized in that, The chemiluminescent immunoassay kit according to any one of claims 1 to 4 comprises the following steps: The sample to be tested is mixed with the first target protein recognition molecule and incubated for the first time to obtain the first incubation product. The first incubation product and the second target protein recognition molecule are mixed and subjected to a second incubation to obtain the second incubation product; The second incubation product, norepinephrine or its soluble salt solution, and hydrogen peroxide solution are mixed and subjected to a third incubation to obtain the third incubation product. The third incubation product is mixed with a chemiluminescent reagent and subjected to a fourth incubation to obtain a fourth incubation product; The fourth incubation product, hydrogen peroxide, and alkaline pH adjuster are mixed, and the relative luminescence value is read using a fully automated chemiluminescence analyzer. The calibration curve is automatically fitted, and the concentration of the target protein is calculated.

7. The method according to claim 6, characterized in that, The first incubation temperature is 37℃, and the time is 5~15min; The second incubation temperature is 37°C, and the time is 5-15 minutes. The third incubation was carried out at a temperature of 37°C for 5-15 minutes. The fourth incubation temperature is 37°C, and the time is 5~15 min; During the third incubation, the concentration of the hydrogen peroxide solution is 1M; the concentration of norepinephrine in the norepinephrine or its soluble salt solution is 5mg / mL; and the volume ratio of the hydrogen peroxide solution to the norepinephrine or its soluble salt solution is 1:

50. The working concentration of the chemiluminescent reagent is 1 nM.

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