CN113075406A

CN113075406A - Composition for pregnancy-associated plasma protein A detection, magnetic microsphere electrochemiluminescence immunoassay kit and detection method

Info

Publication number: CN113075406A
Application number: CN202110407481.8A
Authority: CN
Inventors: 秦军; 谢元东; 谢良思; 曹鹏; 周璟
Original assignee: Jiangsu Youni Taike Biotechnology Co ltd; Beijing Unidiag Tech Inc
Current assignee: Jiangsu Youni Taike Biotechnology Co ltd; Beijing Unidiag Tech Inc
Priority date: 2021-04-15
Filing date: 2021-04-15
Publication date: 2021-07-06

Abstract

The invention relates to the field of electrochemical detection, in particular to a composition and application for the detection of pregnancy-related plasma protein A (PAPP-A), a magnetic microsphere electrochemiluminescence immunodetection kit and a detection method. The method used in the present invention is electrochemiluminescence, and the use of ruthenium pyridine as the chemiluminescence marker has obvious advantages, which are mainly manifested in: better sensitivity, better stability, ruthenium is a metal ion, and has a small molecular weight, which does not affect the steric hindrance of the antibody. . The production process is short, the repeatability is good, and the detection range is wide. The electrochemiluminescence reaction is controllable, reducing the difficulty of signal acquisition.

Description

Composition for pregnancy-associated plasma protein A detection, magnetic microsphere electrochemiluminescence immunoassay kit and detection method

Technical Field

The invention relates to the field of electrochemical detection, in particular to ｃA composition for detecting pregnancy associated plasmｃA protein A (PAPP-A), application of the composition, ｃA magnetic microsphere electrochemiluminescence immunoassay kit containing the composition, and ｃA magnetic microsphere electrochemiluminescence immunoassay method based on the composition or the kit.

Background

PAPP-A is Pregnancy-associated plasmｃA protein A (Pregnancy-associated plasmｃA protein A), ｃA macromolecular glycoprotein with ｃA molecular weight of 200kDｃA, which is ｃA member of the zinc peptidase superfamily. PAPP- ｃA first appears in the serum of pregnant women, rising steadily during pregnancy until term.

The concentration of the serum PAPP-A becomes ｃA reliable detection index for detecting aneuploid fetuses. Many studies have demonstrated that in the first trimester (weeks 8-14) of pregnancy, PAPP- ｃA as an alternative serum marker, combined with β HCG and ultrasound detection of cervical translucency (NT) identifies high risk pregnant women with down's syndrome fetuses. The detection rate of the combined detection of the marker can reach 70 percent (only adopting a serum marker) and 90 percent (combined neck translucency NT detection), and the false positive rate is 5 percent.

The median value of the serum PAPP-A concentration of the pregnant women carrying the fetus with the Down syndrome is higher than that of normal pregnant women. Depending on the age of the pregnant woman, a particular algorithm (e.g., likelihood ratio) may be employed to calculate the risk of Down syndrome.

The method adopted by the invention is an electrochemical luminescence method, and the method adopting the terpyridyl ruthenium as the chemiluminescent marker has obvious advantages, which are mainly shown in that: the sensitivity is better, the stability is better, ruthenium is metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. Short production process, wide detection range and good repeatability. The electrochemical luminescence reaction is controllable, and the signal acquisition difficulty is reduced.

Disclosure of Invention

The invention provides ｃA composition for detecting pregnancy related plasmｃA protein A (PAPP-A), application thereof, ｃA magnetic microsphere electrochemiluminescence immunoassay kit and ｃA detection method, and has the advantages of high production efficiency, short detection time, suitability for full-automatic detection, higher sensitivity, wide linear range and the like.

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

the invention provides ｃA composition for detecting pregnancy-associated plasmｃA protein A (PAPP-A), which comprises ｃA PAPP-A reagent RｃA, ｃA PAPP-A reagent Rb and streptavidin superparamagnetic microspheres;

the PAPP-A reagent RｃA comprises an anti-PAPP-A monoclonal antibody containing ｃA biotin label;

the PAPP-A reagent Rb comprises an anti-PAPP-A monoclonal antibody marked by terpyridyl ruthenium;

the streptavidin superparamagnetic microspheres comprise superparamagnetic microspheres with streptavidin coated on the surfaces.

In some embodiments of the invention, the superparamagnetic microspheres have a particle size of 1.5 to 5.0 μm.

In some embodiments of the invention, the amount of the biotin molecular marker on the surface of each antibody molecule in the PAPP-A reagent RｃA is 2-5; in the PAPP-A reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.

In some embodiments of the invention, the preparation method of the PAPP- ｃA reagent rｃA is: mixing an anti-PAPP-A monoclonal antibody with biotin in the presence of ｃA buffer solution to prepare ｃA PAPP-A reagent RｃA; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.

In some embodiments of the invention, the preparation method of the PAPP- ｃA reagent rｃA is: 2.0mg of the antibody for labeling biotin pregnancy-associated plasmｃA protein ｃA (PAPP- ｃA) was taken, the buffer was changed to phosphate buffer (pH 7.8) using desalting column PD10, the concentration was adjusted to 2.0mg/mL after concentration using an ultrafiltration tube, 80 μ g of biotin (dissolved in DMF) was added, the mixture was mixed for 30 minutes, and unlabeled biotin was removed using desalting column PD 10.ｃA pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody labeled biotin was diluted to 1mg/L with ｃA phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as ｃA PAPP- ｃA reagent rｃA.

In some embodiments of the invention, the PAPP- ｃA reagent Rb is prepared by: mixing an anti-PAPP-A monoclonal antibody with terpyridyl ruthenium in the presence of ｃA buffer solution to prepare ｃA PAPP-A reagent Rb; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.

In some embodiments of the invention, the PAPP- ｃA reagent Rb is prepared by: 2.0mg of pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody for labeling ruthenium terpyridine was taken, the buffer was changed to phosphate buffer (pH 7.8) using desalting column PD10, the mixture was concentrated using an ultrafiltration tube and adjusted to ｃA concentration of 2.0mg/mL, 80 μ g of ruthenium succinamide terpyridine (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled ruthenium was removed using desalting column PD 10.ｃA pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody labeled with ruthenium was diluted to 1mg/L using ｃA phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as ｃA PAPP- ｃA reagent Rb.

In some embodiments of the invention, the compositions provided herein further comprise a taggant and/or a cleaning solution; the cleaning solution comprises tripropylamine with the concentration of 150-200 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L; or dibutylethanolamine with the concentration of 80-100 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L.

In some embodiments of the present invention, the cleaning solution includes, but is not limited to, a tripropylamine cleaning solution, a dibutylethanolamine cleaning solution, a pipeline cleaning solution.

In some more specific embodiments of the present invention, the cleaning solution comprises tripropylamine at a concentration of 180mmol/L and a phosphate buffer at a concentration of 300 mmol/L; or dibutylethanolamine at a concentration of 90mmol/L and phosphate buffer at a concentration of 300 mmol/L.

In some embodiments of the invention, the volume ratio of the PAPP- ｃA reagent rｃA, the PAPP- ｃA reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): (20-40).

On the basis of the research, the invention also provides application of the composition in preparing ｃA magnetic microsphere electrochemiluminescence immunoassay kit of pregnancy-associated plasmｃA protein A (PAPP-A).

The invention also provides ｃA magnetic microsphere electrochemiluminescence immunoassay kit for pregnancy related plasmｃA protein A (PAPP-A), which comprises the composition and an acceptable detection reagent.

The invention also provides ｃA magnetic microsphere electrochemiluminescence immunoassay method for pregnancy related plasmｃA protein A (PAPP-A), which is based on the composition or the kit and comprises the following steps:

step 1: taking ｃA sample, sequentially adding ｃA PAPP-A reagent RｃA and ｃA PAPP-A reagent Rb, incubating for 8-12 min at 37 ℃, finally adding streptavidin superparamagnetic microspheres, and incubating for 8-12 min at 37 ℃ to obtain ｃA reaction solution; wherein the volume ratio of the sample, the PAPP-A reagent RｃA, the PAPP-A reagent Rb to the streptavidin superparamagnetic microspheres is 15: (50-80): (50-80): (20-40);

step 2: adsorbing the reaction solution by using a magnet;

and step 3: taking a cleaning solution, cleaning the ruthenium-labeled antibody and the sample which are not bonded to the superparamagnetic microspheres, electrifying, and enabling the terpyridyl ruthenium to emit light under the condition of the presence of the cleaning solution;

and 4, step 4: and recording the luminous value, establishing ｃA standard curve, and obtaining the concentration of the PAPP-A in the sample according to the established standard curve.

In some embodiments of the invention, the incubation is at 37 ℃ for 9 min.

In some embodiments of the present invention, the detection method specifically comprises:

step 1: adding 15 μ l of sample into ｃA reaction tube, sequentially adding ｃA PAPP-A reagent RｃA and ｃA PAPP-A reagent Rb, incubating at 37 ℃ for 9min, finally adding 35 μ l of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9 min;

step 2: sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;

and step 3: and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.

And 4, step 4: and recording the luminous value by the photomultiplier, establishing ｃA standard curve, and calculating the concentration of the PAPP-A in the sample according to the established standard curve.

The magnetic particles can be used as carriers of biological macromolecules, the antibody-coated magnetic particles are called immune magnetic particles, and the immune magnetic particles have the characteristics of antigen combination and magnetism, so that the immune magnetic particles have more advantages in the aspects of separating, purifying and concentrating target microorganisms, cells, biological macromolecules and the like from complex samples, and comprise rapidness, strong specificity, simple and convenient operation, wide application range and the like. The nanometer material is a new material which is rapidly developed after 90 years in the 20 th century, and the nanometer magnetic particles (the particle size is less than 10 nm-100 nm) are greatly different from the common magnetic particles in the aspects of magnetic structure and magnetism: the nano magnetic particles have more particles per unit volume and larger specific surface area; the magnetic material has superparamagnetism, and the magnetic interaction is weak; it can move directionally under the action of external magnetic field to separate, concentrate or purify some special components. The magnetic particle chemiluminescence method established by the invention has the advantages of high sensitivity, strong specificity, accuracy, rapidness, short detection time and higher accuracy and repeatability of a detection result.

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.

FIG. 1 shows the results of a straight line fit of the diluted concentration to the measured concentration in the reaction method of example 8- (1);

FIG. 2 shows the results of straight line fitting of the diluted concentration to the measured concentration in the reaction method of example 8- (2).

Detailed Description

The invention discloses ｃA magnetic microsphere electrochemiluminescence immunoassay kit for detecting pregnancy related plasmｃA protein A (PAPP-A), which can be realized by appropriately improving process parameters 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.

The invention provides the following technical scheme: a PAPP-A magnetic microsphere electrochemiluminescence kit comprises: the kit comprises ｃA PAPP-A reagent RｃA, ｃA PAPP-A reagent Rb, streptavidin superparamagnetic microspheres, ｃA calibration product, ｃA tripropylamine cleaning solution, ｃA dibutylethanolamine cleaning solution and ｃA pipeline cleaning solution.

The PAPP-A magnetic microsphere electrochemiluminescence kit comprises ｃA PAPP-A reagent RｃA, ｃA buffer solution and ｃA magnetic microsphere electrochemiluminescence kit, wherein the PAPP-A reagent RｃA is an anti-PAPP-A monoclonal antibody containing biotin labels, the labeling quantity of biotin molecules on the surface of each antibody molecule is 2-5, the buffer solution is 20 mM-200 mM phosphate buffer solution, the pH value is 7.4-7.8 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and the pH value is 7.4-7.8. The PAPP-A reagent Rb is an anti-PAPP-A monoclonal antibody containing ｃA terpyridyl ruthenium label, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10, and the buffer solution is 20 mM-200 mM phosphate buffer solution with the pH value of 7.4-7.8 or 20 mM-200 mM tris buffer solution with the pH value of 7.4-7.8.

The PAPP-A magnetic microsphere electrochemiluminescence kit comprises ｃA streptavidin superparamagnetic microsphere, ｃA magnetic microsphere and ｃA magnetic particle coating buffer, wherein the surface of the streptavidin superparamagnetic microsphere is coated with the streptavidin superparamagnetic microsphere, the particle size of the magnetic microsphere is 1.5-5.0 micrometers, the magnetic particle coating buffer is 20 mM-200 mM phosphate buffer, the pH value is 7.4-7.8 or 20 mM-200 mM tris buffer, and the pH value is 7.4-7.8.

The PAPP-A magnetic microsphere electrochemiluminescence kit comprises ｃA kit body, ｃA kit body and ｃA kit body, wherein the kit body is used for carrying out electrochemiluminescence on PAPP-A magnetic microspheres, and the kit body is used for carrying out electrochemiluminescence on PAPP-A magnetic microspheres.

The detection method of the PAPP-A magnetic microsphere electrochemiluminescence kit comprises the following steps of (1) taking tripropylamine with the concentration of 180mmol/L as ｃA cleaning solution, wherein the tripropylamine with the concentration of 300mmol/L contains ｃA phosphate buffer solution; or 90mmol/L dibutylethanolamine containing phosphate buffer solution with concentration of 300 mmol/L.

The invention provides ｃA detection method of ｃA PAPP-A magnetic microsphere electrochemiluminescence kit, which comprises the following steps:

1) adding 15 μ l of sample into ｃA reaction tube, sequentially adding 70 μ l of PAPP-A reagent RｃA and 70 μ l of PAPP-A reagent Rb, incubating at 37 deg.C for 9min, adding 40 μ l of streptavidin superparamagnetic microsphere, and incubating at 37 deg.C for 9 min;

2) sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;

3) and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.

4) And recording the luminous value by the photomultiplier, establishing ｃA standard curve, and calculating the concentration of the PAPP-A in the sample according to the established standard curve.

The streptavidin and the biotin have high-specificity binding capacity, and the streptavidin and the biotin-labeled high-purity antibody are specifically bound through non-covalent bonds, so that the streptavidin-labeled high-purity antibody has the effect of cascade amplification, and the reaction is highly specific. Therefore, the sensitivity is improved, non-specific interference is not increased, and the binding property is not affected by the high dilution of the reaction reagent, so that the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application.

The invention combines the high specificity of antibody-antigen reaction with the high sensitivity of ruthenium terpyridyl luminescence, utilizes the photons generated by ruthenium terpyridyl under tripropylamine or DBAE to detect the product concentration, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.

The magnetic microsphere electrochemiluminescence immunoassay kit for detecting pregnancy related plasmｃA protein A (PAPP-A) provided by the invention is commercially available in raw materials and reagents.

All components of the test kit of the present invention can be commercially obtained from biological or chemical reagents companies. The device used was a full-automatic chemiluminescence immunoassay analyzer (model UD90DT) manufactured by tek technologies ltd, yokyo.

The invention is further illustrated by the following examples:

example 1: preparation of biotin-labeled pregnancy-associated plasmｃA protein A (PAPP-A) antibody and reagent RｃA

The pregnancy associated plasmｃA protein A (PAPP-A) antibody for labeling biotin is purchased from Beijing edge Tianxin wild science and technology Limited, with the product number of YT-PAPP-A-002 and the clone number of 5D 6.

2.0mg of the antibody for labeling biotin pregnancy-associated plasmｃA protein ｃA (PAPP- ｃA) was taken, the buffer was changed to phosphate buffer (pH 7.8) using desalting column PD10, the concentration was adjusted to 2.0mg/mL after concentration using an ultrafiltration tube, 80 μ g of biotin (dissolved in DMF) was added, the mixture was mixed for 30 minutes, and unlabeled biotin was removed using desalting column PD 10.ｃA pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody labeled biotin was diluted to 1mg/L with ｃA phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as ｃA PAPP- ｃA reagent rｃA. The number of biotin molecular markers on the surface of each antibody molecule is 2-3.

Example 2: preparation of ruthenium-labeled pregnancy-associated plasmｃA protein A (PAPP-A) antibody and reagent Rb

The pregnancy associated plasmｃA protein A (PAPP-A) antibody for labeling biotin was purchased from Beijing edge Tianxin field science and technology Co., Ltd, with the product number YT-PAPP-A-003 and the clone number 3E 11.

2.0mg of pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody for labeling ruthenium terpyridine was taken, the buffer was changed to phosphate buffer (pH 7.8) using desalting column PD10, the mixture was concentrated using an ultrafiltration tube and adjusted to ｃA concentration of 2.0mg/mL, 80 μ g of ruthenium succinamide terpyridine (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled ruthenium was removed using desalting column PD 10.ｃA pregnancy associated plasmｃA protein ｃA (PAPP- ｃA) antibody labeled with ruthenium was diluted to 1mg/L using ｃA phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as ｃA PAPP- ｃA reagent Rb. The number of the ruthenium molecular markers on the surface of each antibody molecule is 5-6.

Example 3: preparation of the calibration articles

The antigen for preparing the calibration sample is purchased from Ji Tech Co Ltd of Beijing edge Tian Xin, and has ｃA product number of YT-PAPP-A-001. For recombinant expression of the protein.

The antigen was diluted to 75.0mIU/L and 2500mIU/L at the indicated concentrations using a phosphate buffer containing 1% bovine serum albumin (pH 7.4). Used as a calibrator for establishing a standard curve.

Example 4 preparation of tripropylamine cleaning solution and dibutylethanolamine cleaning solution

300mmol/L phosphate buffer solution is prepared, tripropylamine is added to 180mmol/L, and the mixture is mixed and dissolved. As a tripropylamine cleaning solution.

Preparing 300mmol/L phosphate buffer solution, adding dibutyl ethanolamine to 90mmol/L, and mixing and dissolving. As a cleaning solution of dibutylethanolamine.

Example 5:

the pregnancy related plasmｃA protein A (PAPP-A) determination adopts ｃA sandwich method, and the detection method comprises the following steps:

1) adding 15 μ l of sample into ｃA reaction tube, sequentially adding 80 μ l of the PAPP-A reagent RｃA prepared in example 1 and 75 μ l of the PAPP-A reagent Rb prepared in example 2, incubating at 37 deg.C for 9min, adding 35 μ l of streptavidin magnetic microsphere (particle size of 3.0 μm), and incubating at 37 deg.C for 9 min;

3) and (3) sucking a cleaning solution (tripropylamine) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium in the presence of the tripropylamine.

4) And recording the luminous value by using ｃA photomultiplier, and calculating the concentration of the PAPP-A in the sample according to ｃA standard curve established after the calibration is carried out by using the luminous value of the calibration object.

Example 6:

1) adding 15 μ l of sample into ｃA reaction tube, sequentially adding 80 μ l of the PAPP-A reagent RｃA prepared in example 1 and 75 μ l of the PAPP-A reagent Rb prepared in example 2, incubating at 37 deg.C for 9min, adding 35 μ l of streptavidin superparamagnetic microsphere (particle size of 3.0 μm), and incubating at 37 deg.C for 9 min;

2) adding a streptavidin-coated superparamagnetic microsphere for incubation, and allowing the formed immune complex to be bound to the superparamagnetic microsphere through the interaction between biotin and streptavidin;

3) after incubation, absorbing the reaction mixture into a measuring cell, adsorbing the superparamagnetic microspheres onto an electrode through a magnet, absorbing the cleaning solution (dibutylethanolamine) by a liquid absorbing steel needle, and absorbing a mark Ru (bpy) which is not combined with the superparamagnetic microspheres₃ ²⁺After the antibody and the sample were washed, the flow cell was charged, and Ru (bpy) was performed in the presence of dibutylethanolamine₃ ²⁺And (4) emitting light.

Example 7: margin test

(1) The reaction method in example 5 was used, and the RLU value (relative luminescence value) of 20 measurements was obtained using the zero-concentration diluent as the sample, and the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, and at the same time, samples of adjacent concentrations were repeatedly tested 2 times, and two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU value of M +2SD was substituted into the above equation to obtain the corresponding concentration value, which was the margin.

TABLE 1

(2) The reaction method in example 6 was used, and a zero-concentration diluent was used as a sample to obtain RLU values (relative luminescence values) of 20 measurements, and the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, and samples of adjacent concentrations were repeatedly tested 2 times, and two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU values of M +2SD were substituted into the above equation to obtain the corresponding concentration value, which was the blank limit.

TABLE 2

Example 8: verification of linear range

(1) Using the reaction method of example 5, high-value samples close to the upper limit of the linear range (10000mIU/ml) were diluted to at least 5 concentrations in a certain proportion, wherein the low-value samples had to be close to 4.0 mIU/ml. And (3) repeatedly detecting the samples with each concentration for 2 times, calculating the average value of the samples to obtain the measured concentration, performing straight line fitting on the diluted concentration and the measured concentration by using a least square method, and calculating a linear correlation coefficient r, wherein r is not less than 0.99. The results of a straight line fit of the diluted concentrations to the measured concentrations are shown in figure 1.

TABLE 3

(2) The reaction method of example 6, high value samples near the upper end of the linear range (10000mIU/ml) were diluted to at least 5 concentrations in a certain proportion, wherein the low value samples were close to 4.0 mIU/ml. And (3) repeatedly detecting the samples with each concentration for 2 times, calculating the average value of the samples to obtain the measured concentration, performing straight line fitting on the diluted concentration and the measured concentration by using a least square method, and calculating a linear correlation coefficient r, wherein r is not less than 0.99. The results of a straight line fit of the diluted concentrations to the measured concentrations are shown in figure 2.

TABLE 4

Summary of the comparative kit conditions:

TABLE 5

	Examples 7 to 8	Comparative example 1	Comparative example 2
				Methodology of	Electrochemiluminescence sandwich method	Enzymatic chemiluminescence method	Enzyme linked immunosorbent assay
Sensitivity of the probe	4mIU/L	10mIU/L	10mIU/L
				Linear range	10-10000mIU/L	10-5000mIU/L	10-3000mIU/L
Time of detection	18 minutes	20 minutes	120 minutes
				Antibody treatment	60 minutes	Greater than 10 hours	Greater than 10 hours

The electrochemical luminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, safety, no toxicity, no pollution and the like. Luminol, isoluminol and its derivatives are the first type of chemiluminescent species used, but their application to chemiluminescent immunoassays requires the use of catalysts and enhancers, which leads to an increase in background luminescence, thereby limiting the sensitivity of this technology and its application and development. The acridinium ester luminescent system is simple, does not need a catalyst and is placed in H₂O₂The acridinium ester can emit light in the solution without a catalytic process or an enhancer, so background light emission is reduced, sensitivity is improved, interference effect is small, but the acridinium ester is easy to hydrolyze, and the light emission of the acridinium ester is released rapidly. The peak value of luminescence is 0.4s, so in-situ sample injection is needed, and the requirement on equipment is high. The ruthenium terpyridyl is easy to be connected with protein, has small molecular weight, has small influence on the conformation of the connected antibody, and has good stability because the marker is metal ions and controllable luminescence because the luminescence is required under the condition of electrification. Therefore, the electrochemical method applied to the detection of the PAPP-A can improve the sensitivity of the product, shorten the process marking time, improve the linear range and shrinkShort test time and provides a basis for clinically dealing with the treatment of the brain trauma in time.

The property of the electrochemiluminescence marker ruthenium pyridine is very stable, and the luminous efficiency of the electrochemiluminescence marker ruthenium pyridine is not influenced by factors such as temperature, pH and ionic strength. The signal value of the electrochemiluminescence reagent is reduced within 3 percent compared with that of a fresh reagent. The bottle opening period is three months, and the bottle can be stabilized at 2-8 ℃ for more than 15 months.

TABLE 6

Light-emitting system	Horseradish enzyme-luminol	Alkaline phosphatase	Electrochemiluminescence
				Time stamping	Greater than 24 hours	Greater than 24 hours	60 minutes
Test time	60 minutes	30 minutes	18 minutes
				Expiration date of reagent	12 months old	12 months old	More than 15 months

The electrochemical labeling reaction is rapid, and the whole reaction only needs half an hour. The marking efficiency reaches 70%. The proportion of the marks can be controlled by the feed ratio, and the productivity is improved by over 50 percent. Ruthenium has small molecular weight (800D), small steric hindrance and good antibody activity. An absorption peak at 455nm, the feed ratio can be controlled to control the batch-to-batch difference.

The steps show that the reaction mode of the sandwich method adopted by the invention utilizes the principle of magnetic microsphere electrochemistry to quantitatively detect the pregnancy related plasmｃA protein A (PAPP-A) content in human serum or plasmｃA samples, thereby ensuring the detection sensitivity. And is suitable for use in fully automatic equipment. The detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. The composition for detecting pregnancy associated plasmｃA protein A (PAPP-A) is characterized by comprising ｃA PAPP-A reagent RｃA, ｃA PAPP-A reagent Rb and streptavidin superparamagnetic microspheres;

2. The composition of claim 1, wherein the superparamagnetic microspheres have a particle size of 1.5 to 5.0 μm.

3. The composition of claim 1, wherein the amount of biotin molecular labels per antibody molecule surface in the PAPP- ｃA reagent rｃA is 2-5; in the PAPP-A reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.

4. The composition according to any one of claims 1 to 3, wherein the PAPP- ｃA reagent rｃA is prepared by: mixing an anti-PAPP-A monoclonal antibody with biotin in the presence of ｃA buffer solution to prepare ｃA PAPP-A reagent RｃA; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.

5. The composition according to any one of claims 1 to 3, wherein said PAPP- ｃA reagent Rb is prepared by: mixing an anti-PAPP-A monoclonal antibody with terpyridyl ruthenium in the presence of ｃA buffer solution to prepare ｃA PAPP-A reagent Rb; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.

6. A composition according to any one of claims 1 to 3, further comprising a standard and/or a cleaning solution; the cleaning solution comprises tripropylamine with the concentration of 150-200 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L; or dibutylethanolamine with the concentration of 80-100 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L.

7. The composition of any one of claims 1 to 3, wherein the volume ratio of the PAPP-A reagent RｃA, the PAPP-A reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): (20-40).

8. Use of ｃA composition according to any one of claims 1 to 7 for the preparation of ｃA magnetic microsphere electrochemiluminescence immunoassay kit for pregnancy related plasmｃA protein ｃA (PAPP- ｃA).

9.ｃA magnetic microsphere electrochemiluminescence immunoassay kit for pregnancy related plasmｃA protein ｃA (PAPP- ｃA), comprising the composition of any one of claims 1 to 7 and ｃA reagent acceptable for detection.

10.ｃA magnetic microsphere electrochemiluminescence immunoassay method for pregnancy related plasmｃA protein ｃA (PAPP- ｃA), based on the composition according to any one of claims 1 to 7 or the kit according to claim 9, comprising the steps of:

step 2: adsorbing the reaction solution by using a magnet;