CN114236115A

CN114236115A - Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method and preparation method thereof

Info

Publication number: CN114236115A
Application number: CN202111537069.4A
Authority: CN
Inventors: 邵飞飞; 刘振世
Original assignee: Taizhou Zecen Biotechnology Co ltd
Current assignee: Taizhou Zecen Biotechnology Co ltd
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-03-25

Abstract

The invention discloses a magnetic particle chemiluminescence alpha tumor necrosis factor determination kit and a preparation method thereof. Relates to the technical field of biological reagents. Comprises the following components: streptavidin magnetic particles, biotinylated TNF alpha antibody, TNF alpha derivative enzyme labeling reagent, calibrator and quality control product. The method has the advantages of short reaction time, avoidance of radioactive pollution, prolonged reagent expiration date, high sensitivity, accurate test result and simplified experimental operation process. The binding between avidin and biotin has very high affinity and the reaction is highly specific. The sensitivity is improved, and meanwhile, nonspecific interference is not increased. In addition, the binding characteristics are not influenced by the high dilution of the reaction reagent, and the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application.

Description

Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method and preparation method thereof

Technical Field

The invention relates to the technical field of biological reagents, in particular to a magnetic particle chemiluminescence alpha tumor necrosis factor determination kit and a preparation method thereof.

Background

Tumor Necrosis Factor (TNF) is a cytokine naturally produced by macrophages in response to bacterial infection or other immune sources. Is a small molecule protein secreted by macrophages. Is a kind of cell factor capable of directly causing tumor cell death. Tumor necrosis factor acts synergistically with interferon to kill tumor cells. They are classified into two types, TNF-alpha and TNF-beta, depending on their origin and structure. The former is mainly produced by macrophages, LPS is a strong stimulator inducing the production of the LPS, and T cells and NK cells can also secrete TNF-alpha under the action of certain stimulating factors (such as PMA); the latter are mainly produced by activated T cells, which produce high levels of TNF- β upon stimulation by antigens, mitogens and the like. TNF-. beta.is known as lymphotoxin (1 ymphotox-in, LT). TNF- α is secreted primarily by monocyte-macrophages; TNF-beta is secreted primarily by activated T lymphocytes, both of which have similar pyrogenicity. A small dose is unimodal heat and a large dose is bimodal heat; TNF stimulates the production of IL-1 in vitro and in vivo. It is heat-labile and deactivated at 70 deg.C for 30 min.

However, the existing detection method has long reaction time or radioactive hazards, and the sensitivity and the detection accuracy rate need to be improved.

Therefore, how to more accurately and efficiently measure the alpha-TNF is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the invention provides a magnetic particle chemiluminescence alpha tumor necrosis factor assay kit and a preparation method thereof

In order to achieve the purpose, the invention adopts the following technical scheme:

a kit for measuring alpha tumor necrosis factor by a magnetic particle chemiluminescence method comprises the following components: streptavidin magnetic particles, biotinylated TNF alpha antibody, TNF alpha derivative enzyme labeling reagent, calibrator and quality control product.

Further, streptavidin magnetic particles were purchased from beijing zecheng biotechnology limited.

Preferably: biotinylated TNF α antibodies were prepared as follows:

1) measuring a TNF alpha antibody;

2) calculating according to the molecular weight of the TNF alpha antibody and the molecular weight of biotin-NHS, and weighing the biotin-NHS;

3) weighing biotin-NHS, and dissolving with DMSO;

4) adding biotin dissolved in DMSO into the TNF alpha antibody, fully and uniformly mixing, and reacting at room temperature for 2 h;

5) dialyzing the reaction product in the step 4) to obtain a mixture;

6) and diluting the mixture by using an anti-reagent buffer solution to obtain the biotinylated TNF alpha antibody.

Further, TNF α antibody, anti-reagent buffer was purchased from beijing zecheng biotechnology limited.

Preferably: the TNF alpha antibody is a TNF alpha monoclonal antibody;

step 2) calculating the molecular weight of the anti-TNF alpha antibody according to 30000, calculating the molecular weight of biotin-NHS according to 587, and performing the following steps according to a molar ratio of 1: weighing biotin-NHS at a feed rate of 20;

step 3) dissolving the mixture to the extent that the final concentration of biotin-NHS is 5 mg/ml;

step 4), the reaction time is 2 hours;

step 5), dialysis is carried out by adopting a dialysis bag with molecular weight cutoff of 500; the dialysis buffer solution is 0.15M PBS buffer solution with pH7.5;

step 6) dilution to the extent that the final concentration of the mixture was 0.5. mu.g/ml.

Preferably: the TNF alpha derivative enzyme labeling reagent is prepared by the following steps:

1) after the TNF alpha derivative is dialyzed, activating;

2) activating alkali phosphatase;

3) activating an antigen containing TNF alpha and an antibody to obtain a reactant;

4) purifying the reactant by a chromatographic column, collecting a peak I and a peak II, and mixing to obtain an alkaline phosphatase-labeled TNF alpha derivative;

5) diluting the alkaline phosphatase-labeled TNF alpha derivative to 0.1 mu g/ml by using an anti-reagent buffer solution to obtain the TNF alpha derivative enzyme-labeled reagent.

Further, TNF α derivatives, antibodies (mouse antibodies) were purchased from beijing zecheng biotechnology limited.

Preferably: step 1) dialysis was performed using 0.1 MPBS; activating by adopting an activating agent 2-Iminothiolane.HCl;

step 2), activating by adopting SMCC, wherein the mass-to-volume ratio of the alkali phosphatase to the SMCC is 5 mg/ml;

step 3) activation: respectively mixing the antigen and the antibody containing the TNF alpha with alkaline phosphatase according to the mass-volume ratio of 1:1mg/ml, and reacting for 18 hours at the temperature of 2-8 ℃;

step 4), the mobile phase is Tris buffer solution; the flow rate is 0.75 ml/min; collection of I peak time: the 70 th min; collection II peak time: at 90 min.

Preferably: the preparation method of the calibrator comprises the following steps:

1) preparing a TNF α calibrator buffer: 0.05M TRIS buffer solution with pH7.4 is added with bovine serum albumin with volume ratio of 0.5% and preservative with volume ratio of 0.2%;

2) diluting the pure TNF alpha product with TNF alpha calibrator buffer solution to obtain the calibrator with the calibrator concentrations of 0pg/ml, 10pg/ml, 100pg/ml, 300pg/ml, 500pg/ml and 1000 pg/ml.

Has the advantages that: the calibrator is in liquid state and has good stability.

The kit has simple and convenient preparation method, higher detection sensitivity and good application prospect.

Further: quality control product: the antibody containing TNF alpha was present in two concentrations, one at higher concentrations (100pg/ml and one at lower concentrations (500 pg/ml), within the range of the calibrator.

The invention also provides a method for detecting alpha tumor necrosis factor for non-diagnostic therapeutic purposes by using the kit, which is characterized by comprising the following steps:

1) mixing and incubating a sample, a biotinylated TNF alpha antibody and a TNF alpha derivative enzyme-labeled reagent;

2) adding streptavidin magnetic particles, and incubating;

3) magnetic separation and supernatant removal;

4) washing;

5) add the luminescent substrate solution and measure.

Has the advantages that: the biotinylated TNF α antibody binds to the TNF α derivative enzyme-labeled reagent and the TNF α antigen in the sample, calibrator or quality control to form a "sandwich" complex. Subsequently, streptavidin-linked magnetic particles are added, and the specific binding of the biotinylated TNF alpha antibody to the enzyme label of the TNF alpha derivative allows the antigen-antibody immune complex to bind to the magnetic particles. Separating the complex formed by immunoreaction from other unbound substances under the action of an external magnetic field, washing the complex, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction.

Preferably: the volume ratio of the sample, the biotinylated TNF alpha antibody, the TNF alpha derivative enzyme labeling reagent and the streptavidin magnetic particles is 1:1:1: 1;

step 1), incubating at 37 ℃ for 30 min;

step 2), incubating at 37 ℃ for 5 min;

step 3), the magnetic field is 2000GS, and the magnetic separation time is 2 min;

step 4), washing: 3 times, 300. mu.l of wash solution each time.

Furthermore, the washing solution and the luminescent substrate solution are purchased from Beijing Zhencheng Biotechnology Limited.

According to the technical scheme, compared with the prior art, the magnetic particle chemiluminescence alpha tumor necrosis factor detection kit and the preparation method thereof have the advantages that the reaction time of a reaction system is short, radioactive pollution is avoided, the effective period of reagents is prolonged, the sensitivity is high, the test result is accurate, and the experimental operation process is simplified. The binding between avidin and biotin has very high affinity and the reaction is highly specific. The sensitivity is improved, and meanwhile, nonspecific interference is not increased. In addition, the binding characteristics are not influenced by the high dilution of the reaction reagent, and the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application.

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 embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 the accompanying drawing provides a system 1 curve fit representation of the present invention.

FIG. 2 is a graph showing the curve fitting of system 2 according to the present invention.

FIG. 3 is a graph showing the correlation between the serum in the system 1 test according to the present invention.

FIG. 4 is a graph showing the correlation between the serum in the system 2 test provided by the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious 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 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 embodiment of the invention discloses a kit for measuring alpha tumor necrosis factor by a magnetic particle chemiluminescence method.

Example 1

Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method

Comprises the following components: magnetic separation reagent (streptavidin magnetic particle), biotinylated TNF alpha monoclonal antibody, TNF alpha derivative enzyme labeling reagent, calibrator and quality control product.

The biotinylated TNF α monoclonal antibody was prepared as follows:

1) measuring 1mgTNF alpha monoclonal antibody;

2) calculated from the antibody molecular weight (30000) and biotin-NHS molecular weight (587), in a molar ratio of 1: weighing biotin with the feeding amount of 20;

3) 0.4mg of biotin-NHS was weighed and dissolved in DMSO to a final concentration of 5 mg/ml;

4) adding biotin dissolved in DMSO into the TNF alpha monoclonal antibody, fully and uniformly mixing, and reacting for 2 hours at room temperature (22 +/-3 ℃);

5) dialyzing the reaction product in step 4) with a dialysis bag having a molecular weight cut-off of 500 to obtain a mixture, wherein the dialysis buffer is 0.15M PBS buffer with pH of 7.5;

6) the mixture was diluted to 0.5. mu.g/ml with an anti-reagent buffer and used as a working solution.

The TNF alpha derivative enzyme labeling reagent is prepared by the following steps:

1) dialyzing the TNF alpha derivative to be detected with 0.1MPBS, and then activating with 2-Iminothiolane.HCl;

2) activating alkaline phosphatase with SMCC (the mass-volume ratio of the alkaline phosphatase to the SMCC is 5 mg/ml);

3) respectively mixing and activating an antigen and an antibody containing TNF alpha with alkaline phosphatase (the mass-volume ratio is 1:1mg/ml), and reacting for 18 hours at 2-8 ℃;

4) the reaction was purified on a chromatographic column (Tris buffer, flow rate 0.75 ml/min; collection of I peak time: the 70 th min; collection II peak time: 90min), collecting the peak I and the peak II, and mixing to obtain the TNF alpha derivative marked by the alkaline phosphatase;

5) diluting the antibody-alkali phosphatase to 0.1 mu g/ml by using an anti-reagent buffer solution to obtain the TNF alpha derivative enzyme-labeled reagent.

The preparation method of the calibrator comprises the following steps:

1) preparing a TNF α calibrator buffer: 0.05M TRIS buffer pH7.4 was added with Bovine Serum Albumin (BSA) in an amount of 0.5% by volume and a preservative (preservative) in an amount of 0.2% by volume.

2) The pure TNF alpha product is diluted by TNF alpha calibrator buffer, the calibrator concentration is TNF alpha solution of 0pg/ml, 10pg/ml, 100pg/ml, 300pg/ml, 500pg/ml and 1000pg/ml respectively, and the calibrator is in liquid state and has good stability.

Example 2

The reaction steps of the method for detecting the alpha tumor necrosis factor for the non-diagnostic therapeutic purpose by using the kit are as follows:

1) incubating the sample with 30 ul of biotinylated TNF alpha monoclonal antibody and 30 ul of TNF alpha derivative enzyme-labeled reagent for 30min (37 ℃);

2) adding 30 μ l of streptavidin magnetic beads, and incubating for 5min (37 ℃);

3) performing magnetic separation for 2min, and removing supernatant;

4) washing 3 times, 300 mul washing liquid each time;

5) substrate 200. mu.l was added and measured.

Comparative experiment

Control group: carboxylated magnetic microparticles coated with NF α monoclonal antibodies were purchased from beijing zecheng biotechnology limited.

Simultaneously with example 1 (system 1), the control (system 2), TNF α samples of 0pg/ml, 10pg/ml, 100pg/ml, 300pg/ml, 500pg/ml and 1000pg/ml were tested in two systems, respectively, and the same set of samples was tested simultaneously and compared to the given values, as shown in table 1 below:

TABLE 1 comparison of measured values with given values and comparison between measured values for different systems

From the results, it was found that the correlation between the measured values of the different systems and the given value was 0.9 or more, indicating that the correlation was good, and that the correlation between the measured values of the different systems was more than 0.9, indicating that the measured values of the 2 systems were the same (fig. 1 to 4).

Therefore, the technical solution of the present invention is fully feasible.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for measuring alpha tumor necrosis factor by a magnetic particle chemiluminescence method is characterized by comprising the following components: streptavidin magnetic particles, biotinylated TNF alpha antibody, TNF alpha derivative enzyme labeling reagent, calibrator and quality control product.

2. The kit of claim 1, wherein the biotinylated TNF α antibody is prepared by the steps of:

1) measuring a TNF alpha antibody;

3) weighing biotin-NHS, and dissolving with DMSO;

4) adding biotin dissolved in DMSO into the TNF alpha antibody, fully and uniformly mixing, and reacting;

5) dialyzing the reaction product in the step 4) to obtain a mixture;

3. The kit of claim 1, wherein the TNF α antibody is a TNF α monoclonal antibody;

the dissolution degree of the step 3) is to the final concentration of biotin-NHS of 5 mg/ml;

step 4), the reaction time is 2 hours;

step 5), dialysis is carried out by adopting a dialysis bag with molecular weight cutoff of 500; the dialysis buffer was 0.15M PBS buffer pH 7.5;

step 6) the degree of dilution was such that the final concentration of the mixture was 0.5. mu.g/ml.

4. The kit of claim 1, wherein the TNF alpha derivative enzyme-labeled reagent is prepared by the following steps:

1) after the TNF alpha derivative is dialyzed, activating;

2) activating alkali phosphatase;

5. The kit of claim 4, wherein the dialysis of step 1) is performed using 0.1M PBS; the activation adopts an activating agent 2-Iminothiolane.HCl;

step 2), activating by adopting SMCC, wherein the mass-volume ratio of the alkaline phosphatase to the SMCC is 5 mg/ml;

step 3) the activation: respectively mixing the antigen and the antibody containing the TNF alpha with alkaline phosphatase according to the mass-volume ratio of 1:1mg/ml, and reacting for 18 hours at the temperature of 2-8 ℃;

6. The kit of claim 1, wherein the calibrator is prepared by the steps of:

7. A method for detecting alpha tumor necrosis factor for non-diagnostic therapeutic purposes using the kit of any one of claims 1 to 6, comprising the steps of: