CN111208290A - Magnetic particle chemiluminescence aldosterone determination kit and use method thereof - Google Patents
Magnetic particle chemiluminescence aldosterone determination kit and use method thereof Download PDFInfo
- Publication number
- CN111208290A CN111208290A CN202010124924.8A CN202010124924A CN111208290A CN 111208290 A CN111208290 A CN 111208290A CN 202010124924 A CN202010124924 A CN 202010124924A CN 111208290 A CN111208290 A CN 111208290A
- Authority
- CN
- China
- Prior art keywords
- aldosterone
- antigen
- magnetic particle
- prepared
- particle chemiluminescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229960002478 aldosterone Drugs 0.000 title claims abstract description 109
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 title claims abstract description 108
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 239000006249 magnetic particle Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 33
- 102000036639 antigens Human genes 0.000 claims abstract description 33
- 108091007433 antigens Proteins 0.000 claims abstract description 33
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 32
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 19
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 19
- 238000003908 quality control method Methods 0.000 claims abstract description 11
- 238000007885 magnetic separation Methods 0.000 claims abstract description 9
- 230000006287 biotinylation Effects 0.000 claims abstract description 4
- 238000007413 biotinylation Methods 0.000 claims abstract description 4
- 238000010790 dilution Methods 0.000 claims abstract description 4
- 239000012895 dilution Substances 0.000 claims abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 48
- 239000007853 buffer solution Substances 0.000 claims description 34
- 238000000502 dialysis Methods 0.000 claims description 25
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 19
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 17
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 17
- 239000003513 alkali Substances 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 16
- 238000007865 diluting Methods 0.000 claims description 15
- 238000003149 assay kit Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 11
- 239000000376 reactant Substances 0.000 claims description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 10
- 230000003213 activating effect Effects 0.000 claims description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims description 10
- 239000003755 preservative agent Substances 0.000 claims description 10
- 230000002335 preservative effect Effects 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 claims description 5
- 101000652736 Homo sapiens Transgelin Proteins 0.000 claims description 5
- 102100031013 Transgelin Human genes 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 5
- 239000007795 chemical reaction product Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- 241001529936 Murinae Species 0.000 claims description 2
- 150000001325 aldosterones Chemical class 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 108090001008 Avidin Proteins 0.000 abstract description 2
- 229960002685 biotin Drugs 0.000 abstract description 2
- 235000020958 biotin Nutrition 0.000 abstract description 2
- 239000011616 biotin Substances 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000003904 radioactive pollution Methods 0.000 abstract description 2
- 210000003722 extracellular fluid Anatomy 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000009103 reabsorption Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108700017442 18-Hydroxylase deficiency Proteins 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- 208000012904 Bartter disease Diseases 0.000 description 1
- 208000010062 Bartter syndrome Diseases 0.000 description 1
- 208000016998 Conn syndrome Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000019025 Hypokalemia Diseases 0.000 description 1
- 206010021137 Hypovolaemia Diseases 0.000 description 1
- 208000026709 Liddle syndrome Diseases 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 229910018830 PO3H Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010037113 Pseudoaldosteronism Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010039808 Secondary aldosteronism Diseases 0.000 description 1
- 208000026928 Turner syndrome Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 208000016106 familial hyperreninemic hypoaldosteronism type 1 Diseases 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005857 malignant hypertension Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 208000024896 potassium deficiency disease Diseases 0.000 description 1
- 208000013846 primary aldosteronism Diseases 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008060 renal absorption Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses the medical apparatus and instruments biology immune in vitro diagnosis related technical field, especially relate to a magnetic particle chemiluminescence method aldosterone determination kit and its operation method; comprises a magnetic separation reagent, a biotinylation aldosterone antibody, an alkaline phosphatase marked aldosterone antigen, a calibrator and a quality control product which are packaged independently; the whole reaction system avoids radioactive pollution, prolongs the effective period of reagents and simplifies the experimental operation process; the combination between the avidin and the biotin has extremely high affinity, the reaction is highly specific, and the nonspecific interference is not increased while the sensitivity is improved; the binding characteristics are not affected by the high dilution of the reagents, so that the non-specific effects of the reagents can be reduced to the maximum extent in practical application.
Description
Technical Field
The invention relates to the technical field related to biological immune in-vitro diagnosis of medical instruments, in particular to a magnetic particle chemiluminescence aldosterone determination kit and a use method thereof.
Background
Aldosterone is a hormone in the human body that regulates blood volume and maintains water balance by regulating the reabsorption of sodium by the kidneys. Aldosterone is a hormone that regulates extracellular fluid volume and electrolytes, and the secretion of aldosterone is accomplished through the aldosterone-angiotensin system. When the extracellular fluid capacity is reduced, the periglomerular cells are stimulated to secrete aldosterone, an aldosterone-angiotensin-aldosterone system is activated, aldosterone secretion is increased, and the reabsorption of sodium by the kidney is increased, so that the reabsorption of water is increased and the extracellular fluid capacity is increased; conversely, when the extracellular fluid volume increases, the secretion of aldosterone decreases, the renal reabsorption of sodium decreases, and the extracellular fluid volume decreases by the reverse mechanism described above. Blood sodium is reduced and blood potassium is increased to stimulate adrenal cortex and increase aldosterone secretion.
Aldosterone increases are mostly seen in: primary aldosteronism, pseudo-aldosteronism (bilateral adrenal bulbar hyperplasia), secondary aldosteronism caused by diuretic, heart failure, liver cirrhosis, renal failure, nephrotic syndrome, etc., primary periodic edema, Bartter's syndrome, proliferation of perirenal bulb, postoperative hypovolemia, hypokalemia caused by various reasons, partial malignant hypertension, and progressive hypertension.
Aldosterone reduction is seen in: hypoadrenocortical function, hypoaldosterone-hypoaldosterone syndrome, 18-hydroxylase deficiency, diabetes, Turner's syndrome, acute alcoholism, and the like.
Therefore, the development of an aldosterone detection product with simple operation and high reaction sensitivity plays an important role in the prior art.
Disclosure of Invention
Aiming at the technical problem that the prior art needs an aldosterone detection product with simple operation and high reaction sensitivity, the invention provides a magnetic particle chemiluminescence method aldosterone determination kit and a use method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a magnetic particle chemiluminescence method aldosterone assay kit comprises a magnetic separation reagent, a biotinylation aldosterone antibody, an alkaline phosphatase labeled aldosterone antigen, a calibrator and a quality control product which are packaged independently;
A. the biotinylated aldosterone antibody is prepared by the operation comprising the following steps:
sa 1: accurately weighing 0.3-0.5 mg of biotin-NHS, and dissolving the biotin-NHS in DMSO until the final concentration is 4.5-5.5 mg/ml to prepare a DMSO solution of the biotin-NHS;
sa 2: accurately weighing 0.9-1.1 mg of aldosterone antibody, adding the aldosterone antibody into the DMSO solution of biotin-NHS, fully and uniformly mixing, and reacting at room temperature for 1.5-2.5 h;
sa 3: performing dialysis treatment on the reaction product in Sa2 by using a dialysis bag;
sa 4: diluting the mixture prepared by Sa3 to 0.45-0.55 mu g/ml by using an anti-reagent buffer solution to obtain the reagent;
the anti-reagent buffer solution is a PBS system with the pH value of 5-6; the anti-reagent buffer solution comprises purified water 1L, Na2PO3H·12H2O10～20g、NaPO3H2·12H2O1-2 g, sheep serum 1-5 g, newborn bovine serum 3-10 g and horse serum 1-5 g;
B. the alkaline phosphatase labeled aldosterone antigen is prepared by the following operation steps:
sb 1: carrying out dialysis treatment on the detected aldosterone antigen and then activating the aldosterone antigen by using 2-IT;
sb 2: activating alkali phosphatase by using SMCC;
sb 3: mixing the activated aldosterone detection antigen and the alkaline phosphatase, and reacting at 2-8 ℃ for 16-20 h;
sb 4: purifying a reactant prepared from Sb3 by using a chromatographic column, collecting a peak I and a peak II, and mixing to prepare antigen-alkali phosphatase;
sb 5: diluting the antigen-alkali phosphatase to 0.09-0.11 mu g/ml by using an anti-reagent buffer solution to obtain the antigen-alkali phosphatase;
C. the calibration material and the quality control material have the same components and are prepared by diluting the antigen to be detected with aldosterone calibration material buffer solution.
Preferably, the aldosterone antibodies are murine monoclonal antibodies.
Preferably, the molar ratio of the aldosterone antibody to the biotin-NHS is 1: 20; wherein the molecular weight of the aldosterone antibody is calculated as 1500000, and the molecular weight of the biotin-NHS is calculated as 587.
Preferably, the dialysis bag described in Sa3 has a molecular weight cut-off of 500.
Preferably, the dialysis buffer used for the dialysis treatment in Sa3 is 0.15M PBS buffer with pH 7.5.
Preferably, the dialysis buffer used for the dialysis treatment in Sb1 is 0.1M PBS buffer.
Preferably, the aldosterone calibrator buffer is prepared by adding bovine serum albumin and a preservative to 0.05M TRIS buffer with PH of 7.4, wherein the volume percentage of the bovine serum albumin in the aldosterone calibrator buffer is 0.5%, and the volume percentage of the preservative is 0.2%.
Preferably, the final concentration of the dilution of the antigen to be tested is 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000 pg/ml.
Preferably, the magnetic separation reagent is streptavidin magnetic beads.
A method for using the magnetic particle chemiluminescence method aldosterone assay kit of any one of the above, comprising the following steps:
sd 1: accurately weighing 30 mu l of sample to be detected, 30 mu l of biotinylated aldosterone antibody and 30 mu l of alkaline phosphatase labeled aldosterone antigen, fully mixing and incubating for 15 min;
sd 2: adding 30 mu l of the magnetic separation reagent into the reactant prepared by Sd1, fully mixing and incubating for 5 min;
sd 3: magnetically separating the reactant prepared from Sd2 for 2min, removing the supernatant and retaining the substrate;
sd 4: the substrate was washed 3 times with 300. mu.l of wash solution each time;
sd 5: the substrate was diluted with 200. mu.l of the aldosterone calibrator buffer and measured.
The invention has the following advantages:
1. the whole reaction system avoids radioactive pollution, prolongs the effective period of reagents and simplifies the experimental operation process;
2. the combination between the avidin and the biotin has extremely high affinity, the reaction is highly specific, and the nonspecific interference is not increased while the sensitivity is improved;
3. the binding characteristics are not affected by the high dilution of the reagents, so that the non-specific effects of the reagents can be reduced to the maximum extent in practical application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only 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 system 1 curve fit;
figure 2 is a system 2 curve fit.
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
A kit for measuring aldosterone by a magnetic particle chemiluminescence method comprises streptavidin magnetic beads, a biotinylated aldosterone antibody, an alkaline phosphatase labeled aldosterone antigen, a calibrator and a quality control product which are packaged independently;
A. the biotinylated aldosterone antibody is prepared by an operation comprising the steps of:
sa 1: accurately weighing 0.3mg of biotin-NHS and dissolving the biotin-NHS in DMSO until the final concentration is 4.5mg/ml to prepare a DMSO solution of the biotin-NHS;
sa 2: accurately weighing 0.9mg of mouse monoclonal aldosterone antibody and adding the mouse monoclonal aldosterone antibody into a DMSO solution of biotin-NHS, wherein the mouse monoclonal aldosterone antibody and the DMSO solution are reacted at room temperature for 1.5h after being fully and uniformly mixed;
sa 3: dialyzing the reaction product in Sa2 with a dialysis bag with a molecular weight cut-off of 500 and a dialysis buffer solution of 0.15M PBS buffer solution with pH 7.5;
sa 4: diluting the mixture prepared by Sa3 to 0.45 mu g/ml by using an anti-reagent buffer solution to obtain the reagent;
B. the alkaline phosphatase labeled aldosterone antigen is prepared by the following operation steps:
sb 1: dialyzing the antigen for detecting aldosterone by using 0.1M PBS buffer solution as dialysis buffer solution, and activating by using 2-IT;
sb 2: activating alkali phosphatase by using SMCC;
sb 3: mixing the activated detection aldosterone antigen and alkaline phosphatase, and reacting for 16h at 2 ℃;
sb 4: purifying a reactant prepared from Sb3 by using a chromatographic column, collecting a peak I and a peak II, and mixing to prepare antigen-alkali phosphatase;
sb 5: diluting antigen-alkali phosphatase to 0.09 μ g/ml with anti-reagent buffer solution;
C. the components of the calibrator and the quality control product are the same, and the final concentration of the antigen to be detected is 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000pg/ml by diluting with aldosterone calibrator buffer;
the aldosterone calibrator buffer solution is prepared by adding bovine serum albumin and a preservative into 0.05M TRIS buffer solution with pH of 7.4, wherein the volume percentage of the bovine serum albumin in the aldosterone calibrator buffer solution is 0.5%, and the volume percentage of the preservative is 0.2%.
Example 2
A kit for measuring aldosterone by a magnetic particle chemiluminescence method comprises streptavidin magnetic beads, a biotinylated aldosterone antibody, an alkaline phosphatase labeled aldosterone antigen, a calibrator and a quality control product which are packaged independently;
A. the biotinylated aldosterone antibody is prepared by an operation comprising the steps of:
sa 1: accurately weighing 0.4mg of biotin-NHS and dissolving the biotin-NHS in DMSO until the final concentration is 5mg/ml to prepare a DMSO solution of the biotin-NHS;
sa 2: accurately weighing 1mg of mouse monoclonal aldosterone antibody and adding the antibody into a DMSO solution of biotin-NHS, wherein the molar ratio of the aldosterone antibody to the biotin-NHS is 1: 20, fully and uniformly mixing, and reacting at room temperature for 2 hours;
sa 3: dialyzing the reaction product in Sa2 with a dialysis bag with a molecular weight cut-off of 500 and a dialysis buffer solution of 0.15M PBS buffer solution with pH 7.5;
sa 4: diluting the mixture prepared by Sa3 to 0.5 mu g/ml by using an anti-reagent buffer solution to obtain the reagent;
B. the alkaline phosphatase labeled aldosterone antigen is prepared by the following operation steps:
sb 1: dialyzing the antigen for detecting aldosterone by using 0.1M PBS buffer solution as dialysis buffer solution, and activating by using 2-IT;
sb 2: activating alkali phosphatase by using SMCC;
sb 3: mixing the activated detection aldosterone antigen and alkaline phosphatase, and reacting at 4 ℃ for 18 h;
sb 4: purifying a reactant prepared from Sb3 by using a chromatographic column, collecting a peak I and a peak II, and mixing to prepare antigen-alkali phosphatase;
sb 5: diluting antigen-alkali phosphatase to 1 μ g/ml with anti-reagent buffer solution;
C. the components of the calibrator and the quality control product are the same, and the final concentration of the antigen to be detected is 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000pg/ml by diluting with aldosterone calibrator buffer;
the aldosterone calibrator buffer solution is prepared by adding bovine serum albumin and a preservative into 0.05M TRIS buffer solution with pH of 7.4, wherein the volume percentage of the bovine serum albumin in the aldosterone calibrator buffer solution is 0.5%, and the volume percentage of the preservative is 0.2%.
Example 3
A magnetic particle chemiluminescence method aldosterone assay kit comprises a magnetic separation reagent, a biotinylation aldosterone antibody, an alkaline phosphatase labeled aldosterone antigen, a calibrator and a quality control product which are packaged independently;
A. the biotinylated aldosterone antibody is prepared by an operation comprising the steps of:
sa 1: accurately weighing 0.5mg of biotin-NHS and dissolving the biotin-NHS in DMSO until the final concentration is 5.5mg/ml to prepare a DMSO solution of the biotin-NHS;
sa 2: accurately weighing 1.1mg of aldosterone antibody and adding the aldosterone antibody into a DMSO solution of biotin-NHS, wherein the aldosterone antibody is reacted for 2.5 hours at room temperature after being fully and uniformly mixed;
sa 3: dialyzing the reaction product in Sa2 with a dialysis bag with a molecular weight cut-off of 500 and a dialysis buffer solution of 0.15M PBS buffer solution with pH 7.5;
sa 4: diluting the mixture prepared by Sa3 to 0.55 mu g/ml by using an anti-reagent buffer solution to obtain the reagent;
B. the alkaline phosphatase labeled aldosterone antigen is prepared by the following operation steps:
sb 1: dialyzing the antigen for detecting aldosterone by using 0.1M PBS buffer solution as dialysis buffer solution, and activating by using 2-IT;
sb 2: activating alkali phosphatase by using SMCC;
sb 3: mixing the activated detection aldosterone antigen and alkaline phosphatase, and reacting at 8 ℃ for 20 h;
sb 4: purifying a reactant prepared from Sb3 by using a chromatographic column, collecting a peak I and a peak II, and mixing to prepare antigen-alkali phosphatase;
sb 5: diluting antigen-alkali phosphatase to 0.11 μ g/ml with anti-reagent buffer solution;
C. the components of the calibrator and the quality control product are the same, and the final concentration of the antigen to be detected is 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000pg/ml by diluting with aldosterone calibrator buffer;
the aldosterone calibrator buffer solution is prepared by adding bovine serum albumin and a preservative into 0.05M TRIS buffer solution with pH of 7.4, wherein the volume percentage of the bovine serum albumin in the aldosterone calibrator buffer solution is 0.5%, and the volume percentage of the preservative is 0.2%.
Example 4
The use method of the magnetic particle chemiluminescence method aldosterone measuring kit (taking the product prepared in example 2 as an example) comprises the following steps:
sd 1: accurately weighing 30 mu l of sample to be detected, 30 mu l of biotinylated aldosterone antibody and 30 mu l of alkaline phosphatase labeled aldosterone antigen, fully mixing and incubating for 15 min;
sd 2: adding 30 mu l of magnetic separation reagent into the reactant prepared from Sd1, fully mixing and incubating for 5 min;
sd 3: magnetically separating the reactant prepared from Sd2 for 2min, removing the supernatant and retaining the substrate;
sd 4: the substrate was washed 3 times with 300. mu.l of wash solution each time;
sd 5: substrate was diluted with 200. mu.l aldosterone calibrator buffer and measured.
The invention simultaneously uses biotinylated aldosterone monoclonal antibody-streptavidin magnetic bead (system 1) and carboxylated magnetic particles (system 2) coated by the aldosterone monoclonal antibody to respectively detect aldosterone samples of 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000pg/ml by using two systems, simultaneously test the same group of samples and compare the samples with given values, and the data are shown in the following table:
TABLE 1 comparison of measured values with given values and comparison between measured values for different systems
The curve fits for system 1 and system 2 are shown in fig. 1 and fig. 2, respectively. From the results, it was found that the correlation between the measured values of the different systems and the given value was 0.9 or more, indicating that the correlation was good, and that the correlation between the measured values of the different systems was more than 0.9, indicating that the measured values of the 2 systems were the same.
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. A magnetic particle chemiluminescence method aldosterone assay kit is characterized in that: comprises a magnetic separation reagent, a biotinylation aldosterone antibody, an alkaline phosphatase marked aldosterone antigen, a calibrator and a quality control product which are packaged independently;
A. the biotinylated aldosterone antibody is prepared by the operation comprising the following steps:
sa 1: accurately weighing 0.3-0.5 mg of biotin-NHS, and dissolving the biotin-NHS in DMSO until the final concentration is 4.5-5.5 mg/ml to prepare a DMSO solution of the biotin-NHS;
sa 2: accurately weighing 0.9-1.1 mg of aldosterone antibody, adding the aldosterone antibody into the DMSO solution of biotin-NHS, fully and uniformly mixing, and reacting at room temperature for 1.5-2.5 h;
sa 3: performing dialysis treatment on the reaction product in Sa2 by using a dialysis bag;
sa 4: diluting the mixture prepared by Sa3 to 0.45-0.55 mu g/ml by using an anti-reagent buffer solution to obtain the reagent;
B. the alkaline phosphatase labeled aldosterone antigen is prepared by the following operation steps:
sb 1: carrying out dialysis treatment on the detected aldosterone antigen and then activating the aldosterone antigen by using 2-IT;
sb 2: activating alkali phosphatase by using SMCC;
sb 3: mixing the activated aldosterone detection antigen and the alkaline phosphatase, and reacting at 2-8 ℃ for 16-20 h;
sb 4: purifying a reactant prepared from Sb3 by using a chromatographic column, collecting a peak I and a peak II, and mixing to prepare antigen-alkali phosphatase;
sb 5: diluting the antigen-alkali phosphatase to 0.09-0.11 mu g/ml by using an anti-reagent buffer solution to obtain the antigen-alkali phosphatase;
C. the calibration material and the quality control material have the same components and are prepared by diluting the antigen to be detected with aldosterone calibration material buffer solution.
2. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the aldosterone antibodies are murine monoclonal antibodies.
3. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the molar ratio of the aldosterone antibody to the biotin-NHS is 1: 20.
4. the magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the cut-off molecular weight of the dialysis bag in Sa3 was 500.
5. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the dialysis buffer used for the dialysis treatment in Sa3 was 0.15M PBS buffer with pH 7.5.
6. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the dialysis buffer used for the dialysis treatment in Sb1 was 0.1M PBS buffer.
7. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the aldosterone calibrator buffer solution is prepared by adding bovine serum albumin and a preservative into 0.05M TRIS buffer solution with pH of 7.4, wherein the volume percentage of the bovine serum albumin in the aldosterone calibrator buffer solution is 0.5%, and the volume percentage of the preservative is 0.2%.
8. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the final concentrations of the dilutions of the antigen to be tested were 0pg/ml, 50pg/ml, 250pg/ml, 500pg/ml, 1000pg/ml and 2000 pg/ml.
9. The magnetic particle chemiluminescence aldosterone assay kit of claim 1, wherein: the magnetic separation reagent is streptavidin magnetic beads.
10. A method of using the magnetic particle chemiluminescence aldosterone assay kit of any one of claims 1 to 9, wherein: the method comprises the following steps:
sd 1: accurately weighing 30 mu l of sample to be detected, 30 mu l of biotinylated aldosterone antibody and 30 mu l of alkaline phosphatase labeled aldosterone antigen, fully mixing and incubating for 15 min;
sd 2: adding 30 mu l of the magnetic separation reagent into the reactant prepared by Sd1, fully mixing and incubating for 5 min;
sd 3: magnetically separating the reactant prepared from Sd2 for 2min, removing the supernatant and retaining the substrate;
sd 4: the substrate was washed 3 times with 300. mu.l of wash solution each time;
sd 5: the substrate was diluted with 200. mu.l of the aldosterone calibrator buffer and measured.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010124924.8A CN111208290A (en) | 2020-02-27 | 2020-02-27 | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010124924.8A CN111208290A (en) | 2020-02-27 | 2020-02-27 | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111208290A true CN111208290A (en) | 2020-05-29 |
Family
ID=70785613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010124924.8A Pending CN111208290A (en) | 2020-02-27 | 2020-02-27 | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111208290A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990008957A1 (en) * | 1989-01-30 | 1990-08-09 | Epitope, Inc. | Avidin-biotin assisted immunoassay |
| CN102998442A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit |
| CN108548924A (en) * | 2018-03-30 | 2018-09-18 | 泰州泽成生物技术有限公司 | AngiotensinⅡ Magnetism particulate immuno chemistry luminescence method detection kit and detection method |
| CN108982880A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of testosterone magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
-
2020
- 2020-02-27 CN CN202010124924.8A patent/CN111208290A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990008957A1 (en) * | 1989-01-30 | 1990-08-09 | Epitope, Inc. | Avidin-biotin assisted immunoassay |
| CN102998442A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit |
| CN108548924A (en) * | 2018-03-30 | 2018-09-18 | 泰州泽成生物技术有限公司 | AngiotensinⅡ Magnetism particulate immuno chemistry luminescence method detection kit and detection method |
| CN108982880A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of testosterone magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6750066B2 (en) | Solution for dissociating vitamin D from vitamin D binding protein, associated detection method and use thereof | |
| CA1139203A (en) | Method and reagent for counteracting lipemic interference | |
| JP2019060891A (en) | Immunoagglutination assay | |
| JPH03504276A (en) | Analysis method | |
| CN111381024B (en) | Immunocapture composition, preparation method, kit and application | |
| JPH03502244A (en) | Test method and reagent kit | |
| CN112816694A (en) | Abnormal prothrombin detection kit and preparation method thereof | |
| CN111551730B (en) | Fluorescent microsphere sealing liquid and kit using same | |
| CN108444988A (en) | Thyroglobulin chemiluminescence detection kit | |
| CN112625145A (en) | 1, 3-beta-D glucan derivative, kit, preparation method and method for determining content of 1, 3-beta-D glucan | |
| US5506151A (en) | Non-specific reaction suppressor | |
| EP2786148A1 (en) | One-step immunoassay detection of analytes | |
| CN111208290A (en) | Magnetic particle chemiluminescence aldosterone determination kit and use method thereof | |
| JP2010530966A (en) | Analyte detection method | |
| WO2002048711A1 (en) | Immunological assay reagents and assay method | |
| AU761498B2 (en) | Cobalamin assay | |
| CN101680865A (en) | A rapid homogeneous immunoassay using electrophoresis | |
| CN111175493A (en) | Magnetic particle chemiluminescence method human cortisol determination kit and use method thereof | |
| LU500306B1 (en) | Method for detecting DNASE1L3 based on magnetic particle chemiluminescence immunoassay | |
| CN111487413A (en) | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method | |
| CN118243936B (en) | N-terminal brain natriuretic peptide precursor detection kit | |
| CN116106559A (en) | Biotin-antibody coupling ratio detection kit and application thereof | |
| CA2104596A1 (en) | Multi-test immunochemical reagent and method to use same | |
| EP4481384A1 (en) | Nonspecific reaction inhibitor | |
| EP0903582B1 (en) | Ligand binding surfaces |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200529 |
|
| RJ01 | Rejection of invention patent application after publication |