阅读说明：本技术 一种甲氧基去甲肾上腺素发光免疫检测试剂盒 (Methoxy norepinephrine luminescent immunoassay kit ) 是由 许君艳 庄路阳 王丹 杨敏 于 2020-12-11 设计创作，主要内容包括：本发明涉及生物技术领域,特别涉及甲氧基去甲肾上腺素发光免疫检测试剂盒。包括样本前处理试剂和检测试剂；样本前处理试剂包含：酸化液、酰化缓冲液、酰化剂溶液；酰化剂溶液中的酰化剂为乙酸酐、琥珀酸酐、丁酸酐、己酸酐、辛酸酐、乙酰氯、冰醋酸中的一种或几种；检测试剂包含：包被有甲氧基去甲肾上腺素的磁微粒混悬液,甲氧基去甲肾上腺素的特异性抗体溶液,含有辣根过氧化物酶标记抗抗体的酶结合物溶液,校准品；或者：包被有甲氧基去甲肾上腺素的磁微粒混悬液,含有辣根过氧化物酶标记的甲氧基去甲肾上腺素特异性抗体的酶结合物溶液,校准品。本发明提供的试剂盒大幅提高检测效率；本发明具有更高的灵敏度和特异性,大大提高临床符合率。(The invention relates to the technical field of biology, in particular to a methoxy norepinephrine luminescence immunoassay kit. Comprises a sample pretreatment reagent and a detection reagent; the sample pretreatment reagent comprises: acidifying solution, acylation buffer solution and acylating agent solution; the acylating agent in the acylating agent solution is one or more of acetic anhydride, succinic anhydride, butyric anhydride, hexanoic anhydride, caprylic anhydride, acetyl chloride and glacial acetic acid; the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, a specific antibody solution of the methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase labeled anti-antibody, and a calibrator; or: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase-labeled methoxy norepinephrine specific antibody, and a calibrator. The kit provided by the invention greatly improves the detection efficiency; the invention has higher sensitivity and specificity, and greatly improves the clinical compliance rate.)

1. A methoxy norepinephrine luminescence immunoassay kit is characterized by comprising a sample pretreatment reagent and a detection reagent;

the sample pretreatment reagent comprises: acidifying solution, acylation buffer solution and acylating agent solution; the acylating agent in the acylating agent solution is one or more of acetic anhydride, succinic anhydride, butyric anhydride, hexanoic anhydride, caprylic anhydride, acetyl chloride and glacial acetic acid;

the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, a specific antibody solution of the methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase labeled anti-antibody, and a calibrator;

alternatively, the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase-labeled methoxy norepinephrine specific antibody, and a calibrator.

2. The kit according to claim 1, wherein the acidizing fluid is a strong acid solution containing a phenol red indicator and having a pH of less than 2, and the strong acid is one of hydrochloric acid and sulfuric acid and has a concentration of 0.1-0.5M; the content of the phenol red indicator in the acidizing fluid is 0.0001 vt-0.01 vt%.

3. The kit of claim 1, wherein the acylation buffer is one of Bis-Tris, Tris and PBS, and has a concentration of 0.1-10M and a pH of 8.0-9.0.

4. The kit according to claim 1, wherein the solvent in the acylating agent solution is one or more of DMF, DMSO and absolute ethyl alcohol; the volume ratio of the solvent to the acylating agent in the acylating agent solution is (50-70): 1.

5. the kit according to claim 1, wherein the concentration of the antibody specific to methoxynoradrenaline in the solution of the antibody specific to methoxynoradrenaline is 0.001 to 1% by volume.

6. The kit according to claim 1, wherein in the antibody solution specific to methoxynorepinephrine, an antibody diluent is used which consists of a dilution buffer, a protective protein, and a preservative; the concentration of a dilution buffer solution in the antibody diluent is 0.01-0.1M, the mass volume percentage of the protective protein is 0.5-5%, and the volume percentage of the preservative is 0.01-1%;

the dilution buffer solution is one of Bis-Tris, Tris-NaCl and PBS, and the pH value is 3.0-8.0;

the protective protein is one or two of bovine serum albumin, bovine serum, human serum, gelatin and casein;

the antiseptic is ProClin300, Bronidox, MIT, NaN₃Any combination of (a).

7. The kit of claim 1, wherein the enzyme conjugate solution consists of horseradish peroxidase-labeled anti-antibody, enzyme diluent;

or the enzyme conjugate solution consists of a horse radish peroxidase-labeled methoxy norepinephrine specific antibody and an enzyme diluent;

the enzyme diluent consists of a dilution buffer solution and protective protein;

the dilution buffer solution is one of Bis-Tris, Tris-NaCl and PBS, and the pH value is 3.0-8.0;

the mass volume percentage of the protective protein is 0.5-5%, and the protective protein is one or two of bovine serum albumin, bovine serum, human serum, gelatin and casein.

8. The kit according to claim 1, wherein the methoxy noradrenaline used for preparing the magnetic particle suspension coated with methoxy noradrenaline is coupled with carrier protein, and the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin and egg white albumin;

the concentration of the methoxyl norepinephrine used in the preparation of the magnetic particle suspension is 0.5-5 mug/mL;

the calibrator is a PBS (phosphate buffer solution) containing methoxy norepinephrine, and the concentration of the methoxy norepinephrine in the calibrator is 0-5000 ng/mL; and controlling the pH value of the PBS solution in the calibrator to be 2-4.

9. The kit according to any one of claims 1 to 8, wherein the step of performing a luminescent immunoassay using the sample pretreatment reagent comprises:

adding an acidizing fluid into a sample to be tested, uniformly mixing, and incubating for 20-40 min at 80-100 ℃; after the incubation is finished, cooling the acidified sample to room temperature, adding an acylation buffer solution, and uniformly mixing; adding acylating agent, mixing uniformly, standing at room temperature for at least 15 min.

10. The kit according to claim 9, wherein the volume ratio of the sample to be detected, the acidification liquid, the acylation buffer solution and the acylating agent is (50-150): (200-300): (200-300): (20-30).

Technical Field

The invention relates to the technical field of biology, in particular to a methoxy noradrenaline luminescence immunoassay kit.

Background

At present, the mainstream market detection method for methoxy norepinephrine (NMN) mainly comprises High Performance Liquid Chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and the like, but the HPLC takes longer time, the cost of a kit instrument is high, batch measurement is difficult to realize, the technical requirement on operators is high, the enzyme-linked immunosorbent assay lacks accuracy and reproducibility, and meanwhile, the method has the advantages of low automation degree, easiness in environmental influence, low reaction rate, long detection time and low sensitivity.

In recent years, with the popularization of magnetic particle coupling technology and chemiluminescence technology, a full-automatic chemiluminescence immunoassay method is also adopted for detecting the methoxy norepinephrine, namely, the magnetic particle coupling technology, the immunoreaction technology and the chemiluminescence technology are combined, so that the automatic detection is realized, and the method has the characteristics of high sensitivity, high specificity, high accuracy and high stability.

The magnetic microsphere is a spherical composite material with the diameter of nanometer or micron. The core of the magnetic microsphere is made of superparamagnetic materials such as ferroferric oxide or ferric oxide, and the periphery of the magnetic microsphere is coated with polymer materials such as polystyrene or glucan. The core material has superparamagnetism, so that the magnetic beads can move towards the direction of the magnetic field under the action of an external magnetic field to achieve the purpose of separation; the peripheral high molecular material can be activated by physical or chemical method to generate amino (NH)₂-), carboxyl (COOH-), epoxy (-CH (O)), etc., and can be coupled with bioactive molecules such as proteins. The coupled magnetic beads are widely applied to the fields of biological markers, biomolecule separation, protein purification, antibody purification, molecular diagnosis and the like. The chemiluminescence immunoassay comprises two parts, namely an immunoreaction technology and a chemiluminescence technology. The basic principle is that the general enzyme of the immune reaction acts on a luminescent substrate, so that the luminescent substrate is subjected to chemical reaction and releases a large amount of energy to generate an excited intermediate. When the excited state intermediate returns to a stable ground state, photons can be emitted simultaneously. The light quantum yield can be measured by using a light-emitting signal detection instrument, and is in direct proportion to the amount of the substance to be measured in the sample, so that a standard curve can be established and the content of the substance to be measured in the sample can be calculated. Chemical hairThe light immunoassay technique usually adopts reaction modes such as a double antibody sandwich method, a competition method, an indirect method and the like.

The publication No. CN109444416A discloses a methoxy noradrenaline luminescence immunoassay kit, which comprises a detection system and a sample pretreatment system, wherein the detection system comprises a solid phase carrier directly or indirectly coated with a methoxy noradrenaline antibody, a methoxy noradrenaline antibody solution, an avidin connected tracer solution and a calibrator; or the detection system comprises a magnetic particle suspension directly or indirectly coated with the methoxy noradrenaline, an enzyme conjugate solution containing a horseradish peroxidase-labeled methoxy noradrenaline specific antibody, and a calibrator. The sample pretreatment system comprises an acidification liquid, an acylating agent and an alkaline buffer liquid. However, the product performance of the detection kit in the aspects of precision, sensitivity, detection gradient and the like is still to be improved.

Disclosure of Invention

In view of the above, the invention provides a methoxy norepinephrine luminescent immunoassay kit. According to the kit provided by the invention, the first result detection needs 30min, so that the detection efficiency is greatly improved; the invention has higher sensitivity and specificity, and greatly improves the clinical compliance rate.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a methoxy noradrenaline luminescence immunoassay kit, which comprises a sample pretreatment reagent and a detection reagent;

the sample pretreatment reagent comprises: acidifying solution, acylation buffer solution and acylating agent solution; the acylating agent in the acylating agent solution is one or more of acetic anhydride, succinic anhydride, butyric anhydride, hexanoic anhydride, caprylic anhydride, acetyl chloride and glacial acetic acid;

the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, a specific antibody solution of the methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase labeled anti-antibody, and a calibrator;

alternatively, the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase-labeled methoxy norepinephrine specific antibody, and a calibrator.

The invention adopts the principle of a competition method, namely, the magnetic particle coated antigen and the antigen in a sample are competitively combined with the antibody in the antibody solution, the competition result is a solid phase antigen-antibody compound, then enzyme-labeled anti-antibody is added, and finally enzymatic chemiluminescence substrate is added. Or: the magnetic particle coated antigen and the antigen in the sample are competitively combined with the enzyme-labeled antibody in the enzyme conjugate solution, the competition result is a solid phase antigen-enzyme-labeled antibody compound, and finally, an enzymatic chemiluminescence substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, when the excited state intermediate reaches the ground state, photons are emitted to form a luminescence reaction, photon energy is recorded through a photon reading system, the light energy intensity is converted into the concentration of the antigen to be detected on a standard curve through a computer processing system, and the result is reported.

The invention adopts a special sample pretreatment system, firstly carries out acidification and acylation pretreatment on methoxy noradrenaline in urine or plasma, adopts an acylation agent acylation scheme of acid anhydride, organic acid or ester, leads the acylated methoxy noradrenaline and immobilized methoxy adrenaline to competitively combine with an antibody, and leads the immobilized antibody to be combined with an enzyme-labeled anti-antibody, thereby improving the sensitivity and specificity of the kit. The principle is that MN in the organism is a small molecular hormone with molecular weight of about 200 and belongs to hapten; most of MN in the blood or urine of the organism exists in a sulfonated state; the reactogenicity of the NMN molecule can be improved through the pretreatment of acidification and acylation.

Preferably, the acidizing fluid is a strong acid solution which has pH <2 and contains a phenol red indicator, and the strong acid is one of hydrochloric acid and sulfuric acid, and the concentration of the strong acid is 0.1-0.5M.

Preferably, the concentration of the strong acid in the acidified liquid is 0.25M.

Preferably, the content of the phenol red indicator in the acidizing fluid is 0.0001 vt-0.01 vt%.

Preferably, the content of the phenol red indicator in the acidified liquid is 0.001 vt%.

Preferably, the acylation buffer is one of Bis-Tris, Tris and PBS, the concentration is 0.1-10M, and the pH value is 8.0-9.0.

Preferably, the concentration of the acylation buffer is 1M.

Preferably, the solvent in the acylating agent solution is one or more of DMF, DMSO and absolute ethyl alcohol; the volume ratio of the solvent to the acylating agent in the acylating agent solution is (50-70): 1.

preferably, the volume ratio of solvent to acylating agent in the acylating agent solution is 60: 1.

in the invention, the magnetic particles coated with methoxy noradrenaline are used, and the immobilized methoxy noradrenaline (NMN) antigen includes but is not limited to NMN molecules and derivatized NMN molecules.

Preferably, the concentration of the antibody specific to methoxynoradrenaline in the solution of the antibody specific to methoxynoradrenaline is 0.001% to 1% by volume.

Preferably, the concentration of the antibody specific for methoxynorepinephrine is 0.01% by volume.

Preferably, in the specific antibody solution of the methoxy norepinephrine, the used antibody diluent consists of a dilution buffer, a protective protein and a preservative; the concentration of a dilution buffer solution in the antibody diluent is 0.01-0.1M, the mass volume percentage of the protective protein is 0.5-5%, and the volume percentage of the preservative is 0.01-1%;

preferably, the concentration of the dilution buffer in the antibody diluent is 0.05M, the mass volume percentage of the protective protein is 2%, and the volume percentage of the preservative is 0.1%;

the dilution buffer solution is one of Bis-Tris, Tris-NaCl and PBS, and the pH value is 3.0-8.0;

the protective protein is one or two of bovine serum albumin, bovine serum, human serum, gelatin and casein;

the antiseptic is ProClin300, Bronidox, MIT, NaN₃Any combination of (a).

Preferably, the enzyme conjugate solution consists of one of horseradish peroxidase-labeled anti-antibody or horseradish peroxidase-labeled methoxy norepinephrine specific antibody and enzyme diluent;

preferably, the dilution buffer is one of Bis-Tris, Tris-NaCl and PBS, and the pH value is 3.0-8.0;

preferably, the mass volume percentage of the protective protein is 0.5-5%, and the protective protein is one or two of bovine serum albumin, bovine serum, human serum, gelatin and casein.

Preferably, the methoxy noradrenaline used in the preparation of the magnetic particle suspension coated with the methoxy noradrenaline is coupled with carrier protein, and the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin and ovalbumin;

preferably, the concentration of methoxynorepinephrine used in the preparation of the magnetic particle suspension is 0.3 to 5 μ g/mL.

Preferably, the concentration of methoxynorepinephrine used in the preparation of the magnetic microparticle suspension is 0.3 μ g/mL.

Preferably, the calibrator is a PBS (phosphate buffer solution) containing methoxy-noradrenaline, and the concentration of the methoxy-noradrenaline in the calibrator is 0-5000 ng/mL.

Preferably, the concentration of the methoxy norepinephrine in the calibrator is 0-2000 ng/mL.

Preferably, the pH of the PBS solution in the calibrator is controlled to be 2-4.

Preferably, the step of performing a luminescent immunoassay using the sample pretreatment reagent comprises:

adding an acidizing fluid into a sample to be tested, uniformly mixing, and incubating for 20-40 min at 80-100 ℃; after the incubation is finished, cooling the acidified sample to room temperature, adding an acylation buffer solution, and uniformly mixing; adding acylating agent, mixing uniformly, standing at room temperature for at least 15 min.

In the specific embodiment provided by the invention, the steps of performing the luminescence immunoassay by using the sample pretreatment reagent are as follows:

adding an acidizing fluid into a sample to be detected, uniformly mixing, and incubating for 30min at 90 ℃; after the incubation is finished, cooling the acidified sample to room temperature, adding an acylation buffer solution, and uniformly mixing; adding acylating agent, mixing uniformly, standing at room temperature for at least 15 min.

Preferably, the volume ratio of the sample to be detected, the acidizing fluid, the acylation buffer solution and the acylating agent is (50-150): (200-300): (200-300): (20-30).

In the specific embodiment provided by the invention, the volume ratio of the sample to be detected, the acidification liquid, the acylation buffer solution and the acylating agent is 100: 250: 250: 25.

the invention provides a methoxy noradrenaline luminescence immunoassay kit. The kit comprises a sample pretreatment reagent and a detection reagent; the sample pretreatment reagent comprises: acidifying solution, acylation buffer solution and acylating agent solution; the acylating agent in the acylating agent solution is one or more of acetic anhydride, succinic anhydride, butyric anhydride, hexanoic anhydride, caprylic anhydride, acetyl chloride and glacial acetic acid; the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, a specific antibody solution of the methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase labeled anti-antibody, and a calibrator; alternatively, the detection reagent comprises: the kit comprises a magnetic particle suspension coated with methoxy norepinephrine, an enzyme conjugate solution containing horseradish peroxidase-labeled methoxy norepinephrine specific antibody, and a calibrator. The invention has the following technical effects:

1. the invention has the advantages of short detection time and high sensitivity;

2. the method adopts a reaction mode of a two-step method, and reports the result 30min after the first sample is added; the detection can be realized by 200 per hour by matching with a matched instrument, and a large amount of time for clinical examination can be saved.

3. The kit is matched with a full-automatic immunoassay instrument for an ampere chart, and the instrument adopts a special cleaning mode, so that common interference factors are reduced, and the detection sensitivity is improved.

4. The PH range of the acidizing fluid in the pretreatment system is further accurate to be less than 2, and the pretreatment efficiency of the sample is improved.

5. According to the invention, through the selection of the coating antigen on the solid phase carrier and the selection of the type of the acylating agent in the sample pretreatment system, the maximum competitiveness between the coating antigen and the treated sample is ensured, and compared with CN109444416A, the product performance in the aspects of precision, sensitivity, detection gradient and the like of the method is obviously improved.

Drawings

FIG. 1 correlation of the kit of the present invention with HPLC-MS/MS.

Detailed Description

The invention discloses a methoxy noradrenaline luminescence immunoassay kit, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Chinese-English comparison:

Bis-Tris: bis (2-hydroxymethyl) amino-tris (hydroxymethyl) methane;

Tris-NaCl: tris-hydroxymethyl aminomethane-sodium chloride;

PBS: phosphate buffer;

DMF: n, N-dimethylformamide;

DMSO, DMSO: dimethyl sulfoxide;

methoxy norepinephrine: normetaneprine, NMN;

BRINIDOX: 5-bromo-5 nitro-1, 3-dioxane;

MIT: 2-methyl-4-isothiazolin-3-one hydrochloride;

NaN₃: sodium azide.

Reagents or instruments used in the methoxy norepinephrine luminescence immunoassay kit provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

The kit comprises the following components:

the invention comprises a sample pretreatment system and a detection system.

1. The sample pretreatment system comprises: acidifying liquid, acylation buffer liquid and acylating agent.

Firstly, the acidizing fluid is hydrochloric acid containing 0.001 percent of phenol red indicator, the concentration is 0.25M, and the pH value is less than 1;

the acylation buffer solution is Tris, and the pH value of the acylation buffer solution is 8.0-9.0;

③ the acylating agent is DMSO solution containing acetic anhydride (volume ratio of DMSO to acetic anhydride is 60: 1).

2. The detection system comprises: the kit comprises magnetic particles coated with methoxy norepinephrine (NMN) antigen, specific antibody solution of NMN molecules, enzyme conjugate solution containing horseradish peroxidase labeled anti-antibody, and a calibrator containing NMN molecules.

The antibody diluent used in the invention is Tris-NaCl containing bovine serum albumin and ProClin300, and the pH value is 7.4;

second, preparation method

1. Preparing a magnetic particle suspension: fully and uniformly mixing magnetic particle stock solution with the particle size of 1-3 mu m and the magnetic content of 40%, taking out 30 mu L, adding 300 mu L of phosphate buffer solution for washing for 5 times, washing for 5min each time, and adsorbing and fixing the magnetic particles by using a magnet during washing to remove supernatant; after washing, adding 100 mu L of EDC (carbodiimide) and NHS (succinimide ester) activators dissolved in sodium acetate buffer solution, wherein the concentration of the activators is 20mg/mL, uniformly mixing, vibrating and activating for 1 h; and after activation, adding 300 mu L of sodium acetate buffer solution for washing for 2 times, washing for 5min each time, then adding a certain amount (0.3 mu g/mL, 0.1mL) of NMN antigen coupled with BSA molecules, uniformly mixing, shaking and coating for 2h, after coating, adding 300 mu L of phosphate buffer solution containing 2% of bovine serum albumin, sealing for 4 times, finally diluting to 3mL by using phosphate buffer solution containing 2% of bovine serum albumin, and storing at 2-8 ℃.

2. Preparing an antibody solution: adding 2% bovine serum albumin into a prepared Tris-NaCl buffer solution (0.05M Tris containing 0.85% NaCl and 0.1% Proclin 300), mixing to obtain an antibody solution diluent, adding a specific antibody of the NMN molecule according to the proportion of 1:10000(1:100-1: 100000), uniformly mixing, and storing at 2-8 ℃.

3. Preparation of enzyme conjugate solution: adding 2% bovine serum albumin into a prepared Tris-NaCl buffer solution, mixing to obtain an enzyme diluent, adding an anti-antibody marked by HRP (horse radish peroxidase) according to the proportion of 1:5000(1:100-1: 100000), mixing uniformly, and storing at 2-8 ℃.

4. Preparing a calibrator: the prepared PBS (pH 3-4) buffer solution is a calibrator diluent, MN antigen is diluted into 6 gradients by the calibrator diluent, and the gradients are 0ng/mL, 20ng/mL, 60ng/mL, 200ng/mL, 600ng/mL and 2000ng/mL, and the gradients are packaged into 1 mL/bottle and stored at the temperature of 2-8 ℃.

5. Preparing an acidizing fluid of a sample pretreatment system: concentrated hydrochloric acid was diluted to 0.25M with purified water and 0.001% phenol red indicator was added.

6. Preparing an acylation buffer solution of a sample pretreatment system: preparing 1M Tris buffer solution, and adjusting the pH value to 8-9 by using 10M NaOH.

7. Preparing an acylating agent: the acylating agent solvent is DMSO, and the acylating agent is obtained after the DMSO and the acetic anhydride are mixed according to the proportion of 60: 1.

Third, detection method

1. The operation steps of the sample pretreatment system are as follows:

adding 250 mul of acidizing fluid into 100 mul of sample to be tested, uniformly mixing, and then putting into a water bath kettle at 90 ℃ for incubation for 30 min; after the incubation is finished, cooling the acidified sample to room temperature, adding 250 mu L of acylation buffer solution into each sample, and mixing uniformly; adding 25 mu L of prepared acylating agent into each sample, uniformly mixing, standing at room temperature for at least 15min, and detecting.

2. A detection step:

and (4) transferring all the calibrator or samples obtained in the pretreatment step into a reaction cup, and detecting by using an AutoLumo full-automatic detection analyzer. Mu.l of magnetic particle suspension, 50. mu.l of sample and 50. mu.l of antibody solution are added to each well, mixed uniformly, incubated at 37 ℃ for 15min, and washed 6 times with washing solution. Adding 100 mu l of enzyme conjugate into each hole, uniformly mixing, incubating for 17min at 37 ℃, washing with washing liquor for 6 times, respectively adding 50 mu l of substrate A liquid and 50 mu l of substrate B liquid into each hole, uniformly mixing, detecting a luminous value within 1-5 min, and calculating a concentration value of the sample through a calibrator curve.

Example 2 performance verification of the present kit

1. Assay sensitivity detection

The analytical sensitivity assessment was carried out according to EP17A protocol for determination of detection limits and quantitative limits, 5 clinical plasma samples with values close to 0 were prepared, each sample was repeated 3 times for a total of 4 days to obtain 60 non-negative data, and if less than 60, the analytical sensitivity was calculated by performing experiments as required and supplementing 60.

TABLE 1 results of sensitivity detection

As can be seen from the results in Table 1, the assay sensitivity of the kit of the present invention is <1.89 pg/mL.

2. Detection of precision

The precision evaluation was carried out according to "precision evaluation of EP05A 2-quantitative determination method", wherein two batches of reagents were used together with high/medium/low value quality control materials, and the variation of the measured concentration was calculated 20 times each, and the measurement results are shown in Table 2.

TABLE 2 detection of the precision of the kit of the invention

Table 2 the data results show that: the coefficient of variation of the kit is less than 5%.

3. Clinical alignment

The kit and HPLC-MS/MS are used for simultaneously measuring 47 urine samples for clinical diagnosis of adrenal cortex hyperplasia, hypertension, pheochromocytoma, primary hyperparathyroidism and the like, and the correlation with HPLC is shown in figure 1: 0.9948x +2.8291, r²0.9885, it is indicated that the kit can be used to provide assistance in the timely diagnosis and treatment of pheochromocytoma.

Example 3 comparison with existing kits

Compared with embodiment 1 of CN109444416A, the technical effect of the scheme of the application is as follows:

1. sensitivity enhancement (5 clinical plasma samples with values close to 0)

TABLE 3

2. Detecting a rise in gradient

TABLE 4

3. Precision improvement

TABLE 5

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.