CN111175496A - Pregnancy-associated protein A magnetic particle chemiluminescence detection kit and use method thereof - Google Patents
Pregnancy-associated protein A magnetic particle chemiluminescence detection kit and use method thereof Download PDFInfo
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- CN111175496A CN111175496A CN202010125618.6A CN202010125618A CN111175496A CN 111175496 A CN111175496 A CN 111175496A CN 202010125618 A CN202010125618 A CN 202010125618A CN 111175496 A CN111175496 A CN 111175496A
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a pregnancy-associated protein A magnetic particle chemiluminescence detection kit and a use method thereof, belonging to the technical field of medical apparatus and biological immune in-vitro diagnosis; the kit comprises ｃA fluorescein-labeled PAPP-A antibody solution, an alkaline phosphatase-labeled PAPP-A antibody solution, ｃA fluorescein antibody-coated magnetic particle suspension, ｃA PAPP-A calibrator and ｃA luminescent substrate solution which are packaged independently; the invention combines the magnetic particle technology and the chemiluminescence technology, and provides a reaction system which tends to be homogeneous; can quantitatively detect pregnancy-associated protein A (PAPP-A) with higher accuracy, specificity, precision and lower cost.
Description
Technical Field
The invention relates to the technical field of medical apparatus and instrument biological immune in-vitro diagnosis, in particular to a pregnancy-associated protein A magnetic particle chemiluminescence detection kit and a using method thereof.
Background
3 serum 3 pregnancy 3 associated 3 plasma 3 protein 3 A 3 ( 3 PAPA 3- 3 A 3) 3 is 3 a 3 macromolecular 3 glycoprotein 3 with 3 a 3 molecular 3 weight 3 of 3 200 3 kDa 3, 3 and 3 is 3 a 3 member 3 of 3 the 3 zinc 3 peptidase 3 superfamily 3. 3 PAPP- ｃA first appears in the serum of pregnant women, with increased stability during pregnancy, until term. PAPP- ｃA is composed of two subunits fed together in ｃA ratio of 2: the 2-heterotetraploid complex forms circulate in the blood. Many studies have demonstrated that the combination of PAPP-P as an optional serum marker, and a super-marker of cervical translucency (NT) detects high risk pregnant women with down's syndrome fetuses in the first trimester of pregnancy (11-14 weeks). 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 concentrations of PAAA-A in the serum of pregnant women carrying fetuses with Down syndrome is lower than that of normal pregnant women. Depending on the age of the pregnant woman, a particular algorithm (e.g. likelihood ratio, etc.) may be used to calculate the risk of having down syndrome.
At present, immunological methods are mostly adopted for detecting pregnancy-associated protein A. The immunological detection technology utilizes the antigen and antibody specific reaction principle to detect the matter to be detected in the organism. The radioimmunoassay, fluorescence immunoassay, enzyme-linked immunoassay and chemiluminescence immunoassay technologies are gradually developed and mature, traditional immunoprecipitation, immunoagglutination and other technologies are gradually replaced, and the development of the immunological detection technology causes great changes in clinical detection and inspection: the method has the advantages of greatly increased detectable items, higher detection sensitivity and specificity and increased automation degree. Especially, the chemiluminescence immunoassay technology developed in the later stage further improves the detection performances such as sensitivity, specificity and the like of detection, and provides visual data for quantitative detection, diagnosis and treatment of diseases.
The pregnancy related protein A is an important detection item developed by a hospital, and has no alternative reference function for calculating the risk diagnosis of Down syndrome. At present, most of pregnancy related protein A detection reagents approved to be on the market in China are made in China, and most of detection methodologies adopted are enzyme immunoassay and chemiluminescence.
Therefore, the development of a method with low production cost; the detection performances such as sensitivity, precision, detection range and the like are good; can conveniently, quickly, simply and batch detect products of the pregnancy related protein A, and has important significance.
Disclosure of Invention
Aiming at the needs of the prior art, the production cost is low; the detection performances such as sensitivity, precision, detection range and the like are good; the invention provides a pregnancy-associated protein A magnetic particle chemiluminescence detection kit and a use method thereof, and aims to solve the technical problem that products of pregnancy-associated protein A can be detected conveniently, quickly, simply and in batches.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the pregnancy related protein A magnetic particle chemiluminescence detection kit comprises ｃA fluorescein-labeled PAPP-A antibody solution, an alkaline phosphatase-labeled PAPP-A antibody solution, ｃA fluorescein antibody-coated magnetic particle suspension, ｃA PAPP-A calibrator and ｃA luminescent substrate solution which are packaged independently.
Further, the PAPP-A antibody marked by alkaline phosphatase is formed by connecting alkaline phosphatase and the PAPP-A antibody through ｃA cross-linking agent of disuccinimidyl suberate.
Further, the concentration of the solution of the fluorescein labeled PAPP-A antibody is 0.8-1. 2 mu g/ml, and the pH value is 7-9.
Further, the concentration of the solution of the alkaline phosphatase-labeled PAPP-A antibody is 0.5-0.8 mu g/ml, and the pH value is 7-9.
Further, the solution of the fluorescein-labeled PAPP-A antibody is prepared by the following steps:
sa 1: preparing a Tris-HCl buffer solution, wherein the Tris-HCl buffer solution contains 4-8 wt% of PEG-6000, 0.7-0.9 wt% of NaCl, 0.08-0.1 wt% of sodium azide, 0.58-0.62 wt% of ProClin-300, 0.8-1 wt% of bovine serum albumin and 19-21 mM of EDTA, and adjusting the pH value of the Tris-HCl buffer solution to 7-9;
sa 2: adding ｃA PAPP-A antibody with the volume of 0.6-0.8% of that of the Tris-HCl buffer solution into the Tris-HCl buffer solution, fully mixing, and standing at room temperature for reaction;
sa 3: and (3) passing the reaction solution prepared by Sa2 through a gel column, and adjusting the concentration and the pH value to obtain the product.
Further, the solution of the alkaline phosphatase-labeled PAPP-A antibody is prepared by the following operation steps:
sb 1: dissolving the PAPP-A antibody into ｃA solution with the concentration of 30-60 mg/ml by using ｃA dimethyl sulfoxide solvent, adding ｃA cross-linking agent of disuccinimidyl suberate, uniformly mixing, and reacting at room temperature for 2-3 h; diluting the concentration of the conjugate of the PAPP-A antibody and disuccinimidyl suberate by 8-10 times by using ｃA dimethyl sulfoxide solvent, and storing at the temperature of 2-8 ℃;
sb 2: uniformly mixing alkaline phosphatase and the conjugate by using an alkaline phosphatase buffer solution containing 1-2 mg/ml of alkaline phosphatase according to the molar ratio of the alkaline phosphatase to the conjugate being 1 (1.8-2.2), and reacting at room temperature for 1-2 hours;
sb 3: and (3) passing the reaction solution prepared from Sb2 through a G-25 gel column, and adjusting the concentration and the pH value to obtain the Sb 2-containing aqueous solution.
Further, the magnetic particle suspension coated with the fluorescein antibody is prepared by the following operation: under the environment of a coupling agent carbodiimide, uniformly mixing magnetic particles containing carboxyl active groups with a fluorescein antibody, reacting at room temperature for 10-20 h, carrying out magnetic separation, removing supernatant, and adjusting concentration and pH to obtain the fluorescent nanoparticle; the magnetic particles have superparamagnetism, and the content of carboxyl active groups carried on each gram of the magnetic particles is not less than 0.5 mml; the fluorescein antibody is a monoclonal antibody, and the dilution titer is more than 1:100 ten thousand.
Further, the luminescent substrate is one or more of Lumi-pHos480, adamantane, luminol and derivatives thereof, isoluminol and derivatives thereof, and acridine ester.
Further, the solution of the luminescent substrate is prepared by the operation comprising the following steps: 2g of TRIS, 6g of NaCl and 0.005g of Na were accurately weighed2SO3And 0.5ml ProClin-300 are respectively added into 600ml purified water and fully dissolved, and the pH value is adjusted to 7.5; adding 200ml luminescent substrate, filtering with 0.2 μm filter, and adding purified water to reach volume of 1000 ml.
The use method of the pregnancy related protein A magnetic particle chemiluminescence detection kit comprises the following steps:
sc 1: immune reaction: adding ｃA sample to be detected into ｃA reaction tube, sequentially adding the solution of the fluorescein-labeled PAPP-A antibody and the solution of the alkaline phosphatase-labeled PAPP-A antibody, uniformly mixing, and incubating at 20-40 ℃ for 30min to prepare ｃA solution of the double-antigen sandwich complex;
adding the magnetic particle suspension coated with the fluorescein antibody into the solution of the double-antigen sandwich compound, uniformly mixing, incubating for 30min at 20-40 ℃, and fixing the double-antigen sandwich compound on magnetic microspheres;
sc 2: washing: adding a cleaning solution into the solution prepared by Sc1, oscillating to enable the magnetic particles to settle in a magnetic field, removing the supernatant and removing the antigen of the connection labeled tracer which does not form a double-antigen sandwich compound through the antibody to be detected; repeating the operation for 0-5 times;
sc 3: and (3) adding a substrate for detection: and adding the solution of the luminescent substrate catalyzed by alkaline phosphatase into the reaction tube, fully and uniformly mixing, detecting a luminescent value, and calculating to obtain the content of the pregnancy-associated protein A.
The invention has the following advantages:
1. combining an immunomagnetic particle technology with ｃA chemiluminescence technology, providing ｃA reaction system close to homogeneous phase, and enabling the PAPP-A antibody connected with ｃA labeled tracer and the PAPP-A antibody connected with ｃA bridging substance to react with an antibody to be detected to form ｃA compound with ｃA double-antigen sandwich structure; in the process of forming the double-antigen sandwich compound, small molecular substances are used together with the labeled antigen, the steric hindrance of the small molecular substances is far smaller than that of the magnetic microspheres, so that the antigen-antibody combination reaction can be promoted to be fully carried out, and the detection performances such as sensitivity, precision, detection range and the like are greatly improved;
2. the PAPP-A antibody and alkaline phosphatase are coupled by using the disuccinimide suberate as ｃA cross-linking agent, so that the coupling efficiency is higher than that of other cross-linking agents, the process is stable, the detection efficiency is improved, and the preparation cost is reduced;
3. the kit can be matched with ｃA full-automatic chemiluminescence immunoassay analyzer (particularly ｃA ZECENCIA series full-automatic chemiluminescence immunoassay analyzer), so that the full automation of sample detection is realized, the detection of the concentration of the PAPP-A antibody can be conveniently, quickly, simply and massively carried out, the system error is reduced to the maximum extent, and the guarantee is provided for the service life and the detection performance of the kit.
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 test calibrator standard curve;
FIG. 2 shows the correlation between the test results of urine samples.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. Moreover, in the following description, descriptions of well-known structures, techniques, and operations are omitted so as to not unnecessarily obscure the concepts of the present invention. In addition, the technical features related to the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the present invention, the proportion and content of the unit are not particularly specified, the solid component is the mass proportion and content, and the liquid component is the volume proportion and content.
Example 1
The pregnancy-associated protein A magnetic particle chemiluminescence detection kit comprises the following components in independent package: ｃA solution of fluorescein-labeled PAPP-A antibody at ｃA concentration of 0.8. mu.g/ml and ｃA pH of 7; ｃA solution of alkaline phosphatase-labeled PAPP-A antibody at ｃA concentration of 0.5. mu.g/ml and ｃA pH of 7; ｃA solution of ｃA magnetic particle suspension coated with ｃA fluorescein antibody, ｃA PAPP-A calibrator and ｃA luminescent substrate;
1. the solution of fluorescein-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sa 1: preparing a Tris-HCl buffer solution, wherein the Tris-HCl buffer solution contains 4 wt% of PEG-6000, 0.7 wt% of NaCl, 0.08 wt% of sodium azide, 0.58 wt% of ProClin-300, 0.8 wt% of bovine serum albumin and 19mM of EDTA, and the pH value of the Tris-HCl buffer solution is adjusted to be 7;
sa 2: adding ｃA PAPP-A antibody with the volume of 0.6 percent of that of the Tris-HCl buffer solution into the Tris-HCl buffer solution, fully mixing, and standing at room temperature for reaction;
sa 3: the reaction solution prepared from Sa2 is prepared by passing through a gel column and adjusting the concentration and pH;
2. the solution of alkaline phosphatase-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sb 1: dissolving the PAPP-A antibody into ｃA solution with the concentration of 30mg/ml by using ｃA dimethyl sulfoxide solvent, adding ｃA cross-linking agent of disuccinimidyl suberate, uniformly mixing, and reacting at room temperature for 2 hours; diluting the concentration of the conjugate of the PAPP-A antibody and disuccinimidyl suberate by 8 times by using ｃA dimethyl sulfoxide solvent, and storing at the temperature of 2 ℃;
sb 2: uniformly mixing alkaline phosphatase and the conjugate according to the molar ratio of 1:1.8 of the alkaline phosphatase and the conjugate by using an alkaline phosphatase buffer solution containing 1mg/ml of alkaline phosphatase, and reacting at room temperature for 1 h;
sb 3: the reaction solution prepared from Sb2 is prepared by passing through a G-25 gel column and adjusting the concentration and the pH value;
3. the magnetic particle suspension coated with the fluorescein antibody is prepared by the following steps: under the environment of coupling agent carbodiimide, uniformly mixing magnetic particles containing carboxyl active groups with a fluorescein antibody, reacting for 10 hours at room temperature, carrying out magnetic separation, removing supernatant, and adjusting concentration and pH to obtain the fluorescent nanoparticle; the magnetic particles have superparamagnetism, and the content of carboxyl active groups carried on each gram of magnetic particles is not less than 0.5 mml; the fluorescein antibody is a monoclonal antibody, and the dilution titer is more than 1:100 ten thousand;
4. the solution of luminescent substrate is prepared by an operation comprising the steps of: 2g of TRIS, 6g of NaCl and 0.005g of Na were accurately weighed2SO3And 0.5ml ProClin-300 are respectively added into 600ml purified water and fully dissolved, and the pH value is adjusted to 7.5; adding 200ml adamantane, filtering with 0.2 μm filter, and adding purified water to 1000 ml.
Example 2
The pregnancy-associated protein A magnetic particle chemiluminescence detection kit comprises the following components in independent package: ｃA solution of fluorescein-labeled PAPP-A antibody at ｃA concentration of 1. mu.g/ml and ｃA pH of 8; ｃA solution of alkaline phosphatase-labeled PAPP-A antibody with ｃA concentration of 0.6. mu.g/ml and ｃA pH of 8; ｃA solution of ｃA magnetic particle suspension coated with ｃA fluorescein antibody, ｃA PAPP-A calibrator and ｃA luminescent substrate;
1. the solution of fluorescein-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sa 1: preparing a Tris-HCl buffer solution, wherein the Tris-HCl buffer solution contains 6 wt% of PEG-6000, 0.8 wt% of NaCl, 0.09 wt% of sodium azide, 0.6 wt% of ProClin-300, 0.9 wt% of bovine serum albumin and 20mM of EDTA, and the pH value of the Tris-HCl buffer solution is adjusted to be 8;
sa 2: adding ｃA PAPP-A antibody with the volume of 0.7 percent of that of the Tris-HCl buffer solution into the Tris-HCl buffer solution, fully mixing, and standing at room temperature for reaction;
sa 3: the reaction solution prepared from Sa2 is prepared by passing through a gel column and adjusting the concentration and pH;
2. the solution of alkaline phosphatase-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sb 1: dissolving the PAPP-A antibody into ｃA solution with the concentration of 50mg/ml by using ｃA dimethyl sulfoxide solvent, adding ｃA cross-linking agent of disuccinimidyl suberate, uniformly mixing, and reacting at room temperature for 2.5 h; diluting the conjugate of the PAPP-A antibody and disuccinimidyl suberate by 9 times with dimethyl sulfoxide solvent, and storing at 4 deg.C;
sb 2: uniformly mixing alkaline phosphatase and the conjugate according to the molar ratio of 1:2 of the alkaline phosphatase and the conjugate by using an alkaline phosphatase buffer solution containing 1.5mg/ml of alkaline phosphatase, and reacting at room temperature for 1.5 h;
sb 3: the reaction solution prepared from Sb2 is prepared by passing through a G-25 gel column and adjusting the concentration and the pH value;
3. the magnetic particle suspension coated with the fluorescein antibody is prepared by the following steps: under the environment of coupling agent carbodiimide, uniformly mixing magnetic particles containing carboxyl active groups with a fluorescein antibody, reacting for 15 hours at room temperature, carrying out magnetic separation, removing supernatant, and adjusting concentration and pH to obtain the fluorescent nanoparticle; the magnetic particles have superparamagnetism, and the content of carboxyl active groups carried on each gram of magnetic particles is not less than 0.5 mml; the fluorescein antibody is a monoclonal antibody, and the dilution titer is more than 1:100 ten thousand;
4. the solution of luminescent substrate is prepared by an operation comprising the steps of: 2g of TRIS, 6g of NaCl and 0.005g of Na were accurately weighed2SO3And 0.5ml ProClin-300 are respectively added into 600ml purified water and fully dissolved, and the pH value is adjusted to 7.5; adding 200ml Lumi-pHos480, filtering with 0.2 μm filter, and adding purified water to 1000 ml.
Example 3
The pregnancy-associated protein A magnetic particle chemiluminescence detection kit comprises the following components in independent package: ｃA solution of fluorescein-labeled PAPP-A antibody at ｃA concentration of 1. 2. mu.g/ml and ｃA pH of 9; ｃA solution of alkaline phosphatase-labeled PAPP-A antibody with ｃA concentration of 0.8. mu.g/ml and ｃA pH of 9; ｃA solution of ｃA magnetic particle suspension coated with ｃA fluorescein antibody, ｃA PAPP-A calibrator and ｃA luminescent substrate;
1. the solution of fluorescein-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sa 1: preparing a Tris-HCl buffer solution, wherein the Tris-HCl buffer solution contains 8 wt% of PEG-6000, 0.9 wt% of NaCl, 0.1 wt% of sodium azide, 0.62 wt% of ProClin-300, 1 wt% of bovine serum albumin and 21mM of EDTA, and the pH value of the Tris-HCl buffer solution is adjusted to 9;
sa 2: adding ｃA PAPP-A antibody with the volume of 0.8 percent of that of the Tris-HCl buffer solution into the Tris-HCl buffer solution, fully mixing, and standing at room temperature for reaction;
sa 3: the reaction solution prepared from Sa2 is prepared by passing through a gel column and adjusting the concentration and pH;
2. the solution of alkaline phosphatase-labeled PAPP-A antibody was prepared by an operation comprising the steps of:
sb 1: dissolving the PAPP-A antibody into ｃA solution with the concentration of 60mg/ml by using ｃA dimethyl sulfoxide solvent, adding ｃA cross-linking agent of disuccinimidyl suberate, uniformly mixing, and reacting at room temperature for 3 hours; diluting the concentration of the conjugate of the PAPP-A antibody and disuccinimidyl suberate by 10 times by using ｃA dimethyl sulfoxide solvent, and storing at 8 ℃;
sb 2: uniformly mixing alkaline phosphatase and the conjugate by using an alkaline phosphatase buffer solution containing 2mg/ml of alkaline phosphatase according to the molar ratio of the alkaline phosphatase to the conjugate being 1:2.2, and reacting at room temperature for 2 hours;
sb 3: the reaction solution prepared from Sb2 is prepared by passing through a G-25 gel column and adjusting the concentration and the pH value;
3. the magnetic particle suspension coated with the fluorescein antibody is prepared by the following steps: under the environment of coupling agent carbodiimide, uniformly mixing magnetic particles containing carboxyl active groups with a fluorescein antibody, reacting for 20 hours at room temperature, carrying out magnetic separation, removing supernatant, and adjusting concentration and pH to obtain the fluorescent nanoparticle; the magnetic particles have superparamagnetism, and the content of carboxyl active groups carried on each gram of magnetic particles is not less than 0.5 mml; the fluorescein antibody is a monoclonal antibody, and the dilution titer is more than 1:100 ten thousand;
4. the solution of luminescent substrate is prepared by an operation comprising the steps of: 2g of TRIS, 6g of NaCl and 0.005g of Na were accurately weighed2SO3And 0.5ml ProClin-300 are respectively added into 600ml purified water and fully dissolved, and the pH value is adjusted to 7.5; adding 200ml of acridinium ester, filtering by using a 0.2 mu m filter, and then adding purified water to reach the constant volume of 1000ml to obtain the product; in specific implementation, the luminescent substrate can be replaced by luminol and derivatives thereof, isoluminol and derivatives thereof.
Example 4
The application method of the pregnancy-associated protein A magnetic particle chemiluminescence detection kit prepared in examples 1-3, in this example, the kit prepared in example 2 is used to demonstrate a method for quantitatively detecting pregnancy-associated protein A, and the method comprises the following steps:
sc 1: immune reaction: adding ｃA sample to be detected into ｃA reaction tube, sequentially adding ｃA solution of ｃA fluorescein-labeled PAPP-A antibody and ｃA solution of an alkaline phosphatase-labeled PAPP-A antibody, uniformly mixing, and incubating at 37 ℃ for 30min to prepare ｃA solution of ｃA double-antigen sandwich compound;
adding a magnetic particle suspension coated with a fluorescein antibody into the solution of the double-antigen sandwich compound, uniformly mixing, incubating for 30min at 37 ℃, and fixing the double-antigen sandwich compound on the magnetic microspheres; in specific implementation, the temperature of the two incubations in the step can be arbitrarily selected between 20 ℃ and 40 ℃;
sc 2: washing: adding a cleaning solution into the solution prepared by Sc1, oscillating to enable the magnetic particles to settle in a magnetic field, removing the supernatant and removing the antigen of the connection labeled tracer which does not form a double-antigen sandwich compound through the antibody to be detected; repeating the operation for 5 times, wherein the operation can be not repeated or repeated for 1 to 4 times during specific implementation;
sc 3: and (3) adding a substrate for detection: adding a solution of a luminescent substrate catalyzed by alkaline phosphatase into the reaction tube, fully and uniformly mixing, detecting a luminescent value, and calculating to obtain the content of the pregnancy-associated protein A.
And establishing a standard curve by adopting a four-parameter fitting mode and taking the concentration value of the calibrator as an X axis and the luminous intensity value of the calibrator as a Y axis. And calculating back the corresponding concentration value according to the luminous intensity value of the sample to be detected.
The kit disclosed by the invention is identified according to methodology, and the performance parameters can reach the following indexes:
the standard curve is linear: r is greater than 0.999 (as shown in figure 1)
Sensitivity: and (3) repeatedly detecting the zero calibration substance for 20 times, calculating the average value (M) and the Standard Deviation (SD) of the luminous intensity, calculating M-2 SD, and calculating a corresponding concentration value according to the corresponding concentration on the standard curve, namely the analysis sensitivity. Wherein, the sensitivity is not more than 0.025 mlU/mL.
Precision: the intra-assay variation, inter-assay variation and inter-batch variation were all less than 10% (as shown in Table 1)
TABLE 1 precision data sheet
a) Intra-and inter-assay variation data sheet
b) Inter-batch variation data sheet
| Q1 | Q2 | Q3 | |
| Variation CV% | 6.09 | 5.25 | 5.06 |
Specificity: the magnetic particle of the invention has two-step specificity identification, and is better improved compared with other detection reagents. The false positive rate of the reagent is 0 percent when the reagent kit is used for detecting 200 clinical negative serum samples.
The accuracy is as follows: fitting concentration values to the luminescence values of the reference substances by adding a recovery method, wherein the average value of the ratio of the fitting concentration to the labeled concentration is 0.85-1.15 (shown in Table 3)
Compared with similar products: 120 clinical urine samples are measured by the kit and the Roche PAPP-A kit simultaneously, the correlation between the measurement results of the kit and the clinical urine samples is better, and the correlation coefficient R is 0.998 (shown in figure 2).
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are within the scope of the invention.
Claims (10)
1. Pregnancy-associated protein A magnetic particle chemiluminescence detection kit, which is characterized in that: the kit comprises ｃA fluorescein-labeled PAPP-A antibody solution, an alkaline phosphatase-labeled PAPP-A antibody solution, ｃA fluorescein antibody-coated magnetic particle suspension, ｃA PAPP-A calibrator and ｃA luminescent substrate solution which are packaged independently.
2. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the PAPP-A antibody marked by alkaline phosphatase is formed by connecting alkaline phosphatase and the PAPP-A antibody through ｃA cross-linking agent of disuccinimidyl suberate.
3. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the concentration of the solution of the fluorescein labeled PAPP-A antibody is 0.8-1. 2 mu g/ml, and the pH value is 7-9.
4. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the solution concentration of the alkaline phosphatase-labeled PAPP-A antibody is 0.5-0.8 mu g/ml, and the pH value is 7-9.
5. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the solution of the fluorescein-labeled PAPP-A antibody is prepared by the following steps:
sa 1: preparing a Tris-HCl buffer solution, wherein the Tris-HCl buffer solution contains 4-8 wt% of PEG-6000, 0.7-0.9 wt% of NaCl, 0.08-0.1 wt% of sodium azide, 0.58-0.62 wt% of ProClin-300, 0.8-1 wt% of bovine serum albumin and 19-21 mM of EDTA, and adjusting the pH value of the Tris-HCl buffer solution to 7-9;
sa 2: adding ｃA PAPP-A antibody with the volume of 0.6-0.8% of that of the Tris-HCl buffer solution into the Tris-HCl buffer solution, fully mixing, and standing at room temperature for reaction;
sa 3: and (3) passing the reaction solution prepared by Sa2 through a gel column, and adjusting the concentration and the pH value to obtain the product.
6. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the solution of the alkaline phosphatase-labeled PAPP-A antibody is prepared by the following operation steps:
sb 1: dissolving the PAPP-A antibody into ｃA solution with the concentration of 30-60 mg/ml by using ｃA dimethyl sulfoxide solvent, adding ｃA cross-linking agent of disuccinimidyl suberate, uniformly mixing, and reacting at room temperature for 2-3 h; diluting the concentration of the conjugate of the PAPP-A antibody and disuccinimidyl suberate by 8-10 times by using ｃA dimethyl sulfoxide solvent, and storing at the temperature of 2-8 ℃;
sb 2: uniformly mixing alkaline phosphatase and the conjugate by using an alkaline phosphatase buffer solution containing 1-2 mg/ml of alkaline phosphatase according to the molar ratio of the alkaline phosphatase to the conjugate being 1 (1.8-2.2), and reacting at room temperature for 1-2 hours;
sb 3: and (3) passing the reaction solution prepared from Sb2 through a G-25 gel column, and adjusting the concentration and the pH value to obtain the Sb 2-containing aqueous solution.
7. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the magnetic particle suspension coated with the fluorescein antibody is prepared by the following steps: under the environment of a coupling agent carbodiimide, uniformly mixing magnetic particles containing carboxyl active groups with a fluorescein antibody, reacting at room temperature for 10-20 h, carrying out magnetic separation, removing supernatant, and adjusting concentration and pH to obtain the fluorescent nanoparticle; the magnetic particles have superparamagnetism, and the content of carboxyl active groups carried on each gram of the magnetic particles is not less than 0.5 mml; the fluorescein antibody is a monoclonal antibody, and the dilution titer is more than 1:100 ten thousand.
8. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the luminescent substrate is one or more of Lumi-pHos480, adamantane, luminol and derivatives thereof, isoluminol and derivatives thereof, and acridine ester.
9. The pregnancy related protein a magnetic particle chemiluminescence detection kit of claim 1, wherein: the solution of the luminescent substrate is prepared by the operation comprising the following steps: 2g of TRIS, 6g of NaCl and 0.005g of Na were accurately weighed2SO3And 0.5ml ProClin-300 are respectively added into 600ml purified water and fully dissolved, and the pH value is adjusted to 7.5; adding 200ml luminescent substrate, filtering with 0.2 μm filter, and adding purified water to reach volume of 1000 ml.
10. The use of the pregnancy-associated protein a magnetic particle chemiluminescence detection kit according to any one of claims 1 to 9, wherein: the method comprises the following steps:
sc 1: immune reaction: adding ｃA sample to be detected into ｃA reaction tube, sequentially adding the solution of the fluorescein-labeled PAPP-A antibody and the solution of the alkaline phosphatase-labeled PAPP-A antibody, uniformly mixing, and incubating at 20-40 ℃ for 30min to prepare ｃA solution of the double-antigen sandwich complex;
adding the magnetic particle suspension coated with the fluorescein antibody into the solution of the double-antigen sandwich compound, uniformly mixing, incubating for 30min at 20-40 ℃, and fixing the double-antigen sandwich compound on magnetic microspheres;
sc 2: washing: adding a cleaning solution into the solution prepared by Sc1, oscillating to enable the magnetic particles to settle in a magnetic field, removing the supernatant and removing the antigen of the connection labeled tracer which does not form a double-antigen sandwich compound through the antibody to be detected; repeating the operation for 0-5 times;
sc 3: and (3) adding a substrate for detection: and adding the solution of the luminescent substrate catalyzed by alkaline phosphatase into the reaction tube, fully and uniformly mixing, detecting a luminescent value, and calculating to obtain the content of the pregnancy-associated protein A.
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