阅读说明：本技术 一种定量检测地高辛的化学发光免疫检测试剂盒及其制备方法 (Chemiluminescence immunoassay kit for quantitatively detecting digoxin and preparation method thereof ) 是由 李磊 李冬梅 韩美玉 孙成艳 高威 何浩会 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种定量检测地高辛的化学发光免疫检测试剂盒,其特征在于,包括试剂R1、R2和R3,其中：试剂R1为包含链霉亲和素磁颗粒的悬浮液；试剂R2为包含化学发光标记物标记的地高辛单克隆抗体的溶液；试剂R3为包含偶联标记物标记的地高辛衍生物的溶液。本发明提供的试剂盒,针对地高辛是小分子物质,采用竞争法原理进行检测,节约时间及成本,具有免疫反应的高度特异性,又具有发光反应的高敏感性,具有灵敏度高、特异性强、线性范围宽、试剂稳定性好、操作简便、容易实现自动化的优点,本发明提供的制备方法,工艺简单,试剂盒有效期长,成本相对较低；方便实现快速检测,对于仪器的要求简单,便于实现全自动化操作。(The invention discloses a chemiluminescence immunoassay kit for quantitatively detecting digoxin, which is characterized by comprising reagents R1, R2 and R3, wherein: reagent R1 is a suspension comprising streptavidin magnetic particles; reagent R2 is a solution containing a chemiluminescent label-labeled digoxin monoclonal antibody; the reagent R3 is a solution comprising a digoxigenin derivative labelled with a conjugate label. The kit provided by the invention is used for detecting digoxin which is a small molecular substance by adopting a competition method principle, so that the time and the cost are saved, the high specificity of immunoreaction is realized, the high sensitivity of luminescence reaction is realized, and the kit has the advantages of high sensitivity, strong specificity, wide linear range, good reagent stability, simple and convenient operation and easy realization of automation; the rapid detection is conveniently realized, the requirement on the instrument is simple, and the full-automatic operation is convenient to realize.)

1. A chemiluminescent immunoassay kit for the quantitative detection of digoxin, which is characterized by comprising reagents R1, R2 and R3, wherein:

reagent R1 is a suspension comprising streptavidin magnetic particles;

reagent R2 is a solution containing a chemiluminescent label-labeled digoxin monoclonal antibody;

the reagent R3 is a solution comprising a digoxigenin derivative labelled with a conjugate label.

2. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 1, wherein the mass percentage of the streptavidin magnetic particles in the reagent R1 is 0.01% -1%.

3. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 2, wherein the particle size of the streptavidin magnetic particle in the reagent R1 is 0.05-3 μm.

4. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 1, wherein the molar ratio of the digoxin monoclonal antibody to the chemiluminescent label in the reagent R2 is 1: 1-1: 20.

5. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 4, wherein the concentration of the chemiluminescent label-labeled digoxin monoclonal antibody in reagent R2 is 10ng/mL or more.

6. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 4, wherein the chemiluminescent label in reagent R2 is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.

7. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 1, wherein the molar ratio of digoxin derivative to conjugated marker in reagent R3 is 1: 1-1: 20.

8. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 7, wherein the concentration of the digoxin derivative labeled with the conjugate label in the reagent R3 is set as follows

≥5ng/mL。

9. The chemiluminescent immunoassay kit for the quantitative detection of digoxin according to claim 7, wherein the digoxin derivative in the reagent R3 is digoxin-BSA or digoxin-OVA, and the conjugated marker is biotin.

10. The method for preparing a chemiluminescent immunoassay kit for the quantitative detection of digoxin according to any one of claims 1-9, comprising the steps of:

step S1, preparation of reagent R1:

uniformly mixing a streptavidin magnetic particle solution and a TBST solution, placing the mixture on a magnetic separator until a supernatant is not turbid, removing the supernatant, reserving magnetic particles, and preparing a solid phase reagent in a buffer solution after cleaning; wherein the concentration of the streptavidin magnetic particle solution is 50-100 mg/ml; the volume ratio of the streptavidin magnetic particle solution to the TBST solution is preferably (0.5-1): (5-10); the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mMPBS, 0.1% Tween-20, 0.1% Proclin300, pH7.2; the concentration of the solid phase reagent is preferably 0.01-1%;

step S2, preparation of reagent R2:

placing the digoxin monoclonal antibody into a centrifuge tube for centrifugation, then adding a phosphate buffer solution, uniformly mixing, adding a chemiluminescent marker solution for centrifugation, sealing the centrifuge tube, then placing the centrifuge tube into a dark box, uniformly mixing, adding a sealing solution, uniformly mixing, purifying and collecting the sealed antibody, then placing the antibody into the buffer solution for dilution, and storing; wherein the molar ratio of the digoxin monoclonal antibody to the chemiluminescent marker is 1: 1-1: 20; the concentration of the chemiluminescent marker solution is 2-2.5 mg/mL; the confining liquid is lysine, and the mass fraction is 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1%

Proclin300，pH6.0；

Step S3, preparation of reagent R3:

putting the digoxin derivative into a centrifugal tube for centrifugation, then adding TRIS buffer solution, adding the coupling marker solution for centrifugation after uniform mixing, adding the confining liquid after uniform mixing, uniformly mixing, purifying and collecting the confined antibody, then putting the antibody into the buffer solution for dilution, and storing; the molar ratio of the digoxin derivative to the coupling marker is 1: 1-1: 20; the concentration of the coupling marker solution is preferably 2-3 mg/mL; the confining liquid is preferably lysine with the mass fraction of 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH6.0.

Technical Field

The invention relates to the technical field of in-vitro detection, in particular to a chemiluminescence immunoassay kit for quantitatively detecting digoxin and a preparation method thereof.

Background

Digoxin (Digoxin, or DIG) is a middle-acting cardiac glycoside drug extracted from digitalis, and is white crystal or crystalline powder, odorless, and bitter. During treatment, the effect on the heart is shown as positive inotropic effect, the heart rate is slowed down, the heart conduction is inhibited, and the traditional Chinese medicine is suitable for low-output congestive heart failure, atrial fibrillation, atrial flutter and paroxysmal supraventricular tachycardia. The positive inotropic effect makes digoxin the first clinically preferred effective medicine for treating heart failure and controlling rapid ventricular rate.

However, the effective blood concentration of digoxin is narrow, the concentration of digoxin in patients without medicines is less than 0.5ng/ml, and the treatment range of digoxin is recommended to be 0.6-1.2 ng/m according to the ESC diagnosis and treatment guidelines of acute and chronic heart failure in 2008. In addition, digoxin has a narrow safety range, a low therapeutic index, large individual difference in pharmacokinetics and pharmacodynamics, and pharmacokinetic characteristics that therapeutic dose and toxic dose overlap each other to a certain extent, and is likely to cause toxic reaction. Therefore, digoxin is one of the main drugs clinically needing monitoring of blood concentration. Timely monitoring of the blood level of digoxin during clinical treatment is a major method for adjusting the dosage regimen and preventing drug poisoning.

The common monitoring scheme for the digoxin blood concentration mainly comprises a liquid phase-mass spectrum combined analysis method, a radioimmunoassay, an enzyme-linked immunosorbent assay method and a chemiluminescence immunoassay method. The liquid phase-mass spectrometry analysis method requires expensive instruments, is complex to operate, has poor sensitivity and takes long detection time, so that the clinical application of the method is limited; the radioimmunoassay has the defects of longer detection time, short half-life of an antigen marker, easy interference of metabolites, radioactive pollution of different degrees and the like, and is difficult to be used for clinical instant determination; the defects of the enzyme-linked immunosorbent assay are that human factors interfere the detection result, the incubation time is long, the enzyme stability is reduced, and the accuracy of the detection result is influenced.

Chemiluminescence immunoassay (CLIA) is an emerging immunoassay technology developed after enzyme immunoassay, radioimmunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Because it has high specificity of immunoreaction and high sensitivity of luminous reaction, it has been widely used in monitoring and analysis of various hormones, special proteins and medicines in recent years by clinical laboratories and scientific research units at home and abroad. The method has the advantages of high sensitivity, strong specificity, wide linear range, simple operation, good reagent stability, simple operation and easy realization of automation, and is an ideal clinical trace biochemical test analysis means.

At present, the detection principle of a clinical detection Digoxin (DIG) kit which is approved to be on the market in China mainly adopts an enzyme-linked immunosorbent assay (ELISA) and horseradish peroxidase in combination with luminol compounds for chemiluminescence. Although the price is low, the sensitivity and the detection reliability can not be guaranteed, and foreign detection systems are expensive and difficult to popularize. Therefore, it is required to develop a Digoxin (DIG) kit which has high detection sensitivity and precision, is simple and convenient for the instrument, and can realize rapid detection.

Disclosure of Invention

The invention aims at the technical problems and provides a chemical luminescence immunoassay kit for quantitatively detecting digoxin, which is used for quantitatively detecting Digoxin (DIG) in serum or plasma, has high sensitivity, good repeatability and low cost, provides useful information for clinicians to adjust the dosage of patients so as to obtain the optimal treatment effect, and has wide market prospect.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the invention firstly provides a chemiluminescence immunoassay kit for quantitatively detecting digoxin, which comprises reagents R1, R2 and R3, wherein:

reagent R1 is a suspension comprising streptavidin magnetic particles;

reagent R2 is a solution containing a chemiluminescent label-labeled digoxin monoclonal antibody;

the reagent R3 is a solution containing digoxin derivative labeled with conjugate marker;

preferably, the mass percentage of the streptomycin avidin magnetic particles in the reagent R1 is 0.01-1%.

Preferably, the particle size of the streptavidin magnetic particle in the reagent R1 is 0.05-3 μm.

Preferably, the mole ratio of the digoxin monoclonal antibody to the chemiluminescent marker in the reagent R2 is 1: 1-1: 20.

Preferably, the concentration of the chemiluminescent marker labeled digoxin monoclonal antibody in reagent R2 is greater than or equal to 10 ng/mL.

Preferably, the chemiluminescent label in reagent R2 is acridinium ester, luminol, isoluminol or ruthenium terpyridyl.

Preferably, the molar ratio of the digoxin derivative to the conjugated marker in the reagent R3 is 1: 1-1: 20.

Preferably, the concentration of the digoxin derivative labeled with the coupling marker in the reagent R3 is more than or equal to 5 ng/mL.

Preferably, the digoxin derivative in the reagent R3 is digoxin-BSA or digoxin-OVA, and the conjugated marker is biotin.

The invention also provides a preparation method of the chemiluminescence immunoassay kit for quantitatively detecting digoxin, which comprises the following steps:

step S1, preparation of reagent R1:

uniformly mixing a streptavidin magnetic particle solution and a TBST solution, placing the mixture on a magnetic separator until a supernatant is not turbid, removing the supernatant, reserving magnetic particles, and preparing a solid phase reagent in a buffer solution after cleaning; wherein the concentration of the streptavidin magnetic particle solution is 50-100 mg/ml; the volume ratio of the streptavidin magnetic particle solution to the TBST solution is preferably (0.5-1): (5-10); the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mMPBS, 0.1% Tween-20, 0.1% Proclin300, pH7.2; the concentration of the solid phase reagent is preferably 0.01-1%;

step S2, preparation of reagent R2:

placing the digoxin monoclonal antibody into a centrifuge tube for centrifugation, then adding a phosphate buffer solution, uniformly mixing, adding a chemiluminescent marker solution for centrifugation, sealing the centrifuge tube, then placing the centrifuge tube into a dark box, uniformly mixing, adding a sealing solution, uniformly mixing, purifying and collecting the sealed antibody, then placing the antibody into the buffer solution for dilution, and storing; wherein the molar ratio of the digoxin monoclonal antibody to the chemiluminescent marker is 1: 1-1: 20; the concentration of the chemiluminescent marker solution is 2-2.5 mg/mL; the confining liquid is lysine, and the mass fraction is 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH6.0;

step S3, preparation of reagent R3:

putting the digoxin derivative into a centrifugal tube for centrifugation, then adding TRIS buffer solution, adding the coupling marker solution for centrifugation after uniform mixing, adding the confining liquid after uniform mixing, uniformly mixing, purifying and collecting the confined antibody, then putting the antibody into the buffer solution for dilution, and storing; the molar ratio of the digoxin derivative to the coupling marker is 1: 1-1: 20; the concentration of the coupling marker solution is preferably 2-3 mg/mL; the confining liquid is preferably lysine with the mass fraction of 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH6.0.

Compared with the prior art, the invention has the technical effects that:

the kit for quantitatively detecting digoxin by chemiluminescence immunoassay provided by the invention aims at the fact that digoxin is a small molecular substance, streptavidin magnetic particles are used as solid phase carriers, detection is carried out by a competition method principle, DIG is marked by combining chemiluminescence substances with higher luminous intensity and sensitivity, a hydrogen peroxide chemiluminescence system is adopted, quantitative detection of DIG is realized by a competition method, acridinium is selected as a marking material of a chemiluminescence immunoassay system, the material has energy transition generated when an excited state returns to a ground state to be direct chemiluminescence, enzyme participation is not needed, and time and cost are saved; the streptavidin magnetic beads and the analog marked by the biotin can be firmly combined together, so that nonspecific adsorption is reduced, the accuracy of a test sample is improved, and the anti-interference capability is strong. The chemiluminescence immunoassay method has the advantages of high specificity of immunoreaction and high sensitivity of luminescence reaction, has high sensitivity, strong specificity, wide linear range, good reagent stability, simple and convenient operation and easy realization of automation, can be matched with a full-automatic chemiluminescence immunoassay analyzer for detection, directly gives numerical values, reduces artificial operation errors, realizes unattended operation, shortens the time required by clinical detection, has high detection precision, forms a closed system by matching the reagent with the instrument, and has small system errors.

The preparation method of the quantitative detection digoxin chemiluminescence immunoassay kit provided by the invention has the advantages of simple acridinium ester labeling process, stable labeling substance luminescence, long effective period of the kit and relatively low cost; the light is flash, the light is fast, concentrated and strong, the fast detection is convenient to realize, the sensitivity and the precision of the detection are high, the requirement on the instrument is simple, and the full-automatic operation is convenient to realize; and secondly, the acridinium ester luminescent system is simple, the alkaline-hydrogen peroxide can directly emit light without a reinforcing agent or a catalyst, interference factors are few, the background is extremely low, and the signal-to-noise ratio is high.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 is a graph comparing standard curves for the test using the kit and the comparative reagent prepared in example 1 of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The invention firstly provides a chemiluminescence immunoassay kit for quantitatively detecting digoxin, which comprises reagents R1, R2 and R3, wherein:

reagent R1 is a suspension comprising streptavidin magnetic particles;

reagent R2 is a solution containing a chemiluminescent label-labeled digoxin monoclonal antibody;

the reagent R3 is a solution containing digoxin derivative labeled with conjugate marker;

preferably, the mass percentage of the streptomycin avidin magnetic particles in the reagent R1 is 0.01-1%, and more preferably 0.072%. The particle size of the streptavidin magnetic particles in the reagent R1 is 0.05-3 μm, and more preferably 3 μm. When the particle size of the streptavidin magnetic particles is less than 0.05 μm, the binding rate of the antigen or antibody and the magnetic beads is low, which may cause the overall quantum number of light to be low; when the particle size of the streptavidin magnetic particle is higher than 3 μm, the nonspecific binding effect is significant, which may cause a decrease in the sensitivity of the kit.

Preferably, the molar ratio of the digoxin monoclonal antibody to the chemiluminescent marker in the reagent R2 is 1: 1-1: 20, and more preferably 1: 5. The concentration of the digoxin monoclonal antibody marked by the chemiluminescent marker in the reagent R2 is more than or equal to 10 ng/mL. The chemiluminescent label in reagent R2 is acridinium ester, luminol, isoluminol or ruthenium terpyridyl, more preferably acridinium ester.

Preferably, the molar ratio of the digoxin derivative to the conjugated marker in the reagent R3 is 1: 1-1: 20, and more preferably 1: 10. The concentration of the coupling label labeled digoxin derivative in the reagent R3 is more than or equal to 5 ng/mL. The digoxin derivative in the reagent R3 is digoxin-BSA or digoxin-OVA, and the coupling marker is biotin.

The invention also provides a preparation method of the chemiluminescence immunoassay kit for quantitatively detecting digoxin, which comprises the following steps:

step S1, preparation of reagent R1:

uniformly mixing a streptavidin magnetic particle solution and a TBST solution, placing the mixture on a magnetic separator until a supernatant is not turbid, removing the supernatant, reserving magnetic particles, and preparing a solid phase reagent in a buffer solution after cleaning; wherein the concentration of the streptavidin magnetic particle solution is 50-100 mg/ml; the volume ratio of the streptavidin magnetic particle solution to the TBST solution is preferably (0.5-1): (5-10); the mixing time is preferably 10-15 min; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH7.2; the concentration of the solid phase reagent is preferably 0.01-1%, and more preferably 0.05%; the streptavidin magnetic particle solution is commercially available from Agilent under the trade designation PL 6827-1006.

Step S2, preparation of reagent R2:

placing the digoxin monoclonal antibody into a centrifugal tube for centrifugation, preferably centrifuging for 10-30 s at room temperature to ensure that the antibody is positioned at the bottom of the centrifugal tube, then adding a phosphate buffer solution, uniformly mixing, adding a chemiluminescent marker solution, and centrifuging, wherein the centrifugation temperature is preferably room temperature, and the centrifugation time is preferably 0.5-3 min; sealing the centrifuge tube, putting the centrifuge tube into a light-resistant cassette, putting the cassette into a gas bath constant temperature oscillator (25 ℃) for uniformly mixing, wherein the uniformly mixing time is preferably 2-4 h, adding a sealing liquid, putting the mixture into the gas bath constant temperature oscillator for uniformly mixing, purifying and collecting the sealed antibody, then putting the antibody into a buffer solution for dilution, and storing; wherein the molar ratio of the digoxin monoclonal antibody to the chemiluminescent marker is 1: 1-1: 20, and more preferably 1: 5; the concentration of the chemiluminescent marker solution is 2-2.5 mg/mL; the confining liquid is lysine, and the mass fraction is 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH6.0;

step S3, preparation of reagent R3:

placing the digoxin derivative into a centrifugal tube for centrifugation, ensuring that the antibody is located at the bottom of the centrifugal tube, preferably centrifuging for 10-30 s at room temperature, then adding TRIS buffer solution, mixing uniformly, adding a conjugate marker solution for centrifugation, centrifuging for 30s at room temperature by using a centrifuge, mixing uniformly at 2-8 ℃, preferably for 2-4 h, adding confining liquid, placing into a gas bath constant temperature oscillator for mixing uniformly (25 ℃), preferably for 1-2 h, purifying and collecting the confined antibody, then placing into the buffer solution for dilution, and storing; the molar ratio of the digoxin derivative to the coupling marker is 1: 1-1: 20, and more preferably 1: 10; the concentration of the coupling marker solution is preferably 2-3 mg/mL; the confining liquid is preferably lysine with the mass fraction of 20-25%; the buffer solution is 50mM MES, 0.05% Tween-20, 0.05% Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20, 0.1% Proclin300, pH6.0.

In addition, the kit also comprises a chemiluminescence substrate solution, wherein the chemiluminescence substrate solution comprises a solution A and a solution B; the solution A is hydrogen peroxide and nitric acid solution, and the solution B is sodium hydroxide solution.

The kit of the invention also includes a Digoxin (DIG) calibrator. The Digoxin (DIG) calibrator is preferably prepared by preparing a DIG pure product into Digoxin (DIG) standard solutions with the concentrations of 0.00ng/mL, 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 4.0ng/mL and 5.0ng/mL respectively by using a standard dilution solution.

When the chemiluminescence immunoassay kit for quantitatively detecting digoxin is used for detecting Digoxin (DIG), a full-automatic chemiluminescence immunoassay analyzer (CM180) of DIRII medical company is utilized to detect a Digoxin (DIG) calibrator, a standard curve is drawn, and the standard curve is built in computer software; then testing a clinical sample according to the requirement, and calculating the concentration of Digoxin (DIG) according to the number of light quanta of the sample; finally, the performance (sensitivity, linearity, anti-interference/specificity) of the kit of the invention is evaluated. The present invention will be described in further detail with reference to specific examples.