阅读说明：本技术 用于二抗检测系统的聚酰胺胺结构多聚物及其制备方法 (Polyamide-amine structure polymer for secondary antibody detection system and preparation method thereof ) 是由 谭瑞政 于 2019-09-25 设计创作，主要内容包括：本发明属于二抗检测系统技术领域,具体涉及用于二抗检测系统的聚酰胺胺结构多聚物及其制备方法,其利用树枝状聚酰胺胺结构上的-NH<Sub>2</Sub>官能团与结合酶蛋白、免疫球蛋白上的-COOH官能团进行酰胺化反应,使蛋白分子紧密结合在聚酰胺胺骨架结构上,增加单位体积蛋白的浓度,制备出了相比市面产品具有更高的检测灵敏度,更低的背景和更低成本的二抗聚合物,具有广阔的市场应用前景,实质性特点显著。(The invention belongs to the technical field of secondary antibody detection systems, and particularly relates to a polyamide-amine structure polymer for a secondary antibody detection system and a preparation method thereof, wherein-NH on a dendritic polyamide-amine structure is utilized 2 The amidation reaction between the functional group and the-COOH functional group on the conjugated enzyme protein and immunoglobulin makes the protein molecule combined closely to the polyamide-amine skeleton structure to increase the concentration of the protein in unit volume, and the prepared secondary antibody polymer has higher detection sensitivity, lower background and lower cost.)

1. The polyamide-amine structure polymer for the secondary antibody detection system is characterized by comprising 2-13% of dendritic polyamide-amine, 75-95% of conjugated enzyme and 2-13% of immunoglobulin, and the structural formula of the polyamide-amine structure polymer is as follows:

wherein n is more than or equal to 1.

2. The polyamidoamine structural polymer for secondary antibody detection system as claimed in claim 1, wherein the molecular weight of the dendritic polyamidoamine is (1500-₂，-NH₂The number of groups is 32-128.

3. The polyamidoamine structural polymer for secondary antibody detection system as claimed in claim 2, wherein the molecular weight of the bound enzyme is (25000-44000U) and the RZ value is 2.1-3.5.

4. The polyamidoamine structural polymer for secondary antibody detection system as claimed in claim 3, wherein the molecular weight of the immunoglobulin is (70000-.

5. The polyamidoamine structural polymer for use in a secondary antibody detection system according to claim 4, wherein the binding enzyme is preferably peroxidase and the immunoglobulin is preferably goat anti-rabbit immunoglobulin.

6. A process for preparing the polyamidoamine structural polymer according to any one of claims 1 to 5, comprising the steps of:

(1) weighing the conjugated enzyme, dissolving the conjugated enzyme into a sodium bicarbonate solution, adding a proper amount of a diphenyl sulfoxide solution, reacting for 1 hour, filtering, analyzing and purifying the reacted solution by using a sephadex column, wherein the purified solution is the sodium bicarbonate solution, and collecting the dark filtrate;

(2) carrying out low-temperature centrifugal concentration on the filtrate collected in the step (1), putting the concentrated solution into a test tube, refrigerating at the temperature of 2-8 ℃ for standby, adding dendritic polyamidoamine into the concentrated solution, stirring at a low speed at the temperature of 30 ℃ for reaction for 24 hours, filtering, analyzing and purifying the reaction solution on a sephadex column through a sodium bicarbonate solution, and collecting a dark substance solution;

(3) carrying out low-temperature centrifugal concentration on the dark substance solution purified in the step (2), putting the concentrated solution into a test tube, adding diphenyl sulfoxide, reacting for 4 hours, then filtering, analyzing and purifying the reaction solution on a sephadex column by using a PBS solution, collecting the dark substance, and carrying out low-temperature concentration for later use;

(4) and dissolving the immunoglobulin into a PBS solution, fully and uniformly stirring, adding the concentrated solution obtained in the step (3), magnetically stirring uniformly, incubating for 48 hours, adding an antigen blocking agent, namely bovine serum albumin, fully and uniformly stirring, and finally storing the synthesized polyamide-amine structure polymer at 2-8 ℃ for later use.

Technical Field

The invention relates to the technical field of secondary antibody detection systems, in particular to a polyamide-amine structure polymer for a secondary antibody detection system and a preparation method thereof.

Background

At present, there are two main types of immunohistochemical ready-to-use secondary antibody detection systems used by domestic scientific research institutions and clinical diagnosis institutions, and one type is a secondary antibody detection system (biotin detection system) which amplifies a detection signal through high affinity between avidin-biotin or streptomycin-biotin; the other is a secondary antibody detection system ("enzyme-polymer-antibody" detection system) that amplifies the signal by coupling multiple antibodies and enzymes to a polymer. The biotin detection system is interfered by endogenous biotin of a tissue to be detected sometimes to generate a false positive result, so that the accuracy of the result is influenced, and therefore, the application of the secondary antibody detection system in China is gradually reduced, particularly the application of the secondary antibody detection system in clinical auxiliary diagnosis is gradually reduced; the enzyme-polymer-antibody detection system is free from the interference of endogenous biotin of a tissue to be detected, has low staining background and less operation steps and time, and integrates the advantages of accuracy, convenience and quickness, so that the enzyme-polymer-antibody detection system is more and more widely applied to clinical auxiliary diagnosis.

With the annual increase of the incidence of tumors, the organization of the pathology department and the epidemic department becomes more important, and the number of the items increases by 15% every year, so that the market potential is huge.

Secondary antibody usage market data (including instrumental secondary antibodies): about 1192 three hospitals (779 in the three hospitals, statistics in 2018) and 1888 two hospitals all over the country. Basically, each three-level hospital and 60% of two-degree hospitals develop immunohistochemical projects, the trial amount of the manual immunohistochemical staining secondary antibody is 40ml on average according to incomplete data of 2018 survey, the trial amount is about 413 three-level hospitals and 1132 two-degree hospitals nationwide according to 80 yuan/ml, and the annual sales amount is 4900 ten thousands.

The third hospital is basically dyed by instruments, and the annual sales volume is 93 hundred million yuan calculated according to 80 yuan per piece and 15000 pieces per hospital per year.

At present, the companies supplying the secondary antibody products of the enzyme-polymer-antibody detection system in the domestic market mainly comprise:

dako Gene corporation (Envision secondary antibody), which accounts for about 35% of the market share;

fujianmei (Maxvision secondary antibody) which accounts for about 50% of the market share;

beijing China fir gold bridge (PV6000 secondary antibody) accounting for about 15% of market share;

distribution of instrument secondary antibody market share at present:

roche diagnostic instruments account for approximately 65% of market share;

the Leica diagnostic instrument accounts for about 20% of the market share;

the rest of the brands add up to about 15% of market share;

most of domestic secondary antibody products are imported sub-packages, and the polymer structure design and preparation process are not greatly improved, and the secondary antibody products are specifically represented as follows: the number of enzymes and antibodies in unit volume is not obviously increased; the steric hindrance of the polymer is not obviously reduced, so that the price of the domestic secondary antibody product is not obviously reduced and the sensitivity is not improved compared with the imported product.

Disclosure of Invention

In order to solve the problems that most of enzyme-polymer-antibody detection systems (secondary antibodies) used in China at present depend on import, are expensive, have high use cost and cannot improve sensitivity, the polyamide-amine structure polymer for the secondary antibody detection system and the preparation method thereof are provided.

Polyamidoamine structural polymer (i.e., SL-polytrp polymer, hereinafter collectively referred to by this name as polyamidoamine structural polymer described above) for use in secondary antibody detection system, comprising dendritic polyamidoamine 2-13%, conjugated enzyme 75-95% and immunoglobulin 2-13%, having the structural formula:

wherein n is more than or equal to 1.

Further, the molecular weight of the dendritic polyamidoamine is (1500-₂，-NH₂The number of groups is 32-128.

Further, the molecular weight of the bound enzyme (25000-44000U) was 2.1-3.5.

Further, the molecular weight of the immunoglobulin is (70000-100000U).

Further, the binding enzyme is preferably peroxidase, and the immunoglobulin is preferably goat anti-rabbit immunoglobulin.

A method for preparing a polyamide-amine structure polymer, which is characterized by comprising the following steps:

(1) weighing the conjugated enzyme, dissolving the conjugated enzyme into a sodium bicarbonate solution, adding a proper amount of a diphenyl sulfoxide solution, reacting for 1 hour, filtering, analyzing and purifying the reacted solution by using a sephadex column, wherein the purified solution is the sodium bicarbonate solution, and collecting the dark filtrate;

(2) carrying out low-temperature centrifugal concentration on the filtrate collected in the step (1), putting the concentrated solution into a test tube, refrigerating at the temperature of 2-8 ℃ for standby, adding dendritic polyamidoamine into the concentrated solution, stirring at a low speed at the temperature of 30 ℃ for reaction for 24 hours, filtering, analyzing and purifying the reaction solution on a sephadex column through a sodium bicarbonate solution, and collecting a dark substance solution;

(3) carrying out low-temperature centrifugal concentration on the dark substance solution purified in the step (2), putting the concentrated solution into a test tube, adding diphenyl sulfoxide, reacting for 4 hours, then filtering, analyzing and purifying the reaction solution on a sephadex column by using a PBS solution, collecting the dark substance, and carrying out low-temperature concentration for later use;

(4) dissolving immunoglobulin into PBS solution, fully and uniformly stirring, adding the concentrated solution obtained in the step (3), magnetically stirring uniformly, incubating for 48 hours, adding bovine serum albumin serving as an antigen blocking agent, fully and uniformly stirring, and finally storing the synthesized polyamide-amine structure polymer at 2-8 ℃ for later use.

The invention has the advantages that:

1. the SL-PolyHRP polymer adopts the latest preparation process, has fully activated molecules and good affinity, and is convenient for combining more protein molecules.

2. The cyclic branched structure provided by the synthesis of the polyamidoamine is utilized, the problem of large steric hindrance of the traditional polymer is solved, and the sensitivity of the SL-PolyHRP polymer is obviously better than that of the existing market products.

3. Under the condition that the background is not increased, the SL-PolyHRP polymer secondary antibody reagent has obviously better dyeing intensity than the products on the current market.

4. The product is independently researched, developed and synthesized in China, and has low cost and strong market competitiveness.

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 the structural formula of the dendritic polyamidoamine in the example;

FIG. 2 shows that 128 protein molecules are combined to have 128-NH₂Structural schematic diagram of SL-PolyHRP polymer on dendritic polyamidoamine molecule of surface modifying group;

FIG. 3 is an infrared spectrum of SL-PolyHRP polymer.

Detailed Description

In order to solve the problems that most of enzyme-polymer-antibody detection systems (secondary antibodies) used in China at present depend on import, are expensive, have high use cost and cannot improve sensitivity, the polyamide-amine structure polymer for the secondary antibody detection system and the preparation method thereof are provided.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, 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 some embodiments of the present invention, but not all 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.

In the embodiment, dendritic polyamidoamine with the structural formula shown in figure 1 is preferably purchased (a new molecular material from Waishaham can be adopted, the product number is CYD-150A, and the product link is http:// www.whcyd.com/product _ data/19. html); a SL-PolyHRP polymer preparation experiment was carried out with horseradish peroxidase of molecular weight 30000U (reference: Hongweie, Zhanghui, Luzhong. horseradish peroxidase structure and mechanism of action [ J ] chemistry of life, 2005, 25(01)) and goat anti-rabbit immunoglobulin of molecular weight 80000U.

The preparation method comprises the following steps:

(1) dissolving 5.5G of horseradish peroxidase (30000U, purified) into 10ml of sodium bicarbonate (0.1M, PH ═ 7.0) solution, adding 0.5ml of diphenyl sulfoxide solution (5G of diphenyl sulfoxide is dissolved into 10ml of ethanol solution, shaking at 30 ℃), reacting for 1 hour, filtering, analyzing and purifying the reacted solution by using a sephadex column (G50), wherein the purified solution is sodium bicarbonate solution (0.1M, PH ═ 7.0), and collecting dark filtrate;

(2) and (2) performing low-temperature centrifugal concentration on the filtrate collected in the step (1), putting the concentrated solution into a 5ml test tube, refrigerating at 2-8 ℃ for standby, adding 0.2G of dendritic polyamidoamine (G5, analytical purity) into the concentrated solution, reacting at 30 ℃ for 24 hours (stirring at a low speed), filtering, analyzing and purifying the reaction solution on a cross-linked dextran gel column (G50) through a sodium bicarbonate solution (0.1M, PH ═ 7.0), and collecting a dark substance, wherein the component is the molecular structure of the enzyme-polyamidoamine.

(3) Performing low-temperature centrifugal concentration on the enzyme-polyamide-amine solution purified in the step (2), taking the concentrated solution to be filled into a 5ml test tube, adding 0.5ml of diphenyl sulfoxide (5G of diphenyl sulfoxide is dissolved in 10ml of ethanol solution and is vibrated at 30 ℃), reacting for 4 hours, filtering, analyzing and purifying the reaction solution by using a PBS (phosphate buffer solution) solution with the pH value of 7.4 through a sephadex column (G50), collecting a dark substance, and performing low-temperature concentration for later use;

(4) dissolving 0.2g of goat anti-rabbit immunoglobulin (80000U) (purified) into a 1LPBS solution (pH 7.4), stirring uniformly, adding the concentrated solution obtained in step (3), stirring uniformly by magnetic force, incubating for 48 hours, adding 1g of bovine serum albumin serving as an antigen blocking agent, stirring uniformly, and finally storing the synthesized "enzyme-polyamine acyl-amine-antibody" polymer (SL-PolyHRP polymer) at 2-8 ℃ for later use.

The reaction principle is as follows:

the detection system of 'enzyme-polyamidoamine macromolecule-antibody' (SL-PolyHRP) is developed by adopting a polyamidoamine molecular structure. The polyamidoamine molecule is a dendritic polymer structure, and the terminal functional group of the polyamidoamine molecule can chemically react with the functional group of the zymoprotein molecule under specific conditions (reference: Caoweiman, Juhong. dendritic polymer Polyamidoamine (PAMAM) to study on the solubilization of salicylic acid [ J]2007, No.13) of chemical engineering, canObviously improves the unit density of enzyme molecules. In particular, the use of-NH on dendritic polyamidoamine structures₂The amidation reaction between the functional group and the-COOH functional groups on the conjugated enzyme protein and the second antibody protein makes the protein molecule combined closely to the polyamine acyl-amine skeleton structure to increase the concentration of protein in unit volume.

And (3) performance detection:

1. analysis of Polymer Structure

As shown in fig. 2, the black particles represent protein molecules (including enzymes and immunoglobulins), and the middle white particles are dendrimer polyamidoamine backbone molecules; for example, a dendrimer-polyamidoamine backbone molecule contains 128-NH groups₂Functional groups, in theory, are capable of binding 128 protein molecules. Because of the ring space configuration, the molecules are closely arranged and the space steric hindrance is small, the active fragment Fab of the protein molecule is fully exposed (the reference document: Tang Si Yuan, Dong Qiang, Ching Fei, Yaojing. human immunoglobulin G is used for preparing Fab and Fc fragments by enzymolysis and separating and purifying [ J]Chemical bulletin, 2013, 5, 64(5)), increasing the permeability and sensitivity of polymer molecules.

To further analyze the chemical structure of the polymer, infrared spectroscopy was performed on the primary functional groups, as shown in fig. 3.

As seen from the figure, the polymer has no-CO stretching vibration peak peculiar to-COOH between 1700-1725, and has no free carboxyl. The polymer has-NH stretching vibration around 3270, CO vibration around 1642 and composite vibration peak of NH deformation vibration and C-N stretching vibration around 1563, comprehensive analysis shows that the amidation reaction is complete, protein molecules (namely enzyme and immunoglobulin) and dendritic polyamidoamine skeleton are fully combined, and the effect of synthesizing the secondary antibody polymer is good.

2. Analysis of dyeing Properties of Polymer

Three kinds of commonly used secondary antibody polymer staining reagents on the market are selected and named as: the secondary antibody A (Shanghai gene science, Commodity number: GP016029), the secondary antibody B (Fuzhou mai new reagent, Commy number: KIT-5004), the secondary antibody C (American Saimer Feishell science, Commy number: A32731), and the SL-PolyHRP secondary antibody polymer developed by the self are respectively subjected to immunohistochemical experiments according to the following experimental design, and positive tablets corresponding to the primary antibody reagents (working solutions) ki67, P63, SMA and CK7 are tested, wherein the dyeing intensity and the dyeing background are shown in the following table I:

table one:

secondary antibody reagent	Ki67 reagent	P63 reagent	SMA agents	CK7 reagent
					Second antibody A	+++	+++	+++	+++
Second antibody B	+++	++	+++	+++
					Second antibody C	++	++	++	++
SL-PolyHRP	+++++	+++++	+++++	+++++

Note: + indicates positive, more + more positive stronger; -represents negativity

Table two:

secondary antibody reagent	Ki67 reagent	P63 reagent	SMA agents	CK7 reagent
					Second antibody A	+	-	-	-
Second antibody B	-	+	-	+
					Second antibody C	-	-	-	-
SL-PolyHRP	-	-	-	-

Note: + indicates a background, more + the stronger the background; -represents no background

According to the standard guideline of the industry, shown in the photograph of immunohistochemical industry standard, the immunohistochemical staining result should meet the following conditions: sensitivity is more than or equal to 3+, background intensity is less than 1+ or no background is required. It can be seen that the autonomously developed 'enzyme-polyamidoamine-antibody' type SL-PolyHRP polymer reagent has obviously better dyeing strength than the existing market products and completely has substitution capability under the condition that the background is not increased.

In conclusion, the sensitivity of the SL-PolyHRP polymer is obviously better than that of the existing market products. The preparation method adopts the latest preparation process, the molecules are fully activated, the affinity is good, and more protein molecules are conveniently combined; the annular branched structure overcomes the problem of large steric hindrance of the traditional polymer. Through the technical improvement, the synthesized secondary antibody polymer has higher detection sensitivity, lower background and lower cost.

The foregoing is a more detailed description of the invention than is specifically described in connection with the preferred embodiments, and it is not intended to limit the invention to the particular forms disclosed, since all equivalent variations and modifications as fall within the scope of the appended claims are intended to be embraced therein.