阅读说明：本技术 一种生物标志物TK1的Simoa试剂盒及其使用方法 (Simoa kit of biomarker TK1 and use method thereof ) 是由 李朝辉 蔡齐勇 谢磊 屈凌波 于 2021-07-24 设计创作，主要内容包括：本发明涉及免疫检测技术和体外检测领域,特别是指一种生物标志物TK1的Simoa试剂盒及其使用方法。所述Simoa试剂盒包括捕获抗体包被的磁珠、TK1标准品、生物素化的检测抗体、SBG溶液、荧光底物溶液和样本稀释液。本发明提供的基于Simoa平台和双抗体夹心法检测人体液中TK1蛋白含量的Simoa试剂盒,不仅可以定性还可以定量的检测人体液中TK1含量；具有灵敏度高(0.05pg/mL)、高通量、较少样本量(25μL)、花费时间短(1h)、操作简便、检测范围宽(0.3-1000pg/mL)等特点。(The invention relates to the fields of immunodetection technology and in-vitro detection, in particular to a Simoa kit of a biomarker TK1 and a using method thereof. The Simoa kit comprises capture antibody coated magnetic beads, TK1 standard, biotinylated detection antibody, SBG solution, fluorogenic substrate solution, and sample diluent. The Simoa kit for detecting the TK1 protein content in human body fluid based on the Simoa platform and the double-antibody sandwich method provided by the invention can qualitatively and quantitatively detect the TK1 content in human body fluid; has the characteristics of high sensitivity (0.05pg/mL), high flux, less sample amount (25 mu L), short time consumption (1h), simple and convenient operation, wide detection range (0.3-1000pg/mL) and the like.)

1. A Simoa kit of biomarker TK1, characterized in that: the Simoa kit comprises capture antibody coated magnetic beads, TK1 standard, biotinylated detection antibody, streptavidin-beta-galactosidase (SBG) solution, fluorescent substrate solution, and sample diluent.

2. The Simoa kit of the biomarker TK1 according to claim 1, wherein the magnetic beads coated with capture antibody are prepared by the following steps:

(1) displacing the buffer solution by the capture antibody through an ultrafiltration tube;

(2) 2.7 μm magnetic beads were activated with EDC;

(3) mixing the capture antibody processed in the step (1) with the activated magnetic beads processed in the step (2) and incubating for 2-3h at the temperature of 2-8 ℃;

(4) and (4) washing the product incubated in the step (3) twice by using a PBST solution, then sealing by using a sealing solution at 37 ℃, finally washing twice by using a magnetic bead diluent to obtain magnetic beads coated with the capture antibody, and storing in the magnetic bead diluent.

3. Simoa kit of the biomarker TK1 according to claim 2, characterized in that: the capture antibody in the step (1) is a monoclonal antibody M86541; the buffer used for ultrafiltration was 50mmol/L morpholine ethanesulfonic acid buffer, pH 6.2.

4. Simoa kit of the biomarker TK1 according to claim 2, characterized in that: the reaction concentration of the capture antibody in the step (3) is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads was 1.2X 10⁹/mL。

5. Simoa kit of the biomarker TK1 according to claim 2, characterized in that: the PBST solution in the step (4) is 10mmol/L PBS + 1% Tween20, and the pH value is 7.4; the blocking solution was 10mmol/L PBS + 1% BSA, pH 7.4; the magnetic bead dilution was 50mmol/L Tris-HCl +10mmol/L EDTA + 0.1% Tween20+ 1% BSA, pH 7.4.

6. The Simoa kit of biomarker TK1 according to claim 1, wherein the biotinylated detection antibody is prepared by: and (2) carrying out liquid change treatment on the detection antibody by using a PBS solution through an ultrafiltration tube, adding NHS-Biotin or a derivative thereof at room temperature, reacting for 30min to obtain a biotinylated detection antibody, and purifying by using the ultrafiltration tube to obtain the biotinylated detection antibody, wherein the detection antibody is a monoclonal antibody M86542.

7. Simoa kit of the biomarker TK1 according to claim 6, characterized in that: the quantitative ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1:20-1: 60; the PBS solution was 10mmol/L phosphate buffer, pH 7.4.

8. Simoa kit of the biomarker TK1 according to claim 1, characterized in that: the concentration of the SBG solution is 50-400pmol/L, wherein the enzyme diluent is 10mmol/L PBS + 0.5% BSA +1mmol/L MgCl₂pH 7.4, wherein the sample dilution was 10mmol/L PBS +5mmol/L EDTA + 0.1% Tween20+ 2% BSA; the fluorogenic substrate is resorufin-beta-galactoside, and the concentration is 60-150 mu mol/L.

9. Use of the Simoa kit of the biomarker TK1 according to any of claims 1 to 8 in the manufacture of a diagnostic agent for diseases.

10. Use of the Simoa kit of the biomarker TK1 according to any one of claims 1 to 8 for the detection of TK1 content in human body fluids.

Technical Field

The invention relates to the fields of immunodetection technology and in-vitro detection, in particular to a Simoa kit of a biomarker TK1 and application thereof.

Background

Thymokinase (Thymidine Kinase 1, TK1) is an important enzyme in the DNA synthesis process of cancer cells, has close relation with cell division, and when normal people divide cells, the expression level of TK1 in body fluid is quite low, but the expression level is generally remarkably increased in body fluid of tumor patients. Tumor cells can produce large amounts of TK1 upon cell division and enter the blood stream resulting in higher levels in the body fluids of tumor patients than in benign lung disease patients and healthy people. Thus, TK1 can be used as a marker for pulmonary in vitro detection. In addition, TK1 has reliable research value for postoperative monitoring of non-small cell lung cancer. The research shows that the survival time of the patient with the non-small cell lung cancer is shortened due to the fact that TK1 is increased after the operation, and the content of TK1 in body fluid is proved to be a predictor of the survival time of the patient with the non-small cell lung cancer after the operation.

The TK1 detection method used clinically at present mainly comprises an immunoblotting analysis method, a chemiluminescence immunoassay method and an enzyme activity analysis method. Among them, immunoblotting analysis is the most common method. However, the method has low sensitivity, long time consumption and large sample size requirement, and is not favorable for continuous observation of the disease condition. The other two methods can quickly and sensitively detect the result, but the methods have poor repeatability and large coefficient of variation.

Single molecule array (Simoa) technology, also known as digital ELISA, is a magnetic bead-based ultrasensitive detection method for proteins. In a double antibody sandwich ELISA, the fluorescent product of the enzyme-substrate reaction diffuses to a large volume of about 50-100 μ L, relying on millions of enzyme-labeled immune complexes to generate a measurable fluorescent signal above background. In digital ELISA, the fluorescent product of the enzyme-substrate reaction is confined in 46-femtoliter (fL) sized microwells, which are referred to as single molecule arrays (Simoa). Using this method, the presence of a single enzyme molecule can be detected. In Simoa immunoassays, antibody-coated capture beads are added in excess to a sample containing a low concentration of target protein molecules. Based on the poisson distribution principle, one or zero target protein molecules will bind to each magnetic bead. Magnetic beads that bind more than one protein molecule are negligible. The magnetic beads are then incubated with biotinylated detection antibody and streptavidin-beta-galactosidase to form an enzyme-labeled immunocomplex. Next, the beads were loaded onto a 46fL microwell array, where each well was capable of holding only one bead. The microwells were filled with fluorogenic substrate and sealed with perfluorooil. A bead containing a single immunocomplex will result in a large number of substrate molecules being enzymatically catalyzed to produce a large number of fluorescent products. Since the fluorescent product is limited to a very small volume of about 46fL, the fluorescence imaging of a single micropore can be realized by combining the fluorescence microscopic imaging technology, so that the detection of a single protein molecule is realized. In this way, Simoa can quantify protein concentration using a digital readout mode and achieve a detection range of over 4 orders of magnitude.

Therefore, the TK1 detection method which is high in sensitivity, high in automation degree, rapid, good in accuracy and suitable for popularization is developed based on the Simoa technology, and has great significance for rapidly detecting the biomarker TK 1.

Disclosure of Invention

The invention provides a Simoa kit of a biomarker TK1 and application thereof, and aims to solve the technical problems of complex operation, large sample volume requirement, insufficient sensitivity and the like in the prior art

The technical scheme of the invention is realized as follows:

a Simoa kit for biomarker TK1, comprising capture antibody coated magnetic beads, TK1 standard, biotinylated detection antibody, SBG solution, fluorogenic substrate solution, and sample diluent.

The preparation method of the magnetic bead coated with the capture antibody comprises the following steps:

(1) carrying out liquid change treatment on the capture antibody through an ultrafiltration tube;

(2) 2.7 μm magnetic beads were activated with EDC;

(3) mixing the capture antibody processed in the step (1) with the activated magnetic beads processed in the step (2) and incubating for 2-3h at 4 ℃;

(4) and (4) washing the product incubated in the step (3) twice by using a PBST solution, then sealing by using a sealing solution at 37 ℃, finally washing twice by using a magnetic bead diluent to obtain magnetic beads coated with the capture antibody, and storing in the magnetic bead diluent.

The capture antibody in the step (1) is a monoclonal antibody M86541; the buffer used for ultrafiltration was 50mmol/L morpholine ethanesulfonic acid buffer, pH 6.2.

The reaction concentration of the capture antibody in the step (3) is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads was 1.2X 10⁹/mL。

The PBST solution in the step (4) is 10mmol/L PBS + 1% Tween20, and the pH value is 7.4; the blocking solution was 10mmol/L PBS + 1% BSA, pH 7.4; the magnetic bead dilution was 50mmol/L Tris-HCl +10mmol/L EDTA + 0.1% Tween20+ 1% BSA, pH 7.4.

The preparation method of the biotinylated detection antibody comprises the following steps: and (3) treating the detection antibody with PBS (phosphate buffer solution) through an ultrafiltration tube to replace buffer solution, adding NHS-Biotin or a derivative thereof at room temperature, reacting for 30min to obtain a biotinylated detection antibody, and purifying by using the ultrafiltration tube to obtain the biotinylated detection antibody, wherein the detection antibody is a monoclonal antibody M86542.

The quantity ratio of the detection antibody to the reaction substance of the NHS-Biotin or the derivative thereof is 1: 40; the PBS solution was 10mmol/L phosphate buffer, pH 7.4.

The SBG solution was diluted from the original concentration to a concentration of 150pmol/L by an enzyme dilution of 10mmol/L PBS + 0.5% BSA +1mmol/L MgCl₂The pH value is 7.4; the TK1 standard substance is prepared into 1mL by sample diluent, and the rest standard substances can be automatically diluted and prepared according to requirements; wherein the sample diluent is 10mmol/L PBS +5mmol/L EDTA + 0.1% Tween20+ 2% BSA; the fluorogenic substrate is resorufin-beta-galactoside, and the concentration of the fluorogenic substrate solution is 100 mu mol/L;

the application of the Simoa kit of the biomarker TK1 in preparing a disease diagnostic reagent.

The application of the Simoa kit of the biomarker TK1 in detecting the TK1 content in human body fluid.

The invention has the following beneficial effects:

the Simoa kit for detecting the TK1 protein content in human body fluid based on the Simoa platform and the double-antibody sandwich method is a human TK1 double-antibody sandwich digital ELISA detection kit which takes a monoclonal antibody M86541 as a capture antibody and a monoclonal antibody M86542 for detecting the antibody, and can qualitatively and quantitatively detect the TK1 content in the human body fluid; the kit has the characteristics of high sensitivity, high flux, low sample volume of 25 mu L, automation, short time (1h), simple and convenient operation, wide detection range (0.3-1000pg/mL) and the like, and the lowest detection limit of the kit can reach 0.05pg/mL as shown in the attached figure 1. The invention can be used as a non-invasive method for diagnosing and monitoring the course development of the cancer (especially the lung cancer).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a standard graph of the TK1 Simoa kit of the invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Examples

The Simoa kit for detecting TK1 content in human body fluid comprises 2.7 mu m magnetic beads coated by capture antibodies, TK1 standard, biotinylated detection antibodies, streptavidin-beta-galactosidase (SBG) solution, fluorescent substrate solution, sample diluent and experimental method electronic files.

In the present invention, raw materials of magnetic beads, capture antibodies, standards, detection antibodies, biotinylation reagents, SBG, and fluorescent substrates are obtained by purchase; wherein the capture antibody is monoclonal antibody M86541 and the detection antibody is monoclonal antibody M86542.

In the scheme of the invention, the capture antibody coats the magnetic beads, and the preparation process comprises the following steps: first, the stored antibody was subjected to a solution exchange treatment through an ultrafiltration tube using MES buffer, which was 50mmol/L morpholine ethanesulfonic acid buffer at pH 6.2.

Then, the magnetic beads were activated with EDC and the antibody was captured at 4 deg.CAnd the activated magnetic beads for 2 h. And then washing with PBST twice, blocking with blocking solution at 37 ℃, finally washing with magnetic bead diluent twice, and storing in the magnetic bead diluent to obtain the magnetic beads coated with the capture antibody. Wherein the reaction concentration of the capture antibody is 0.1-0.2 mg/mL; the reaction concentration of the activated magnetic beads was 1.2X 10⁹/mL。

The PBST formula comprises: 10mmol/L PBS + 1% Tween20, pH 7.4; the formula of the sealing liquid is as follows: 10mmol/L PBS + 1% BSA, pH 7.4. The magnetic bead diluent formula comprises: 50mmol/L Tris-HCl +10mmol/L EDTA + 0.1% Tween20+ 1% BSA, pH 7.4.

In the biotinylated detection antibody scheme of the invention, the preparation process comprises the following steps: first, the originally stored detection antibody was subjected to buffer exchange treatment with PBS through an ultrafiltration tube. Then, NHS-Biotin or its derivative was added thereto at room temperature and reacted for 30 min. And finally, purifying the biotinylated detection antibody by using an ultrafiltration tube to obtain the biotinylated detection antibody. PBS formulation: 10mmol/L Phosphate Buffered Saline (PBS), pH 7.4. Wherein the quantity ratio of the detection antibody to the reaction substance of NHS-Biotin or the derivative thereof is 1: 40.

Preparation of enzyme and substrate solutions: SBG was diluted from the original concentration to 150pmol/L by enzyme dilution and substrate was diluted to 100. mu. mol/L with PBS as described above; wherein the enzyme diluent is: 10mmol/L PBS + 0.5% BSA +1mmol/L MgCl₂，pH＝7.4。

The final concentrations are all working concentrations for final imaging.

The substrate is: resorufin-beta-galactoside (RGP)

Preparing a standard solution: 100ng/mL TK1 standard was prepared in 1mL sample dilutions. Other standard products can be prepared according to the needs, wherein the sample diluent is as follows: 10mmol/L PBS +5mmol/L EDTA + 0.1% Tween20+ 2% BSA.

The TK1 Simoa kit is used as follows:

firstly, an electronic document of an experimental method is imported to an HD-1/HD-X Simoa analyzer produced by Quanterix, and the electronic document of the experimental method comprises an experimental operation mode. The method on the electronic document of the experimental method can be changed according to the requirements so as to be suitable for the reagent application of the kit.

The experimental method electronic document comprises: reaction time of magnetic beads, sample and detection antibody (35min), amount of magnetic beads (25. mu.L), amount of detection antibody (20. mu.L), amount of standard and sample (150. mu.L), amount of SBG (100. mu.L) and reaction time (5 min). The default reaction time is set to be 35min-5min, and the default method is a Simoa 2.0 two-step method. The standard concentration can be custom set, default set (500, 167, 55.6, 16.5, 6.17, 2.06, 0.68, 0 pg/mL).

Then, the concentration was 2X 10⁷Magnetic beads/mL, detection antibody at a concentration of 1. mu.g/mL, SBG at a concentration of 150pmol/L, fluorogenic substrate RGP at a concentration of 100. mu. mol/L and 96-well plate with standards and samples added were loaded into the analyzer at the indicated positions while scanning the label and setting the positional parameters.

Finally, the experiment was run. After the experiment is finished, the experimental result is derived, the instrument automatically fits the curve and calculates the sample result, as shown in fig. 1.

The fitting equation is: a four parameter Logistic curve fitting equation: y ═ a-D/[ 1+ (x/C)^{^}B]+D。

The application of the Simoa kit in detecting TK1 content in human body fluid also belongs to the protection scope of the invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.