CN111175487A - Composite prostate specific antigen kit and using method thereof - Google Patents
Composite prostate specific antigen kit and using method thereof Download PDFInfo
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- CN111175487A CN111175487A CN202010124957.2A CN202010124957A CN111175487A CN 111175487 A CN111175487 A CN 111175487A CN 202010124957 A CN202010124957 A CN 202010124957A CN 111175487 A CN111175487 A CN 111175487A
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- 108010072866 Prostate-Specific Antigen Proteins 0.000 title claims abstract description 47
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- 238000000034 method Methods 0.000 title claims abstract description 14
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- 102100038358 Prostate-specific antigen Human genes 0.000 claims abstract description 46
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 41
- 239000006249 magnetic particle Substances 0.000 claims abstract description 28
- 239000000758 substrate Substances 0.000 claims abstract description 20
- 238000004140 cleaning Methods 0.000 claims abstract description 19
- 238000003908 quality control method Methods 0.000 claims abstract description 16
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- 239000000427 antigen Substances 0.000 claims abstract description 11
- 102000036639 antigens Human genes 0.000 claims abstract description 11
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 claims abstract description 7
- 241000283707 Capra Species 0.000 claims abstract description 6
- 239000008213 purified water Substances 0.000 claims abstract description 6
- 238000005303 weighing Methods 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000007983 Tris buffer Substances 0.000 claims description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 12
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- 238000003756 stirring Methods 0.000 claims description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- PGBHMTALBVVCIT-VCIWKGPPSA-N framycetin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O2)N)O[C@@H]1CO PGBHMTALBVVCIT-VCIWKGPPSA-N 0.000 claims description 10
- 229940053050 neomycin sulfate Drugs 0.000 claims description 10
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 10
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- 244000309466 calf Species 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 5
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- YLGXILFCIXHCMC-JHGZEJCSSA-N methyl cellulose Chemical compound COC1C(OC)C(OC)C(COC)O[C@H]1O[C@H]1C(OC)C(OC)C(OC)OC1COC YLGXILFCIXHCMC-JHGZEJCSSA-N 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 235000010265 sodium sulphite Nutrition 0.000 claims description 5
- 239000011592 zinc chloride Substances 0.000 claims description 5
- 235000005074 zinc chloride Nutrition 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 239000007788 liquid Substances 0.000 abstract description 7
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- 206010060862 Prostate cancer Diseases 0.000 description 2
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- 238000001574 biopsy Methods 0.000 description 2
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- 101000856199 Homo sapiens Chymotrypsin-like protease CTRL-1 Proteins 0.000 description 1
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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
- G01N33/531—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
- G01N33/543—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
-
- 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 composite prostate specific antigen kit and a using method thereof in the technical field related to medical detection reagents, and the kit comprises a c-PSA calibrator, a c-PSA quality control product, c-PSA pretreatment liquid, a c-PSA anti-reagent, a magnetic particle reagent, chemiluminescent substrate liquid and cleaning liquid, wherein the c-PSA calibrator is prepared by fully mixing c-PSA antigen with a first buffer solution; the c-PSA pretreatment solution is prepared by fully mixing free prostate specific antigen antibody and a second buffer solution; c-PSA anti-reagent is prepared by adding the PSA monoclonal antibody marked by alkaline phosphatase and the PSA monoclonal antibody marked by fluorescein isothiocyanate into a second buffer solution and fully mixing; the magnetic particle reagent is prepared by diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles through a third buffer solution; diluting APLS in a fourth buffer solution in a dark place by using the chemiluminescence substrate solution, and fully mixing to prepare the APLS; the cleaning solution is prepared by diluting a fifth buffer solution by purified water; sensitive result and convenient use.
Description
Technical Field
The invention relates to the technical field related to medical detection reagents, in particular to a composite prostate specific antigen kit and a using method thereof.
Background
Prostate Specific Antigen (PSA), a tissue-specific chymotrypsin-like serine protease synthesized by prostate epithelial cells and secreted into seminal fluid, PSA is present in serum mainly in both free (F-PSA) and bound (c-PSA) forms, the latter including ACT-PSA and α 2M-PSA (without immunoreactivity), so clinically detected T-PSA is usually F-PSA and ACT-PSA.
prostate antigen is a tumor marker with higher clinical value for prostate cancer monitoring and evaluation, while PSA is an organ-specific antigen, but not prostate cancer specific, BPH and PC PSA levels have a larger overlap at 4-10ng/ml, so it is difficult to distinguish them according to PSA level.
Due to the ratio error caused by low F-PSA content and unstable test, most BPH patients need to carry out biopsy to avoid missed detection or misdiagnosis of PC patients, so that the operation is inconvenient and the patients are easy to suffer.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a composite prostate specific antigen kit and a using method thereof.
The invention provides a method for detecting the introduction of a composite prostate specific antigen kit, which is developed on the basis of a magnetic particle chemiluminescence immunoassay platform, wherein free prostate specific antigens in a substance to be detected are removed through immunoreaction between antigen and antibodies, then the composite prostate specific antigens in the substance to be detected are combined by the specific antibodies, the combined substances are separated under the condition of a magnetic field after being combined with a magnetic particle reagent, and the luminous intensity of the combined substances is detected in a chemiluminescence detector through enzymatic luminous reaction. And calculating the content of the composite prostate specific antigen corresponding to the luminous intensity through a concentration and luminous intensity dose curve.
A composite prostate specific antigen kit comprises a c-PSA calibrator, a c-PSA quality control material, a c-PSA pretreatment solution, a c-PSA anti-reagent, a magnetic particle reagent, a chemiluminescence substrate solution and a cleaning solution, wherein the c-PSA calibrator, the c-PSA quality control material, the c-PSA pretreatment solution, the c-PSA anti-reagent, the magnetic particle reagent, the chemiluminescence substrate solution and the cleaning solution are packaged independently;
the c-PSA calibrator is prepared by fully mixing a c-PSA antigen and a first buffer solution;
the c-PSA pretreatment solution is prepared by fully mixing free prostate specific antigen antibody and a second buffer solution;
the c-PSA anti-reagent is prepared by adding an alkaline phosphatase-labeled PSA monoclonal antibody and a fluorescein isothiocyanate-labeled PSA monoclonal antibody into the second buffer solution and fully mixing;
the magnetic particle reagent is prepared by diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles through a third buffer solution;
the chemiluminescent substrate solution is prepared by diluting APLS in a dark place through a fourth buffer solution and fully mixing;
the cleaning solution is prepared by diluting a fifth buffer solution with purified water.
Further, the first buffer solution is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 0.3-0.4 part of monopotassium phosphate, 1.5-2.5 parts of dipotassium phosphate, 0.5-1.5 parts of potassium chloride, 0.5-1.5 parts of neomycin sulfate and 190-210 parts of glycerol, respectively adding 630-650 parts by weight of newborn calf serum, stirring at room temperature for 50-70 min, adjusting the pH to 7.5 +/-0.05, and then adding pure water until the weight of the solution is 1000 parts by weight.
Further, the second buffer solution is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 12-12.22 parts of TRIS, 5.6-6 parts of sodium chloride, 13-13.72 parts of zinc chloride, 0.1-0.3 part of magnesium chloride, 1.5-2.5 parts of animal serum and 4-6 parts of BSA, respectively adding the raw materials into 680-720 parts of pure water by weight, stirring the mixture at room temperature for 16-20 hours, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
Further, the third buffer solution is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 6-6.04 parts of TRIS, 4.95-4.99 parts of methyl cellulose ether, 0.9-1.1 parts of Tween-20, 0.9-1.1 parts of neomycin sulfate and 4.7-5.3 parts of BSA, respectively adding the mixture into 680-720 parts by weight of pure water, stirring the mixture at room temperature for 50-70 min, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
Further, the fourth buffer solution is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 36-36.6 parts of TRIS, 0.008-0.012 part of sodium sulfite, 0.8-1.2 parts of SDS, 3-3.6 parts of lucigenin and 0.2-0.4 part of Tween-20, respectively adding into 790-810 parts of pure water, fully dissolving, and adjusting the pH value to 8.8 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
Further, the fifth buffer is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 12-12.22 parts of TRIS, 312.23-312.63 parts of sodium chloride, 27.54-27.94 parts of Tween-20, 0.9-1.1 parts of Bronidox and 0.9-1.1 parts of TritonX-100, respectively adding 680-720 parts by weight of pure water, standing at room temperature for 16-20 h, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
A method of using the composite prostate specific antigen kit of any one of the preceding claims, comprising the steps of:
s1: taking 3 reaction tubes, and respectively adding 30 mu l of the c-PSA calibrator, 30 mu l of the c-PSA quality control material or 30 mu l of a sample to be detected;
s2: respectively adding 60 mu l of the c-PSA pretreatment solution into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5-15 min;
s3: respectively adding 60 mu l of the c-PSA anti-reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5-15 min;
s4: respectively adding 30 mul of the magnetic particle reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5 min;
s5: standing the 3 reaction tubes treated by the S4 for 2-3 min under the action of a magnetic field, and removing supernatant;
s5: respectively adding 200-300 mul of the cleaning solution into 3 reaction tubes, uniformly mixing, standing for 2-3 min under the action of a magnetic field, and removing supernatant;
s6: and respectively adding 150-200 mu l of the chemiluminescent substrate solution into 3 reaction tubes, uniformly mixing, and detecting the fluorescence value by using a chemiluminescence instrument.
Further, the operation of the step S5 is repeated 2-3 times.
The invention has the following advantages: the invention aims at the problems of low specificity, low F-PSA content and unstable test of T-PSA in differential diagnosis of PC and BPH in the existing clinical detection, and is developed and designed on the basis of the existing magnetic particle chemiluminescence immunoassay; according to the invention, the content of c-PSA in the sample is directly detected by removing F-PSA in the sample; the c-PSA has obvious specificity in PC and BPH samples, and the content of the c-PSA in serum is relatively high and stable, the kit prepared by the test method improves the specificity of clinical diagnosis of PC and BPH, reduces unnecessary biopsy tests of BPH patients, and simultaneously reduces the omission and misdiagnosis of PC.
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: a calibrator concentration fluorescence value curve graph;
FIG. 2: the invention is compared with the imported factory kit clinically.
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 description of the present invention, the terms "first" and "second" in the technical solutions are only used to distinguish corresponding structures having the same or similar structures or having similar functions, and do not refer to the arrangement of the importance of the structures, nor do they have any ordering, comparison of sizes, or other meanings.
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.
A composite prostate specific antigen kit comprises a c-PSA calibrator, a c-PSA quality control product, c-PSA pretreatment liquid, a c-PSA anti-reagent, a magnetic particle reagent, chemiluminescence substrate liquid and cleaning liquid; the preparation method is shown in the following examples:
example 1
The c-PSA calibrator is fully mixed with the first buffer solution through the c-PSA antigen to prepare A-F with concentration points of 0, 1.5, 3, 10, 30 and 100ng/ml, and the label is stored at 2 ℃ for 15 months;
wherein the first buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 0.3 part of monopotassium phosphate, 1.5 parts of dipotassium phosphate, 0.5 part of potassium chloride, 0.5 part of neomycin sulfate and 190 parts of glycerol are respectively added into 630 parts by weight of newborn calf serum, stirred for 50min at room temperature, the pH is adjusted to 7.45, and then pure water is added until the weight of the solution is 1000 parts by weight;
the preparation method of the c-PSA quality control product is the same as that of the c-PSA calibrator, and the preparation concentrations are 3.5 and 30 ng/ml;
the c-PSA pretreatment solution is fully mixed with a second buffer solution through a free prostate specific antigen antibody to prepare 0.5 mu g/ml, the preparation concentration can be properly adjusted according to the titer of the antibody, and the F point is ensured to be increased along with the concentration of the antibody during testing, and the fluorescence value is not changed; mixing, filtering, labeling, and storing at 2 deg.C for 15 months;
wherein the second buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: adding 12 parts of TRIS, 5.6 parts of sodium chloride, 13 parts of zinc chloride, 0.1 part of magnesium chloride, 1.5 parts of animal serum and 4 parts of BSA (bovine serum albumin) into 680 parts by weight of pure water respectively, stirring at room temperature for 16 hours, and adjusting the pH to 7.95; adding pure water until the total weight of the solution is 1000 parts by weight;
c-PSA anti-reagent is respectively added to a second buffer solution through an Alkaline Phosphatase (AP) -labeled PSA monoclonal antibody and a Fluorescein Isothiocyanate (FITC) -labeled PSA monoclonal antibody, the concentration of the prepared two solutions is 0.5 mu g/ml, and the F point luminescence value is ensured to be about 1500 ten thousand; then mixing the two solutions in equal volume; labeling, keeping away from light at 2 deg.C, and preserving for 15 months;
diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles by using a third buffer solution by using the magnetic particle reagent to prepare 1.0 mu g/ml; after fully and uniformly mixing, the label is stored at 2 ℃ and the effective period is 18 months;
wherein the third buffer is prepared by the following steps: accurately weighing the following components in parts by weight: respectively adding 6 parts of TRIS, 4.95 parts of methyl cellulose ether, 0.9 part of Tween-20, 0.9 part of neomycin sulfate and 4.7 parts of BSA (bovine serum albumin), adding 680 parts by weight of pure water, stirring at room temperature for 50min, and adjusting the pH to 7.95; adding pure water until the total weight of the solution is 1000 parts by weight;
diluting APLS in a fourth buffer solution in a dark place by using the chemiluminescence substrate solution, and fully mixing to prepare the solution with the concentration of 120 mg/L; mixing, filtering, labeling, storing at 2 deg.C in dark for 15 months;
wherein the fourth buffer is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 36 parts of TRIS, 0.008 part of sodium sulfite, 0.8 part of SDS, 3 parts of lucigenin and 0.2 part of Tween-20, respectively adding into 790 parts of pure water by weight, fully dissolving, and adjusting the pH value to 8.75; adding pure water until the total weight of the solution is 1000 parts by weight;
the cleaning solution dilutes the fifth buffer solution by 15 times through purified water; wherein the fifth buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 12 parts of TRIS, 312.23 parts of sodium chloride, 27.54 parts of Tween-20, 0.9 part of Bronidox and 0.9 part of TritonX-100, respectively adding 680 parts of pure water, standing at room temperature for 16h, and adjusting the pH value to 7.95; adding pure water until the total weight of the solution is 1000 parts by weight;
and (3) independently packaging the prepared c-PSA calibrator, c-PSA quality control product, c-PSA pretreatment solution, c-PSA anti-reagent, magnetic particle reagent, chemiluminescence substrate solution and cleaning solution to form the composite prostate specific antigen kit.
Example 2
The c-PSA calibrator is fully mixed with the first buffer solution through the c-PSA antigen to prepare A-F with concentration points of 0, 1.5, 3, 10, 30 and 100ng/ml, and the label is stored at 4 ℃ for 15 months;
wherein the first buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 0.35 part of monopotassium phosphate, 2 parts of dipotassium phosphate, 1 part of potassium chloride, 1 part of neomycin sulfate and 200 parts of glycerol are respectively added into 640 parts by weight of newborn calf serum, stirred for 60min at room temperature, the pH is adjusted to 7.5, and then pure water is added until the weight of the solution is 1000 parts by weight;
the preparation method of the c-PSA quality control product is the same as that of the c-PSA calibrator, and the preparation concentrations are 3.5 and 30 ng/ml;
the c-PSA pretreatment solution is fully mixed with a second buffer solution through a free prostate specific antigen antibody to prepare 0.8 mu g/ml, the preparation concentration can be properly adjusted according to the titer of the antibody, and the F point is ensured to be increased along with the concentration of the antibody during testing, and the fluorescence value is not changed; mixing, filtering, labeling, and storing at 4 deg.C for 15 months;
wherein the second buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: adding 12.11 parts of TRIS, 5.8 parts of sodium chloride, 13.36 parts of zinc chloride, 0.2 part of magnesium chloride, 2 parts of animal serum and 5 parts of BSA (bovine serum albumin) into 700 parts by weight of pure water respectively, stirring at room temperature for 18 hours, and adjusting the pH to 8.0; adding pure water until the total weight of the solution is 1000 parts by weight;
c-PSA anti-reagent is respectively added to a second buffer solution through an Alkaline Phosphatase (AP) -labeled PSA monoclonal antibody and a Fluorescein Isothiocyanate (FITC) -labeled PSA monoclonal antibody, the concentration of the prepared two solutions is 0.8 mu g/ml, and the F point luminescence value is ensured to be about 1500 ten thousand; then mixing the two solutions in equal volume; labeling, storing at 4 deg.C in dark place for 15 months;
diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles by using a third buffer solution by using the magnetic particle reagent to prepare 1.0 mu g/ml; after fully and uniformly mixing, the paste is stored at 4 ℃ and the effective period is 18 months;
wherein the third buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 6.02 parts of TRIS, 4.97 parts of methyl cellulose ether, 1 part of Tween-20, 1 part of neomycin sulfate and 5 parts of BSA, which are respectively added into 700 parts by weight of pure water, stirred for 60min at room temperature and then adjusted to pH 8.0; adding pure water until the total weight of the solution is 1000 parts by weight;
diluting APLS in a fourth buffer solution in a dark place by using the chemiluminescence substrate solution, and fully mixing to prepare the solution with the concentration of 120 mg/L; mixing, filtering, labeling, storing at 4 deg.C in dark place for 15 months;
wherein the fourth buffer is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 36.3 parts of TRIS, 0.01 part of sodium sulfite, 1 part of SDS, 3.3 parts of lucigenin and 0.3 part of Tween-20 are respectively added into 800 parts of pure water by weight, and the pH is adjusted to 8.8 after the complete dissolution; adding pure water until the total weight of the solution is 1000 parts by weight;
the cleaning solution dilutes the fifth buffer solution by 15 times through purified water; wherein the fifth buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 12.11 parts of TRIS, 312.43 parts of sodium chloride, 27.74 parts of Tween-20, 1 part of Bronidox and 1 part of TritonX-100, respectively adding into 700 parts by weight of pure water, standing at room temperature for 18h, and adjusting the pH value to 8.0; adding pure water until the total weight of the solution is 1000 parts by weight;
and (3) independently packaging the prepared c-PSA calibrator, c-PSA quality control product, c-PSA pretreatment solution, c-PSA anti-reagent, magnetic particle reagent, chemiluminescence substrate solution and cleaning solution to form the composite prostate specific antigen kit.
Example 3
The c-PSA calibrator is fully mixed with the first buffer solution through the c-PSA antigen to prepare A-F with concentration points of 0, 1.5, 3, 10, 30 and 100ng/ml, and the label is stored at 8 ℃ for 15 months;
wherein the first buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 0.4 part of monopotassium phosphate, 2.5 parts of dipotassium phosphate, 1.5 parts of potassium chloride, 1.5 parts of neomycin sulfate and 210 parts of glycerol are respectively added into 650 parts by weight of newborn calf serum, stirred at room temperature for 70min, the pH is adjusted to 7.55, and then pure water is added until the weight of the solution is 1000 parts by weight;
the preparation method of the c-PSA quality control product is the same as that of the c-PSA calibrator, and the preparation concentrations are 3.5 and 30 ng/ml;
the c-PSA pretreatment solution is fully mixed with a second buffer solution through a free prostate specific antigen antibody to prepare 1.0 mu g/ml, the preparation concentration can be properly adjusted according to the titer of the antibody, and the F point is ensured to be increased along with the concentration of the antibody during testing, and the fluorescence value is not changed; mixing, filtering, labeling, and storing at 8 deg.C for 15 months;
wherein the second buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: adding 12.22 parts of TRIS, 6 parts of sodium chloride, 13.72 parts of zinc chloride, 0.3 part of magnesium chloride, 2.5 parts of animal serum and 6 parts of BSA (bovine serum albumin) into 720 parts by weight of pure water respectively, stirring at room temperature for 20 hours, and adjusting the pH to 8.05; adding pure water until the total weight of the solution is 1000 parts by weight;
c-PSA anti-reagent is respectively added to a second buffer solution through an Alkaline Phosphatase (AP) -labeled PSA monoclonal antibody and a Fluorescein Isothiocyanate (FITC) -labeled PSA monoclonal antibody, the concentration of the prepared two solutions is 1.0 mu g/ml, and the F point luminescence value is ensured to be about 1500 ten thousand; then mixing the two solutions in equal volume; labeling, keeping away from light at 8 deg.C, and preserving for 15 months;
diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles by using a third buffer solution by using the magnetic particle reagent to prepare 1.0 mu g/ml; after fully and uniformly mixing, the label is stored at 8 ℃ and the effective period is 18 months;
wherein the third buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 6.04 parts of TRIS, 4.99 parts of methyl cellulose ether, 1.1 parts of Tween-20, 1.1 parts of neomycin sulfate and 5.3 parts of BSA, respectively adding 720 parts by weight of pure water, stirring at room temperature for 70min, and adjusting the pH to 8.05; adding pure water until the total weight of the solution is 1000 parts by weight;
diluting APLS in a fourth buffer solution in a dark place by using the chemiluminescence substrate solution, and fully mixing to prepare the solution with the concentration of 120 mg/L; mixing, filtering, labeling, keeping in dark at 8 deg.C for 15 months;
wherein the fourth buffer is prepared by the operation comprising the following steps: accurately weighing the following components in parts by weight: 36.6 parts of TRIS, 0.012 part of sodium sulfite, 1.2 parts of SDS, 3.6 parts of lucigenin and 0.4 part of Tween-20 are respectively added into 810 parts of pure water by weight, and the pH value is adjusted to 8.85 after the pure water is fully dissolved; adding pure water until the total weight of the solution is 1000 parts by weight;
the cleaning solution dilutes the fifth buffer solution by 15 times through purified water; wherein the fifth buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 12.22 parts of TRIS, 312.63 parts of sodium chloride, 27.94 parts of Tween-20, 1.1 parts of Bronidox and 1.1 parts of TritonX-100, respectively adding 720 parts by weight of pure water, standing at room temperature for 20 hours, and adjusting the pH value to 8.05; adding pure water until the total weight of the solution is 1000 parts by weight;
and (3) independently packaging the prepared c-PSA calibrator, c-PSA quality control product, c-PSA pretreatment solution, c-PSA anti-reagent, magnetic particle reagent, chemiluminescence substrate solution and cleaning solution to form the composite prostate specific antigen kit.
Example 4: the method for using the composite prostate specific antigen kit, that is, the method for detecting with the composite prostate specific antigen kit, includes the following steps (the kit used in this example is prepared by example 2):
uniformly mixing the components of the kit prepared in the embodiment 2 and a sample to be detected for 30min at room temperature;
s1: respectively adding 30 mu l c-PSA calibrator, 30 mu l c-PSA quality control material or 30 mu l of sample to be tested into 3 reaction tubes;
s2: respectively adding 60 mu l c-PSA pretreatment solution into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37 ℃ for 5min, wherein in the specific implementation, the temperature and the time can be randomly selected between 37-44 ℃ for 5-15 min;
s3: respectively adding 60 mu l c-PSA anti-reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37 ℃ for 5min, wherein in the specific implementation, the temperature and the time can be randomly selected between 37-44 ℃ for 5-15 min;
s4: respectively adding 30 mul of magnetic particle reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37 ℃ for 5min, wherein the temperature can be any one of 37-44 ℃;
s5: standing the 3 reaction tubes treated by the S4 for 2min under the action of a magnetic field, and removing supernatant, wherein the specific implementation time can be any time between 2min and 3 min;
s5: respectively adding 200-300 mul of cleaning solution into 3 reaction tubes, uniformly mixing, standing for 2min under the action of a magnetic field, and removing supernatant, wherein the specific implementation time can be any time between 2-3 min; in order to improve the precision, the operation of the step can be repeated for 2-3 times;
s6: and respectively adding 150 mul of chemiluminescent substrate solution into the 3 reaction tubes, uniformly mixing, and detecting the fluorescence value by using a chemiluminescence apparatus, wherein in specific implementation, the added chemiluminescent substrate solution can be randomly selected from 150-200 mul.
And (3) detection results:
1. and (3) detection curve:
the method comprises the following steps of preparing a c-PSA calibrator, a c-PSA quality control product, a c-PSA pretreatment solution, a c-PSA anti-reagent, a magnetic particle reagent, a chemiluminescence substrate solution and a cleaning solution, and drawing a curve by using a four-parameter equation according to the concentration of the c-PSA calibrator and a fluorescence value to be tested, namely obtaining a detection curve. The linearity of the kit between 0.01-100ng/ml is 0.999835, see FIG. 1.
2. And (3) testing the sensitivity:
and (3) carrying out 20 times of repeated tests on the zero calibration point, calculating the average value (M) and the Standard Deviation (SD) of 20 times of measured values, and substituting M +2SD into a curve to obtain a concentration value, namely the sensitivity of the kit. The sensitivity of the detection kit is less than or equal to 0.01 ng/ml.
| Calibrator A | RLU | Calibrator A | RLU |
| Pipe 1 | 40260 | Pipe 11 | 43139 |
| Pipe 2 | 38780 | Tube 12 | 39960 |
| Pipe 3 | 39937 | Pipe 13 | 40920 |
| Pipe 4 | 37772 | Tube 14 | 37135 |
| Pipe 5 | 42210 | Pipe 15 | 41609 |
| Pipe 6 | 41170 | Tube 16 | 41643 |
| Pipe 7 | 42754 | Pipe 17 | 38015 |
| Pipe 8 | 39132 | Tube 18 | 41764 |
| Pipe 9 | 36863 | Tube 19 | 41542 |
| Pipe 10 | 39625 | |
37219 |
| Average value M | 40072 | Standard deviation SD | 1951 |
| M+2SD | 43975 | Minimum limit of detection | 0.01 |
3. And (3) testing precision:
randomly selecting each sample with low and high values as a sample to be tested, repeatedly testing each sample for 10 times, and calculating the average value (M), Standard Deviation (SD) and precision (CV) of the sample. The precision of the detection kit is less than or equal to 5 percent.
4. Specificity test
The test results of the 150ng/ml CEA sample and the 1200IU/ml AFP sample are respectively less than 0.01. The detection kit has no cross reaction to CEA (150ng/ml) and AFP (1200 IU/ml).
5. And (3) clinical trials:
selecting 100 clinical samples with known import kit measuring values, testing by using the detection kit, and comparing with the test results of the import manufacturer kit. The alignment results are shown in FIG. 2.
6. The performance indexes of the kit are as follows:
the detection range of the kit is 0.01-100ng/ml, and the linear curve in the detection range is more than or equal to 0.99;
the sensitivity of the kit is less than or equal to 0.01 ng/ml;
the test precision is not more than 5%;
the correlation with the imported kit was 0.997, and the compliance rate was 100%.
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 (8)
1. A composite prostate specific antigen kit is characterized in that: the kit comprises a c-PSA calibrator, a c-PSA quality control product, a c-PSA pretreatment solution, a c-PSA anti-reagent, a magnetic particle reagent, a chemiluminescent substrate solution and a cleaning solution, wherein the c-PSA calibrator, the c-PSA quality control product, the c-PSA pretreatment solution, the c-PSA anti-reagent, the magnetic particle reagent, the chemiluminescent substrate solution and the cleaning solution are packaged independently;
the c-PSA calibrator is prepared by fully mixing a c-PSA antigen and a first buffer solution;
the c-PSA pretreatment solution is prepared by fully mixing free prostate specific antigen antibody and a second buffer solution;
the c-PSA anti-reagent is prepared by adding an alkaline phosphatase-labeled PSA monoclonal antibody and a fluorescein isothiocyanate-labeled PSA monoclonal antibody into the second buffer solution and fully mixing;
the magnetic particle reagent is prepared by diluting and fully mixing goat anti-fluorescein isothiocyanate antibody-labeled magnetic particles through a third buffer solution;
the chemiluminescent substrate solution is prepared by diluting APLS in a dark place through a fourth buffer solution and fully mixing;
the cleaning solution is prepared by diluting a fifth buffer solution with purified water.
2. The composite prostate-specific antigen kit of claim 1, wherein: the first buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 0.3-0.4 part of monopotassium phosphate, 1.5-2.5 parts of dipotassium phosphate, 0.5-1.5 parts of potassium chloride, 0.5-1.5 parts of neomycin sulfate and 190-210 parts of glycerol, respectively adding 630-650 parts by weight of newborn calf serum, stirring at room temperature for 50-70 min, adjusting the pH to 7.5 +/-0.05, and then adding pure water until the weight of the solution is 1000 parts by weight.
3. The composite prostate-specific antigen kit of claim 1, wherein: the second buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 12-12.22 parts of TRIS, 5.6-6 parts of sodium chloride, 13-13.72 parts of zinc chloride, 0.1-0.3 part of magnesium chloride, 1.5-2.5 parts of animal serum and 4-6 parts of BSA, respectively adding the raw materials into 680-720 parts of pure water by weight, stirring the mixture at room temperature for 16-20 hours, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
4. The composite prostate-specific antigen kit of claim 1, wherein: the third buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 6-6.04 parts of TRIS, 4.95-4.99 parts of methyl cellulose ether, 0.9-1.1 parts of Tween-20, 0.9-1.1 parts of neomycin sulfate and 4.7-5.3 parts of BSA, respectively adding the mixture into 680-720 parts by weight of pure water, stirring the mixture at room temperature for 50-70 min, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
5. The composite prostate-specific antigen kit of claim 1, wherein: the fourth buffer is prepared by the following steps: accurately weighing the following components in parts by weight: 36-36.6 parts of TRIS, 0.008-0.012 part of sodium sulfite, 0.8-1.2 parts of SDS, 3-3.6 parts of lucigenin and 0.2-0.4 part of Tween-20, respectively adding into 790-810 parts of pure water, fully dissolving, and adjusting the pH value to 8.8 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
6. The composite prostate-specific antigen kit of claim 1, wherein: the fifth buffer solution is prepared by the following steps: accurately weighing the following components in parts by weight: 12-12.22 parts of TRIS, 312.23-312.63 parts of sodium chloride, 27.54-27.94 parts of Tween-20, 0.9-1.1 parts of Bronidox and 0.9-1.1 parts of TritonX-100, respectively adding 680-720 parts by weight of pure water, standing at room temperature for 16-20 h, and adjusting the pH value to 8.0 +/-0.05; pure water was added to make the total weight of the solution 1000 parts by weight.
7. A method of using the composite prostate specific antigen kit of any one of claims 1 to 6, wherein: the method comprises the following steps:
s1: taking 3 reaction tubes, and respectively adding 30 mu l of the c-PSA calibrator, 30 mu l of the c-PSA quality control material or 30 mu l of a sample to be detected;
s2: respectively adding 60 mu l of the c-PSA pretreatment solution into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5-15 min;
s3: respectively adding 60 mu l of the c-PSA anti-reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5-15 min;
s4: respectively adding 30 mul of the magnetic particle reagent into 3 reaction tubes, uniformly mixing, and carrying out warm bath at 37-44 ℃ for 5 min;
s5: standing the 3 reaction tubes treated by the S4 for 2-3 min under the action of a magnetic field, and removing supernatant;
s5: respectively adding 200-300 mul of the cleaning solution into 3 reaction tubes, uniformly mixing, standing for 2-3 min under the action of a magnetic field, and removing supernatant;
s6: and respectively adding 150-200 mu l of the chemiluminescent substrate solution into 3 reaction tubes, uniformly mixing, and detecting the fluorescence value by using a chemiluminescence instrument.
8. The method of using the composite prostate specific antigen kit of claim 7, wherein: repeating the operation of the step S5 for 2-3 times.
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| US5840501A (en) * | 1996-10-25 | 1998-11-24 | Bayer Corporation | Determination of cPSA |
| CN105092834A (en) * | 2014-05-05 | 2015-11-25 | 江苏泽成生物技术有限公司 | Carbohydrate antigen 19-9 (CA 19-9) quantitative assay kit, preparation method and detection method thereof |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN109061203A (en) * | 2018-08-07 | 2018-12-21 | 泰州泽成生物技术有限公司 | A kind of human chorionic gonadotrophin detection kit and preparation method thereof and application method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5840501A (en) * | 1996-10-25 | 1998-11-24 | Bayer Corporation | Determination of cPSA |
| CN105092834A (en) * | 2014-05-05 | 2015-11-25 | 江苏泽成生物技术有限公司 | Carbohydrate antigen 19-9 (CA 19-9) quantitative assay kit, preparation method and detection method thereof |
| CN108205059A (en) * | 2017-11-28 | 2018-06-26 | 泰州泽成生物技术有限公司 | A kind of kit and its test method for measuring calcitonin content |
| CN109061203A (en) * | 2018-08-07 | 2018-12-21 | 泰州泽成生物技术有限公司 | A kind of human chorionic gonadotrophin detection kit and preparation method thereof and application method |
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