CN105353139A - Parathyroid hormone quantitative determination kit - Google Patents
Parathyroid hormone quantitative determination kit Download PDFInfo
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- CN105353139A CN105353139A CN201510626670.9A CN201510626670A CN105353139A CN 105353139 A CN105353139 A CN 105353139A CN 201510626670 A CN201510626670 A CN 201510626670A CN 105353139 A CN105353139 A CN 105353139A
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- parathormone
- kit
- monoclonal antibody
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- 102000003982 Parathyroid hormone Human genes 0.000 title claims abstract description 134
- 108090000445 Parathyroid hormone Proteins 0.000 title claims abstract description 134
- 239000000199 parathyroid hormone Substances 0.000 title claims abstract description 134
- 229960001319 parathyroid hormone Drugs 0.000 title abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 83
- 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 30
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000006249 magnetic particle Substances 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims description 67
- 210000002966 serum Anatomy 0.000 claims description 46
- 239000012530 fluid Substances 0.000 claims description 45
- 239000000047 product Substances 0.000 claims description 33
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 29
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 28
- 229940098773 bovine serum albumin Drugs 0.000 claims description 28
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 27
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 27
- 239000003513 alkali Substances 0.000 claims description 27
- 239000011324 bead Substances 0.000 claims description 25
- 238000013016 damping Methods 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 24
- 239000000872 buffer Substances 0.000 claims description 21
- 238000004140 cleaning Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000011734 sodium Substances 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 241001494479 Pecora Species 0.000 claims description 15
- 241001465754 Metazoa Species 0.000 claims description 14
- 238000003908 quality control method Methods 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 14
- HJCUTNIGJHJGCF-UHFFFAOYSA-N 9,10-dihydroacridine Chemical compound C1=CC=C2CC3=CC=CC=C3NC2=C1 HJCUTNIGJHJGCF-UHFFFAOYSA-N 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 230000002421 anti-septic effect Effects 0.000 claims description 10
- 241000283690 Bos taurus Species 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 239000007795 chemical reaction product Substances 0.000 claims description 8
- 239000012898 sample dilution Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000003223 protective agent Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 5
- 108010071390 Serum Albumin Proteins 0.000 claims description 4
- 102000007562 Serum Albumin Human genes 0.000 claims description 4
- 230000008105 immune reaction Effects 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 abstract description 10
- 108020004774 Alkaline Phosphatase Proteins 0.000 abstract description 10
- 238000003018 immunoassay Methods 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000004020 luminiscence type Methods 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 3
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- 238000007885 magnetic separation Methods 0.000 abstract description 3
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- 238000000926 separation method Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 11
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 11
- 238000005259 measurement Methods 0.000 description 11
- 210000000988 bone and bone Anatomy 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 8
- 239000011575 calcium Substances 0.000 description 8
- 229910052791 calcium Inorganic materials 0.000 description 8
- 239000011859 microparticle Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000011574 phosphorus Substances 0.000 description 6
- 229910052698 phosphorus Inorganic materials 0.000 description 6
- 239000008213 purified water Substances 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000011777 magnesium Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 210000000813 small intestine Anatomy 0.000 description 4
- 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 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000012190 activator Substances 0.000 description 3
- 230000003139 buffering effect Effects 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 210000002990 parathyroid gland Anatomy 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 210000002997 osteoclast Anatomy 0.000 description 2
- 210000004409 osteocyte Anatomy 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 229910000589 SAE 304 stainless steel Inorganic materials 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- MWKXCSMICWVRGW-UHFFFAOYSA-N calcium;phosphane Chemical compound P.[Ca] MWKXCSMICWVRGW-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007233 catalytic pyrolysis Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000012886 linear function Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- WALYXZANOBBHCI-UHFFFAOYSA-K magnesium sodium trichloride hydrate Chemical compound O.[Cl-].[Na+].[Mg+2].[Cl-].[Cl-] WALYXZANOBBHCI-UHFFFAOYSA-K 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000027425 release of sequestered calcium ion into cytosol Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- -1 therefore Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit for quantitatively detecting parathyroid hormone. The invention provides a parathyroid hormone quantitative detection kit, comprising a parathyroid hormone monoclonal antibody solution marked by fluorescein isothiocyanate, a parathyroid hormone monoclonal antibody solution marked by alkaline phosphatase and a magnetic separation reagent; the detection method adopted by the invention is magnetic particle separation chemiluminescence immunoassay, an enzyme labeling technology, a magnetic separation technology and a chemiluminescence technology are combined, the operation is convenient and simple, the reaction condition is mild, the luminescence value is stable, and the influence of external conditions is small. Compared with the existing detection method, the method has the advantages of simple sample processing process, low detection cost, quick detection, accurate test result, good repeatability and the like.
Description
Technical field
The invention belongs to technical field of immune assay, relate to the magnetic microparticle chemiluminescence immune assay kit and method of testing thereof that detect parathyroid gland cellulose content in serum, particularly relate to a kind of parathormone immue quantitative detection reagent box.
Background technology
Parathormone (parathyroidhormone, the PTH) assignment of genes gene mapping is on No. 11 chromosomal galianconism (11p15).PTH is synthesized by chief cell and secretes.Complete people PTH is a single chain polypeptide, is made up of 84 amino acid residues, and relative molecular mass is 9500.In the blood circulation of people, there is multiple PTH fragment.Having bioactively has PTH
1-84with the N end fragment (2000 ~ 3000) of PTH.In addition, the C end fragment (6000 ~ 7000) and the stage casing amino acid sequence segments that have PTH of inactive.The C end fragment half life period comparatively N dististyle segment length of PTH.The PTH detected in blood is stage casing and C end fragment mainly.
PTH plays an important role to maintenance body calcium phosphorus balance and Bone m etabolism, and its first target organs is bone and kidney, is secondly small intestine.
1) total to the effect PTH of bone effect promotes molten bone, raises blood calcium.The reaction velocity of bone tissue to PTH has two kinds.1. quick effect once in extracellular fluid calcium ion concentration reduce, PTH can cause bone calcium mobilization in several minutes to a few hours, and osteocyte is made a response rapidly, made calcium transport in density bone to blood, and this kind of effect is rapidly but not lasting.2. slow tail effect is after osteocyte plays a role, and a few hours, PTH can promote that preosteoclast is converted into osteoclast, osteoclast number is increased, increased activity in a few days, promotes bone salts to dissolve and ossein decomposition.
2) 1. the effect of kidney is promoted that distal convoluted renal tubule and medullary loop are to the heavily absorption of calcium; 2. suppress kidney proximal tubule and distal convoluted tubule to the heavily absorption of phosphorus; 3. kidney 25-(OH)-D is improved
3the activity of-1 α-hydroxylase, promotes 1,25-(OH)
2-D
3generation.In brief, PTH can promote the heavily absorption of phosphorus discharge and calcium, reduces serium inorganic phosphorus, raises blood calcium.
3) 1,25-(OH) can be promoted to the effect PTH of small intestine
2-D
3generation, 1,25-(OH)
2-D
3have the effect promoting that small intestine absorbs calcium and phosphorus, therefore, PTH promotes that small intestine is to the absorption of calcium and phosphorus indirectly.
In a word, the effect that PTH is total raises blood calcium, reduction serium inorganic phosphorus, acidified blood, promotion bone information.
The conventional reagent that detects mainly adopts the analytical approachs such as radioimmunoassay technique, Enzyme-multiplied immune technique, Chemiluminescence Immunoassay in the market.
Summary of the invention
An object of the present invention is to provide a kind of parathormone immue quantitative detection reagent box.
Kit provided by the invention is 1) or 2):
1) the parathormone monoclonal antibody solution of marked by fluorescein isothiocyanate and the parathormone monoclonal antibody solution of alkali phosphatase enzyme mark is comprised;
The parathormone monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark parathormone monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: the mixing of solution A, cow's serum, horse serum, sheep blood serum and antiseptic is obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, animal serum albumin and water composition;
2) the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate, described alkali phosphatase enzyme mark parathormone monoclonal antibody and described anti-reagent buffer is comprised.
In mentioned reagent box,
Described animal serum albumin is bovine serum albumin(BSA), and described antiseptic is ProcLin-300;
The proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000ml:23-28ml:1.5-2.5ml:4.0-5.0ml:0.5-2.0ml;
In described solution A, described Na
+concentration is 0.1-1mol/L, described Mg
2+concentration be 1mmol/L, described Zn
2+concentration be 0.1mmol/L, the mass percentage of described bovine serum albumin(BSA) is 0.2-1.0%, and described Trizmabase concentration is 0.01-0.5mol/L.
In mentioned reagent box,
In described anti-reagent buffer, the proportioning of described solution A, cow's serum, horse serum, sheep blood serum, ProcLin-300 is 1000g:25ml:2ml:4.5ml:1.0ml;
In described solution A, described Na
+come from NaCl, the concentration of described NaCl is 0.1mol/L;
Described Mg
2+come from MgCl
2, described MgCl
2concentration be 1.0mmol/L;
Described Zn
2+come from ZnCL
2, described ZnCL
2concentration is 0.1mmol/L;
The mass percentage of described bovine serum albumin(BSA) is 0.5%;
The concentration of described Trizmabase is 0.1mol/L.
In mentioned reagent box,
The parathormone monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the parathormone monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark parathormone monoclonal antibody solution is made up of alkali phosphatase enzyme mark parathormone monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark parathormone monoclonal antibody is 0.5-1 μ g/mL;
Parathormone monoclonal antibody in the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate is different with parathormone monoclonal antibody in described alkali phosphatase enzyme mark parathormone monoclonal antibody.
In mentioned reagent box,
Mentioned reagent box also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
Mentioned reagent box also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of parathormone antigen and calibration object damping fluid, and described parathormone antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, protide protective agent, antiseptic, Trizmabase, animal blood serum and water composition, described Na
+concentration is 0.1-1mol/L, and the protectant concentration of described protide is 0.2-1.0%, and described concentration of preservatives is 0.05-0.2%, and described Trizmabase concentration is 0.01-0.5mol/L, and the volumn concentration of described animal blood serum is 0.05-1.0%.
In mentioned reagent box,
Described Na
+come from NaCl, the concentration of described NaCl is 0.1mol/L;
Described protide protective agent is bovine serum albumin(BSA), and the mass percentage of described bovine serum albumin(BSA) is 0.5%;
Described antiseptic is Proclin-300, and the volumn concentration of described Proclin-300 is 0.1%;
The concentration of described Trizmabase is 0.1mol/L;
Described animal blood serum is sheep blood serum, and the volumn concentration of described sheep blood serum is 0.2%.
In mentioned reagent box,
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6; Described calibration object is concentration is 0pg/mL, 64pg/mL, 360pg/mL, 600pg/mL, 1800pg/mL, 3500pg/mL parathormone antigenic solution.
Described kit also comprises quality-control product, Sample dilution, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations; The calibration object of described quality-control product to be concentration be 360pg/mL and 1800pg/mL.
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described acridan final concentration is 0.25mg/mL;
Described Sample dilution is made up of bovine serum albumin(BSA), trizmabase and water, and wherein, the mass percentage of described bovine serum albumin(BSA) is 0.2%-5%, and the concentration of described trizmabase is 0.1mol/L.
Another object of the present invention is to provide a kind of method preparing above-mentioned kit.
Method provided by the invention, comprises the steps: 8 kinds of equal independent packagings of component in mentioned reagent box; Entire package is kit again.
The application of above-mentioned kit in parathormone quantitatively detects also is the scope of protection of the invention;
Or above-mentioned kit is also the scope of protection of the invention preparing the application in the quantitative testing product of parathormone;
Or 8 kinds of components are also the scope of protection of the invention preparing the application in the quantitative testing product of parathormone in above-mentioned kit;
Or parathormone quantitative detecting method in a kind of sample to be tested, comprise the steps:
1) calibration object in above-mentioned kit, quality-control product and sample to be tested are mixed with the parathormone monoclonal antibody solution of the marked by fluorescein isothiocyanate in above-mentioned kit and alkali phosphatase enzyme mark parathormone monoclonal antibody solution respectively, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described immune reaction product and above-mentioned kit, reaction, obtain reaction product, by the precipitation in described reaction product and magnetic bead cleaning fluid suspendible, collecting precipitation, is Magneto separate product;
3) by the substrate solution mixing in described Magneto separate product and above-mentioned kit, obtain mix products, detect the luminous intensity of described mix products with Chemiluminescence Apparatus, calculate gamut parathormone in sample to be tested by luminous intensity quantitative;
Described magnetic bead cleaning fluid is cleaning fluid and water in above-mentioned kit are mixed by 1:7, obtains solution;
Or the application of Magneto separate reagent in parathormone quantitatively detects in above-mentioned kit.
The pH value of described anti-reagent buffer is 8.0 ± 0.05.
The pH value of described calibration object damping fluid is 8.0 ± 0.05.
The parathormone monoclonal antibody of described marked by fluorescein isothiocyanate is the product obtained with marked by fluorescein isothiocyanate parathormone monoclonal antibody A;
Described alkali phosphatase enzyme mark parathormone monoclonal antibody is the product obtained with the monoclonal antibody B of alkali phosphatase enzyme mark parathormone.
The present invention has the following advantages:
1, use Magneto separate reagent to be solid phase, make immune response closer to liquid phase, reaction more fully with rapid, and makes the immune complex of combination be more prone to be separated, and reduces non-specific adsorption;
2, utilize magnetic microparticles to combine with chemiluminescence, provide the quantitative detecting method that a kind of sensing range is wide, highly sensitive, precision is good, measured value is stable, simple to operate, detect fast, can meet fast, detect in a large number the demand of sample.
Detection method of the present invention is magnetic microparticle separating chemiluminescence immune detection, enzyme labeling technology, and magnetic separation technique and chemiluminescence combine, and it is convenient and simple for operation, and reaction conditions is gentle, and luminous value is stablized and affected by external condition less.Compared to existing detection method, it is simple that the present invention has sample process process, and testing cost is low, detects fast, the advantages such as test result is accurate, reproducible.
Accompanying drawing explanation
Fig. 1 is that magnetic microparticle separating chemiluminescence immunization detects parathormone principle schematic diagram.
Fig. 2 is that the detection kit of Beckman company measures parathormone result.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
All components in detection kit of the present invention all obtains from biological reagent or chemical reagents corporation's purchase by commercial sources.In the present invention, mark fluorescein isothiocynate monoclonal antibody used is that Fitzgerald company produces, and article No. is: 10R-2227; Mark alkaline phosphatase antibodies is that Fitzgerald company produces, and article No. is: 10-1514; Antigen is that Fitzgerald company produces, and article No. is: 30R-1048; Alkaline phosphatase is purchased from BBI company of Britain, and lot number is: 1693AA; Carboxyl magnetic bead colloidal solution is purchased from merk company; Goat-anti fluorescein isothiocynate antibody serum serum is purchased from animal used as test section of PLA General Hospital first affiliated hospital; Fluorescein isothiocynate (FITC) purchased from SIGMA company, CAT:3326-32-7, article No. is: F7250; SMCC is purchased from thermofisherscientific company, and article No. is: 22360; 2-IT purchased from thermofisherscientific company, CAS:4781-83-3; Article No. is: 26101; NHS purchased from SIMGA company, CAS:6066-82-6, article No.: 130672; EDC purchased from SIMGA company, CAS:25952-53-8, article No.: E7750.Magnetic particle reagent, substrate solution, cleaning fluid are all from Capitalbio Corporation Co., Ltd.'s finished product kit.
Embodiment 1, parathormone immue quantitative detection reagent box component and method thereof
One, the preparation of parathormone immue quantitative detection reagent box
By after following 8 kinds of component independent packagings again entire package be kit, be parathormone immue quantitative detection reagent box: 1, calibration object (containing the PTH of a series of concentration, for Criterion curve); 2, quality-control product (containing certain density PTH); 3, reagent A (the PTH monoclonal antibody solution containing certain density marked by fluorescein isothiocyanate); 4, reagent B (the PTH monoclonal antibody containing certain density alkali phosphatase enzyme mark) 5, Sample dilution (trizmabase (tris) damping fluid containing 0.2-1.0% bovine serum albumin(BSA) (BSA)); 6, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody); 7, cleaning fluid (for preparing magnetic bead cleaning fluid); 8, substrate solution.In kit, 1,2,3,4,5,6,8 is indispensable reagent, and 7 replace using by buying other company's similar products.Mentioned reagent box is placed in the preservation of 4 degree, refrigerator.
Specific as follows:
1, calibration object
1) preparation of calibration object damping fluid
Calibration object damping fluid is by Na
+, protide protective agent, animal blood serum, antiseptic, Trizmabase and water composition, the final concentration of each component is as follows: Na
+come from NaCl, its concentration is 0.1mol/L; Protide protective agent is bovine serum albumin(BSA), and its concentration is 0.5% (mass percentage); Antiseptic is Proclin-300, and its concentration is 0.1% (volumn concentration); Trizmabase concentration is 0.1mol/L; Animal blood serum is sheep blood serum, and its concentration is 0.2% (volumn concentration); The pH value of calibration object damping fluid is 8.0 ± 0.05.
The collocation method of calibration object damping fluid is specific as follows:
A) get Trizmabase12.06g, NaCl5.82g is in 1.0L reagent bottle;
B) get 600mL purified water in 1.0L reagent bottle, fully stir until dissolve completely, regulate PH to 10.0 ± 0.05;
C) adding sheep blood serum 2.0mL, bovine serum albumin(BSA) 5.0g, fully stirring and evenly mixing to dissolving completely;
D) add ProcLin-3001.0mL, then add purified water quantitatively to 1000mL, fully mix 4h;
E) PH most 8.0 ± 0.05 is regulated;
F) use 0.22 μm of membrane filtration and collect filtrate, posting label, 2 ~ 8 DEG C of preservations.
2) preparation of calibration object
PTH antigen (is produced by Fitzgerald company, article No. is: 30R-1048) with above-mentioned 1) alignment savors damping fluid and is diluted to 3500pg/mL, 1800pg/mL, 600pg/mL, 360pg/mL, the calibration object of 64pg/mL, equivalent is distributed into the calibration object that concentration is 0pg/mL, 64pg/mL, 360pg/mL, 600pg/mL, 1800pg/mL, 3500pg/mL.
2, quality-control product (containing certain density PTH)
Using the above-mentioned concentration calibration object that is 360pg/mL and 1800pg/mL as quality-control product;
3, reagent A (the PTH monoclonal antibody solution of marked by fluorescein isothiocyanate)
1), the preparation of anti-reagent buffer
Anti-reagent buffer is prepared as follows: solution A, cow's serum, horse serum, sheep blood serum and ProcLin-300 mixing is obtained anti-reagent buffer; Solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, bovine serum albumin(BSA) and water composition.The proportioning of solution A, cow's serum, horse serum, sheep blood serum, ProcLin-300 is 1000ml:25ml:2ml:4.5ml:1.0ml;
In solution A, Na
+come from NaCl, its concentration is 0.1mol/L; Mg
2+come from MgCl
2, its concentration is 1.0mmol/L; Zn
2+come from ZnCL
2, its concentration is 0.1mmol/L; The mass percentage of bovine serum albumin(BSA) is 0.5%; The concentration of Trizmabase is 0.1mol/L.The collocation method of anti-reagent buffer is specific as follows:
A) get Trizmabase12.06g, NaCl5.82g is in 1L beaker;
B) get 500g purified water in 1L beaker, fully stir until dissolve completely;
C) MgCl is added
2﹒ 6H
2o203.0mg, ZnCL
213.60mg, fully dissolves mixing, regulates PH to 10.0 ± 0.05;
D) add bovine serum albumin(BSA) 5.0g, fully stir and evenly mix, place 1.0 hours;
E) add purified water surely to weigh to 1000g, after fully stirring and evenly mixing, regulate PH to 8.0 ± 0.05, obtain solution A, now liquor capacity is 1000ml;
F) cow's serum 25mL is drawn, horse serum 2.0mL, sheep blood serum 4.5mL, ProcLin-3001.0mL, fully mixing 4 hours;
G) adjust ph to 8.0 ± 0.05;
H) use 0.22 μm of membrane filtration and collect filtrate, posting label, 2 ~ 8 DEG C of preservations.
2), marked by fluorescein isothiocyanate PTH monoclonal antibody
Marked by fluorescein isothiocyanate PTH monoclonal antibody is the product obtained by marked by fluorescein isothiocyanate PTH monoclonal antibody, and concrete grammar is as follows:
Get 1mgPTH monoclonal antibody (Fitzgerald company, article No. is: 10R-2227), upper SephadexG25 pillar exchange buffering liquid (0.2mol/L carbonate buffer solution PH9.5,16.8g sodium bicarbonate is dissolved in 1000g purified water, PH9.5), and by centrifugal concentrating to 5mg/mL, add fluorescein isothiocynate solution that concentration is 0.5mg/mL (solvent is concentration is 0.2mol/L, pH value be 9.5 carbonate buffer solutions) 100 μ L (proportioning of PTH monoclonal antibody and fluorescein isothiocynate is 1mg:0.05mg), mix.Room temperature lucifuge reacts 20 hours, obtains marked by fluorescein isothiocyanate PTH monoclonal antibody.
Re-use SephadexG25 pillar and remove unreacted fluorescein isothiocynate, with 0.2mol/L carbonate buffer solution PH9.5 wash-out, collect yellow liquid and be connector, be stored in-20 DEG C, obtain marked by fluorescein isothiocyanate PTH monoclonal antibody after purifying.
3), the preparation of reagent A
By above-mentioned 2) purifying after marked by fluorescein isothiocyanate PTH monoclonal antibody and above-mentioned 1) anti-reagent buffer mix, obtain reagent A, wherein the concentration of marked by fluorescein isothiocyanate PTH monoclonal antibody is 0.5ug/ml.
4, reagent B (the PTH monoclonal antibody containing certain density alkali phosphatase enzyme mark)
1) alkali phosphatase enzyme mark PTH monoclonal antibody
Alkali phosphatase enzyme mark PTH monoclonal antibody is the product obtained by the another kind of monoclonal antibody of alkali phosphatase enzyme mark PTH, and concrete grammar is as follows:
Get another kind of monoclonal antibody (the Fitzgerald company of 1mgPTH, article No. is: 10-1514), upper SephadexG25 pillar exchange buffering liquid (0.05mol/L triethanolamine aqueous solution, PH8.6), and by centrifugal concentrating to 2.5mg/mL, adding concentration is that (2-IT is purchased from thermofisherscientific company for activator 2-Iminothiolanehydrochloride (2-IT) solution of 13.76mg/mL, article No. is: 26101 solvents are 0.05mol/L triethanolamine solution ph 10.5) 5 μ L (being volume ratio 80:1), room temperature adds glycocoll and stops priming reaction after placing 20 minutes, ambient temperatare puts 10 minutes.Re-use SephadexG25 pillar desalination, with 0.2mol/L carbonate buffer solution PH9.5 wash-out, collect eluted protein and be the rear antibody of activation.
Get 1.5mg alkaline phosphatase (purchased from BBI company, lot number is: 1693AA), upper SephadexG25 pillar exchange buffering liquid (0.05mol/L triethanolamine solution ph 8.6), and by centrifugal concentrating to 5mg/mL, (SMCC is purchased from thermofisherscientific company to add Succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) solution that concentration is 6.69mg/mL, article No. is: 22360, solvent is DMF, purchased from sigma company, article No.: 227056) 12 μ L (being volume ratio 25:1), room temperature places 20 minutes, add glycocoll and stop priming reaction, room temperature places 10 minutes.Use SephadexG25 pillar desalination, collect the rear alkaline phosphatase of activation.
The ratio that PTH antibody after activation and alkaline phosphatase after activation add 1mg alkaline phosphatase in 1mgPTH antibody is mixed, adds 5 μ L1M magnesium chloride brine mixings simultaneously, place 4 DEG C of reactions 20 hours.
Use Supperdex200 gel chromatography column separating purification, remove the PTH antibody and alkaline phosphatase that do not connect, with 0.05mol/L triethanolamine solution ph 7.0 wash-out, elution flow rate is 0.75ml/min, pillar diameter is 1.6/60cm, observe out peak position and connector collected by comparison standard molecule collection of illustrative plates, connector is stored in 4 DEG C, obtains alkali phosphatase enzyme mark PTH monoclonal antibody after purifying.
2), the preparation of reagent B
By above-mentioned 1) purifying after in alkali phosphatase enzyme mark PTH monoclonal antibody and above-mentioned 3 1) anti-reagent buffer mix, obtain reagent B, wherein the concentration of alkali phosphatase enzyme mark PTH monoclonal antibody is 0.5ug/ml.
5, Sample dilution
Sample dilution is bovine serum albumin(BSA) (BSA), trizmabase and water are mixed, and obtains Sample dilution, and the mass percentage of bovine serum albumin(BSA) is the concentration of 0.5%, trizmabase is 0.1mol/L.
6, Magneto separate reagent (being combined with the magnetic particle suspension of anti-fluorescein isothiocynate antibody)
Magneto separate reagent is by covalently bound for goat-anti fluorescein isothiocynate antibody serum (polyclonal antibody, producer: animal used as test section of PLA General Hospital first affiliated hospital article No.: Y11005) surperficial at magnetic particle, obtains Magneto separate reagent; Wherein, the proportioning of anti-fluorescein isothiocynate antibody and magnetic particle is 150 μ g:1mg.
The diameter (producer: merck company, article No.: EM1-100/40) between 0.5-1.5 μm of magnetic particle, has superparamagnetism, and surface is containing carboxyl (COOH-) reactive group.
The concrete preparation method of Magneto separate reagent:
(1) by magnetic bead and activator EDC (purchased from SIMGA company, CAS:25952-53-8, article No.: E7750) and NHS (purchased from SIMGA company, CAS:6066-82-6, article No.: 130672) mix with certain proportion, room temperature reaction 120 minutes, obtains activated magnetic beads; Above-mentioned magnetic bead and activator mix ratio are: add 5mgEDC and 5mgNHS in 1000mg magnetic bead; (2) mixed with certain proportion with goat-anti fluorescein isothiocynate antibody by magnetic bead after activation, blending ratio is: add 150mg goat-anti fluorescein isothiocynate antibody in 1000mg magnetic bead, and 4 degree of oscillating reactionss 10 hours, obtain the magnetic bead of connection albumen; (3) processed by the magnetic bead confining liquid after connection albumen, room temperature closes 1 hour, obtains magnetic bead solution; Confining liquid is the MES damping fluid of pH7.0,0.01M containing 1g/100mLBSA; (4) the magnetic bead solution (3) obtained sedimentation under magnetic fields abandoned supernatant after 20 minutes; The stainless steel sift that 1500 order materials are 304 steel is crossed after magnetic bead being suspended in the MES damping fluid of 100mlpH7.0,0.01M, sieve in process, continuous vibration is also rinsed repeatedly with the MES damping fluid of pH7.0,0.01M, until compass screen surface remains the agglomerated particle that can not sieve, magnetic bead after sieving answers particle homogeneous, without aggegation after suspendible again; (5) the magnetic bead conserving liquid after sieving is diluted to 5mg/ml, room temperature suspendible is 4 DEG C of preservations after 2 hours, obtain the magnetic bead being combined with anti-fluorescein isothiocynate antibody; Conserving liquid is the trizmabase damping fluid containing 1% bovine serum albumin(BSA); Again the magnetic bead being combined with anti-fluorescein isothiocynate antibody is suspended in the trizmabase damping fluid containing 1% bovine serum albumin(BSA), obtains Magneto separate reagent;
Trizmabase damping fluid containing 1% bovine serum albumin(BSA) is bovine serum albumin(BSA), trizmabase and water are mixed, obtain the trizmabase damping fluid containing bovine serum albumin(BSA), wherein, the concentration of bovine serum albumin(BSA) is 1% (for mass percentage), the concentration of trizmabase is 0.1mol/L.
7, cleaning fluid
Cleaning fluid: the Tris-HCl damping fluid mixing of to be 8.0 ± 0.05 concentration by Tween-20, sodium chloride and pH be 0.15M, obtain cleaning fluid, wherein the concentration of Tween-20 is 0.04% (volumn concentration), and the concentration of sodium chloride is 0.25mol/L.
8, substrate solution
Substrate solution: to be 0.25M, pH by acridan and concentration be 8.0 ± 0.05 Tris-HCl damping fluid mix, dissolve, make acridan final concentration be 0.25mg/mL, the solution obtained.
Two, magnetic microparticles chemiluminescence immunoassay kit is utilized to carry out quantitative measurement to parathyroid gland cellulose content in sample
1, principle
Fig. 1 is that magnetic microparticle separating chemiluminescence immunization detects parathormone principle schematic diagram.
The present invention is double antibody sandwich method.Magnetic microparticles chemiluminescence immune analysis method is utilized to carry out quantitative measurement to parathyroid gland cellulose content in sample.The know-why of reaction is: the parathormone antibody that alkaline phosphatase (AP) marks and the antigen of antibody in same sample, calibration object or quality-control product that fluorescein isothiocynate (FITC) marks are combined, form the sandwich immunoassay compound of FITC labelled antibody-antigen-AP labelled antibody, similar " sandwich " structure.Add the magnetic particle being connected with anti-fluorescein antibody subsequently, antigen antibody complex is made to be connected on magnetic particle by the specific binding of anti-fluorescein antibody and fluorescein, Direct precipitation in externally-applied magnetic field, the compound that immune response is formed and other separating substances unconjugated.Abandon washing and precipitating compound after supernatant, add enzymatic chemical substrate solution.Substrate just catalytic pyrolysis under enzyme effect, forms unstable excited state intermediate, just sends photon when excited state intermediate gets back to ground state, forms luminescence-producing reaction.Use the luminous intensity of analyser detection reaction.In luminous intensity and sample, the content of parathormone is proportional.
2, parathormone magnetic microparticle separating chemiluminescence immunologic detection method
(1) immune response: respectively by the PTH calibration object in the kit of 30 μ L above-mentioned, 30 μ L quality-control products, 30 μ L samples to be tested in differential responses pipe, again respectively to adding 30 μ L reagent A and 30 μ L reagent B in each reaction tube, be placed on oscillator and mix, 37 DEG C of incubation 15min;
(2) Magneto separate: add 30 μ L Magneto separate reagent again in each reaction tube processed through (1), be placed on oscillator and mix, 37 DEG C of incubation 5min, make magnetic particle sedimentation 2mim in magnetic field, remove supernatant, add 200 μ L magnetic bead cleaning fluids (cleaning fluid in the kit of above-mentioned one and purified water are hybridly prepared into magnetic bead cleaning fluid in 1:7 ratio), remove magnetic field, concussion makes the abundant suspendible of magnetic particle 30 seconds, and then make magnetic particle sedimentation in magnetic field, remove supernatant; Repetition like this 2-4 time;
(3) read value: to each reaction tube processed through (2) often pipe add 200 μ L substrate solutions, detect luminous intensity with Chemiluminescence Apparatus.
Specific as follows:
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (PTH).
PTH kit is in measurement range, and luminous intensity is directly proportional to PTH concentration in sample to be tested, uses four parameter Logistic equation models of improvement to calculate PTH concentration in sample.
y＝(A1-A0)/(1+(x/X0)^P)+A0
Y: luminous intensity
X: concentration value
A1: asymptotic line valuation on curve
A0: asymptotic line valuation under curve
X0: the concentration value that knee point is corresponding
P: the parameter that rate of curve is relevant
Embodiment 2, kit performance evaluation
According to the feature of external diagnosis reagent, detect the range of linearity of kit prepared by embodiment 1, sensitivity, accuracy, precision as usual.Concrete operation step is as follows:
One, reagent prepares:
Before experiment, first take out reagent A (concentration of marked by fluorescein isothiocyanate PTH monoclonal antibody is 0.5ug/ml), reagent B (concentration of alkali phosphatase enzyme mark PTH monoclonal antibody is 0.5ug/ml), calibration object, Magneto separate reagent, luminous substrate, sample diluting liquid, cleaning fluid, quality-control product, balance to room temperature.
Two, instrument prepares:
This kit adopts the ChemLite of Capitalbio Corporation Co., Ltd.
tM1200 Full-automatic chemiluminescence immunoassay analysis meters.
Specific as follows:
In sample cup, add calibration object, sample or quality-control product, sample single tube reaction application of sample amount is 30 μ L, and according to repeating addition in pipe number calculation sample cup, minimum application of sample amount is not less than 50 μ L, adds the reagent of sufficient quantity.
Each reagent, sample cup and reaction tube are placed in instrument, and the running program in strict accordance with Full-automatic chemiluminescence immunoassay analysis meter is carried out experimental implementation and calculates concentration of specimens.Instrument has possessed the temperature of reaction 37 DEG C ± 1 DEG C required for experiment, the reaction time of this project and operation steps have been input in full automatic running program, each walks the time needed for testing, instrument spectral measurement range 300nm ~ 650nm can to obtain this this project after selected this project (CRP).
Four, performance index testing result:
1, the range of linearity:
Sample to be tested is exceed the sample of range of linearity upper concentration and exceed or equal the sample of range of linearity hypomere, be mixed at least 5 dilute concentrations (Xi), specifically as shown in table 1, the PTH solution (solute: PTH antigen, solvent: calibration object damping fluid) of wherein dilution ratio is the sample to be tested of 1 to be concentration be 3500pg/mL.
With the kit of a preparation in embodiment 1 and the detection method of two, sample to be tested is detected, measure the range of linearity.Each dilute concentration tests 3 times.
Obtain the average (Yi) of measurement result respectively, with dilute concentration (Xi) for independent variable, with measurement result average (Yi) for dependent variable, obtain equation of linear regression, calculate linear regression coeffficient (r) by formula (1).
Use four parameter Logistic equation models of improvement, in 15-3500pg/nL sensing range, dose-response curve correlation coefficient r=0.9993.This sensing range is far superior to the like product that patent report is crossed.
Table 1 range of linearity experimental result
2, accuracy:
Sample to be tested is dilution parathormone (PTH) international standard substance (95/646), make its ultimate density for (360 ± 72) pg/mL and (1800 ± 360) pg/mL, sample step is to specifications it can be used as to detect, after duplicate measurements 3 times, its average results is designated as Cm, according to the relative deviation Cb of formula (1) computation and measurement concentration, deviation should in ± 10% scope.
Cb＝(Cm-Cr)/Cr×100％……………………………(1)
In formula:
Cb: the relative deviation of concentration
Cm: the average measuring concentration
Cr: demarcate concentration
Deviation is all in ± 10% scope, and concrete data are in table 2.
Table 2 accuracy experimental result
3, lowest detectable limit (sensitivity):
With 0 μ g/mL calibration object in kit as sample to be tested, replication 20 times, draw the RLU value (relative light unit) of 20 measurement results, calculate its mean value (M) and standard deviation (SD), draw RLU value corresponding to M+2SD, carry out 2 regression fits according to the concentration-RLU value result between zero-dose calibration object and adjacent calibration object and draw linear function, the RLU value of M+2SD is brought in above-mentioned equation, obtain corresponding concentration value.
By lowest detectable limit detection method in detection scheme, 3 repeated experiments, concrete data are in table 3.
Table 3 lowest detectable limit experimental result
| Sensitivity (ng/mL) | |
| Experiment 1 | 4.36 |
| Experiment 2 | 6.24 |
| Experiment 3 | 10.25 |
| Experiment 4 | 6.01 |
| Experiment 5 | 3.97 |
4, precision:
In batch: use PTH (360 ± 72) pg/mL and the sample in (1800 ± 360) ng/mL two concentration ranges as sample to be tested.The method of operating of by specification, carries out 10 replications to sample to be tested respectively with chemical luminescence detector device.Repeatability (CV) is calculated by formula (3).
measure the mean value of concentration for 10 times
X
i: measure concentration value at every turn
Between batch: use PTH concentration to be that (1800 ± 360) ng/mL is as sample to be tested.Use the kit of 3 different batches, the method for operating of by specification, with chemical luminescence detector device, 30 replications (every batch kit measurement 10 times) are carried out to sample to be tested.Difference between batch (CV) is calculated by formula (4).
In formula:
measure the mean value of concentration for 30 times
Measure the standard deviation of concentration for SD:30 time
Detect precision by precision detection method in detection scheme, concrete data are in table 4.
Table 4 Precision Experiment result
Embodiment 3, kit provided by the invention compare with home and abroad kit
1, kit provided by the invention and home and abroad kit performance index comparison
Same sample is detected with existing kit, as follows with kit testing result of the present invention comparison:
This kit of table 5 and the comparison of external kit performance index
2, kit provided by the invention and external kit carry out the comparison of clinical sample measured value
Respectively by the detection kit of kit provided by the invention and Beckman company 93 parts of human serums (patient knows the inside story) sample is detected to its testing result simultaneously and sees Fig. 2, the serum PTH levels adopting kit provided by the invention to record is horizontal ordinate, with the PTH detection kit of Beckman Ku Erte commerce and trade (China) company limited measure result be that ordinate does regretional analysis, dependent equation is: y=0.9729x+11.141, and correlation coefficient r is: 0.9886.Show through statistical procedures result, the clinical sample measured value correlativity that kit provided by the invention and external kit record is good.
Claims (10)
1. a parathormone immue quantitative detection reagent box is 1) or 2):
1) the parathormone monoclonal antibody solution of marked by fluorescein isothiocyanate and the parathormone monoclonal antibody solution of alkali phosphatase enzyme mark is comprised;
The parathormone monoclonal antibody solution of described marked by fluorescein isothiocyanate and the solvent of described alkali phosphatase enzyme mark parathormone monoclonal antibody solution are anti-reagent buffer;
Described anti-reagent buffer is prepared as follows: the mixing of solution A, cow's serum, horse serum, sheep blood serum and antiseptic is obtained anti-reagent buffer;
Described solution A is by Na
+, Mg
2+, Zn
2+, Trizmabase, animal serum albumin and water composition;
2) the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate, described alkali phosphatase enzyme mark parathormone monoclonal antibody and described anti-reagent buffer is comprised.
2. kit according to claim 1, is characterized in that:
Described animal serum albumin is bovine serum albumin(BSA), and described antiseptic is ProcLin-300;
The proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000ml:23-28ml:1.5-2.5ml:4.0-5.0ml:0.5-2.0ml;
In described solution A, described Na
+concentration is 0.1-1mol/L, described Mg
2+concentration be 1mmol/L, described Zn
2+concentration be 0.1mmol/L, the mass percentage of described bovine serum albumin(BSA) is 0.2-1.0%, and described Trizmabase concentration is 0.01-0.5mol/L.
3. kit according to claim 1 or 2, is characterized in that:
In described anti-reagent buffer, the proportioning of described solution A, described cow's serum, described horse serum, described sheep blood serum, described ProcLin-300 is 1000ml:25ml:2ml:4.5ml:1.0ml;
In described solution A, described Na
+come from NaCl, the concentration of described NaCl is 0.1mol/L;
Described Mg
2+come from MgCl
2, described MgCl
2concentration be 1.0mmol/L;
Described Zn
2+come from ZnCL
2, described ZnCL
2concentration is 0.1mmol/L;
The mass percentage of described bovine serum albumin(BSA) is 0.5%;
The concentration of described Trizmabase is 0.1mol/L.
4., according to described kit arbitrary in claim 1-3, it is characterized in that:
The parathormone monoclonal antibody solution of described marked by fluorescein isothiocyanate is made up of the parathormone monoclonal antibody of marked by fluorescein isothiocyanate and described anti-reagent buffer, and the concentration of the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate is 0.1-0.5 μ g/mL;
Described alkali phosphatase enzyme mark parathormone monoclonal antibody solution is made up of alkali phosphatase enzyme mark parathormone monoclonal antibody and described anti-reagent buffer, and the concentration of described alkali phosphatase enzyme mark parathormone monoclonal antibody is 0.5-1 μ g/mL;
Parathormone monoclonal antibody in the parathormone monoclonal antibody of described marked by fluorescein isothiocyanate is different with parathormone monoclonal antibody in described alkali phosphatase enzyme mark parathormone monoclonal antibody.
5., according to described kit arbitrary in claim 1-4, it is characterized in that:
Described kit also comprises Magneto separate reagent;
Described Magneto separate reagent is by covalently bound in magnetic bead surfaces for anti-fluorescein isothiocynate antibody, obtains Magneto separate reagent;
Described anti-fluorescein isothiocynate antibody is polyclonal antibody;
The proportioning of described anti-fluorescein isothiocynate antibody and described magnetic particle is 30 μ g-200 μ g:1mg;
The diameter of described magnetic particle is 0.5-1.5 μm.
6., according to described kit arbitrary in claim 1-5, it is characterized in that: described kit also comprises calibration object;
Described calibration object is by the calibration object solution composition of n variable concentrations, and described calibration object solution is made up of parathormone antigen and calibration object damping fluid, and described parathormone antigen is variable concentrations in described calibration object;
Described calibration object damping fluid is by Na
+, protide protective agent, antiseptic, Trizmabase, animal blood serum and water composition, described Na
+concentration is 0.1-1mol/L, and the protectant concentration of described protide is 0.2-1.0%, and described concentration of preservatives is 0.05-0.2%, and described Trizmabase concentration is 0.01-0.5mol/L, and the volumn concentration of described animal blood serum is 0.05-1.0%.
7. kit according to claim 6, is characterized in that:
Described Na
+come from NaCl, the concentration of described NaCl is 0.1mol/L;
Described protide protective agent is bovine serum albumin(BSA), and the mass percentage of described bovine serum albumin(BSA) is 0.5%;
Described antiseptic is Proclin-300, and the volumn concentration of described Proclin-300 is 0.1%;
The concentration of described Trizmabase is 0.1mol/L;
Described animal blood serum is sheep blood serum, and the volumn concentration of described sheep blood serum is 0.2%.
8. the kit according to claim 6 or 7, is characterized in that:
Described calibration object is by the calibration object solution composition of n variable concentrations, and n is less than or equal to 6;
Described kit also comprises quality-control product, Sample dilution, cleaning fluid and substrate solution;
Described quality-control product is the calibration object of 2 variable concentrations;
Described cleaning fluid by Tween-20, sodium chloride and concentration to be 0.15M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, the volumn concentration of described Tween-20 is 0.04%, and the concentration of described sodium chloride is 0.25mol/L;
Described substrate solution by acridan and concentration to be 0.25M, pH be 8.0 ± 0.05 Tris-HCl damping fluid form, wherein, described acridan final concentration is 0.25mg/mL;
Described Sample dilution is made up of bovine serum albumin(BSA), trizmabase and water, and wherein, the mass percentage of described bovine serum albumin(BSA) is 0.2%-5%, and the concentration of described trizmabase is 0.1mol/L.
9. prepare a method for arbitrary described kit in claim 1-8, comprise the steps: 8 kinds of equal independent packagings of component in described kit arbitrary in claim 1-8; Entire package is kit again.
10. the arbitrary described application of kit in parathormone quantitatively detects in claim 1-8;
Or arbitrary described kit is preparing the application in the quantitative testing product of parathormone in claim 1-8;
Or in claim 1-8 in arbitrary described kit 8 kinds of components preparing the application in the quantitative testing product of parathormone;
Or parathormone quantitative detecting method in a kind of sample to be tested, comprise the steps:
1) calibration object, quality-control product and the sample to be tested in described kit arbitrary in claim 1-8 is mixed with the parathormone monoclonal antibody solution of the marked by fluorescein isothiocyanate in arbitrary described kit in claim 1-8 and alkali phosphatase enzyme mark parathormone monoclonal antibody solution respectively, reaction, obtains immune reaction product;
2) mixed by the Magneto separate reagent in described kit arbitrary in described immune reaction product and claim 1-8, reaction, obtain reaction product, by the precipitation in described reaction product and magnetic bead cleaning fluid suspendible, collecting precipitation, is Magneto separate product;
3) by the substrate solution mixing in described kit arbitrary in described Magneto separate product and claim 1-8, obtain mix products, detect the luminous intensity of described mix products with Chemiluminescence Apparatus, calculate gamut parathormone in sample to be tested by luminous intensity quantitative;
Described magnetic bead cleaning fluid is cleaning fluid and water in described kit arbitrary in claim 1-8 are mixed by 1:7, obtains solution;
Or the application of Magneto separate reagent in parathormone quantitatively detects in arbitrary described kit in claim 1-8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510626670.9A CN105353139A (en) | 2015-09-28 | 2015-09-28 | Parathyroid hormone quantitative determination kit |
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| CN107677840A (en) * | 2017-09-30 | 2018-02-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of parathyroid hormone detection kit and its application method |
| CN107741504A (en) * | 2017-09-30 | 2018-02-27 | 安徽伊普诺康生物技术股份有限公司 | A kind of preparation method of parathyroid hormone detection kit |
| CN108254548A (en) * | 2017-12-21 | 2018-07-06 | 江苏泽成生物技术有限公司 | A kind of magnetic bead sealer of magnetic microparticle chemiluminescence diagnosis system |
| CN108362688A (en) * | 2018-01-02 | 2018-08-03 | 北京利德曼生化股份有限公司 | A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit |
| CN110128542A (en) * | 2018-02-08 | 2019-08-16 | 深圳市新产业生物医学工程股份有限公司 | PTH fusion protein, preparation method, the detection reagent containing it, kit and application |
| WO2020082720A1 (en) * | 2018-10-25 | 2020-04-30 | 成都博奥新景医学科技有限公司 | Method and system for single-part chemiluminescence immunoassay on basis of magnetic rod method |
| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
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| CN113203867A (en) * | 2021-04-28 | 2021-08-03 | 北京美联泰科生物技术有限公司 | Buffer solution suitable for insulin detection kit |
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