CN108776218A - A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof - Google Patents
A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 47
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical compound IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 title claims abstract description 30
- 229940035722 triiodothyronine Drugs 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 239000011859 microparticle Substances 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 19
- 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 claims abstract description 88
- 229960002685 biotin Drugs 0.000 claims abstract description 44
- 235000020958 biotin Nutrition 0.000 claims abstract description 44
- 239000011616 biotin Substances 0.000 claims abstract description 44
- 239000012895 dilution Substances 0.000 claims abstract description 42
- 238000010790 dilution Methods 0.000 claims abstract description 42
- 239000006249 magnetic particle Substances 0.000 claims abstract description 29
- 238000003908 quality control method Methods 0.000 claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 23
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 22
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims abstract description 21
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims abstract description 21
- 239000003513 alkali Substances 0.000 claims abstract description 20
- 238000002372 labelling Methods 0.000 claims abstract description 20
- 239000000725 suspension Substances 0.000 claims abstract description 16
- 239000007853 buffer solution Substances 0.000 claims description 85
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 44
- 102000004190 Enzymes Human genes 0.000 claims description 39
- 108090000790 Enzymes Proteins 0.000 claims description 39
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 26
- 239000007983 Tris buffer Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 24
- 210000001685 thyroid gland Anatomy 0.000 claims description 23
- 229910021529 ammonia Inorganic materials 0.000 claims description 22
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 20
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 20
- 239000003550 marker Substances 0.000 claims description 20
- 239000000126 substance Substances 0.000 claims description 20
- 239000002253 acid Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 13
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 13
- 210000004907 gland Anatomy 0.000 claims description 13
- 238000003786 synthesis reaction Methods 0.000 claims description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- 239000011248 coating agent Substances 0.000 claims description 10
- 238000000576 coating method Methods 0.000 claims description 10
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 8
- 239000012894 fetal calf serum Substances 0.000 claims description 8
- KKCIOUWDFWQUBT-AWEZNQCLSA-N L-thyronine Chemical compound C1=CC(C[C@H](N)C(O)=O)=CC=C1OC1=CC=C(O)C=C1 KKCIOUWDFWQUBT-AWEZNQCLSA-N 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000011630 iodine Substances 0.000 claims description 7
- 229910052740 iodine Inorganic materials 0.000 claims description 7
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 238000010494 dissociation reaction Methods 0.000 claims description 6
- 230000005593 dissociations Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 150000001615 biotins Chemical class 0.000 claims description 5
- 239000013078 crystal Substances 0.000 claims description 5
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical group NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 claims description 4
- 239000002585 base Substances 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 4
- 150000003587 threonine derivatives Chemical class 0.000 claims description 4
- -1 Methylaminopropyl Chemical group 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 3
- 238000001953 recrystallisation Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 229960002317 succinimide Drugs 0.000 claims description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 claims 1
- 239000001301 oxygen Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 239000000463 material Substances 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000000034 method Methods 0.000 description 10
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 238000003018 immunoassay Methods 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 238000003127 radioimmunoassay Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- HZCBWYNLGPIQRK-LBPRGKRZSA-N 3,3',5'-triiodo-L-thyronine Chemical compound IC1=CC(C[C@H]([NH3+])C([O-])=O)=CC=C1OC1=CC(I)=C(O)C(I)=C1 HZCBWYNLGPIQRK-LBPRGKRZSA-N 0.000 description 3
- XNSAINXGIQZQOO-UHFFFAOYSA-N L-pyroglutamyl-L-histidyl-L-proline amide Natural products NC(=O)C1CCCN1C(=O)C(NC(=O)C1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-UHFFFAOYSA-N 0.000 description 3
- 102100032251 Pro-thyrotropin-releasing hormone Human genes 0.000 description 3
- 239000000627 Thyrotropin-Releasing Hormone Substances 0.000 description 3
- 101800004623 Thyrotropin-releasing hormone Proteins 0.000 description 3
- 102000002248 Thyroxine-Binding Globulin Human genes 0.000 description 3
- 108010000259 Thyroxine-Binding Globulin Proteins 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 229940034199 thyrotropin-releasing hormone Drugs 0.000 description 3
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 3
- 230000003139 buffering effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101100515942 Mus musculus Nbl1 gene Proteins 0.000 description 1
- 241000255964 Pieridae Species 0.000 description 1
- 241000218606 Pinus contorta Species 0.000 description 1
- 108010071690 Prealbumin Proteins 0.000 description 1
- 102000007584 Prealbumin Human genes 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007233 catalytic pyrolysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000009125 negative feedback regulation Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003904 radioactive pollution Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000000673 shore pine Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- 230000001646 thyrotropic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 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
- G01N33/53—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
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- Biotechnology (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof, the kit includes：The coated magnetic particle suspension of Streptavidin, the trilute derivative of alkali phosphatase enzyme mark, the trilute antibody of biotin labeling test dilution, trilute calibration object, trilute quality-control product.The kit of the present invention is compared with available reagent box, and preparation process is simpler, and cost is lower, and detection range is wide, and stability is good.
Description
Technical field
The invention belongs to technical field of immune assay, it is related to detecting total triiodothyronine TT3 in serum
Magnetic microparticle chemiluminescence immune assay kit of content and preparation method thereof.
Background technology
3,5,3 ' triiodo thryonines（Triiodothyronine, T3）It is a kind of weight to work to various target organs
Want thyroid hormone, molecular weight 651, half-life period is 1.5 days in blood plasma.About 80% T3 is formed in peripheral tissues, is existed by T4
Iodine is taken off under the action of de- iodine enzyme to be transformed, remaining T3 is to be released into blood due to the hydrolysis of thyroglobulin and followed
Ring.The secretion of T3 is by thyrotropic hormone（TSH）And thyrotropin-releasing hormone (TRH)（TRH）Adjusting, and T3 level it is right
There is also negative-feedback regu- lations by TSH.
T3 be by with the transporter in serum in conjunction with by be transported, mainly have thyroxine-binding globulin
（TBG）, prealbumin and albumin, only about 0.4% T3 is free, but free T3（FT3）Just really has the life of hormone
Object activity.The concentration of internal TT3 is influenced by the concentration of thyroxine-binding globulin, and FT3 is unaffected, and TT3 and FT3 are protected
Dynamic equilibrium is held, normal thyroid function is maintained, so the quantitative detection of TT3 has the diagnosis of thyroid illness of early stage very much
Value.
The main method measured for internal total triiodothyronine has radio immunoassay, Enzyme-linked Immunosorbent Assay
Analytic approach, chemiluminescence immunoassay etc..Currently used radio immunoassay（RIA）It is to use I125Mark triiodo first shape
Gland original ammonia acid haptens is come what is realized, and synthesis technology is complicated, and the term of validity is short, has certain pollution to environment, influences testing result
Factor it is more.In recent years, chemiluminescence immunoassay technology was quickly grown, sensitivity, specificity and the degree of automation
Met or exceeded RIA level, especially the stability of marker and it is free from environmental pollution be that RIA methods are incomparable.
Current major Medium Sized Hospitals equal import automatic chemiluminescence immunoassay system, but instrument and reagent price
It is expensive.
Invention content
The problem to be solved in the present invention is to provide the chemiluminescence immunoassay quantitative detecting reagent of total triiodothyronine
Box and preparation method thereof, the reagent term of validity that avoids radioimmunoassay is short, there are radioactive pollution, cumbersome etc. to lack
Point, and solve that sensitivity is low, and stability is poor, defect of high cost.It is simple, at low cost that the invention discloses a kind of preparation processes
Honest and clean, the good total triiodothyronine of stability chemiluminescence immunoassay immue quantitative detection reagent box and preparation method.
The technical solution adopted by the present invention is：
A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, including Streptavidin coating
Magnetic particle suspension, the trilute derivative of alkali phosphatase enzyme mark, the triiodo thyroid gland of biotin labeling
Former propylhomoserin antibody tests dilution, total triiodothyronine calibration object, total triiodothyronine quality-control product.
Further, the coated magnetic particle suspension of the Streptavidin is four of surface package with Streptavidin
Fe 3 O is suspended in the suspension formed in magnetic particle coating object buffer solution, and the coated magnetic particle of Streptavidin is outstanding
A concentration of 0.1mg/mL~1.0mg/mL of supernatant liquid, the magnetic particle coating object buffer solution is 20mM~200mM Tris（Three hydroxyls
Aminomethane）Buffer solution, PH ranging from 6.5~8.0.
The trilute derivative of the alkali phosphatase enzyme mark is that alkaline phosphatase and triiodo thyroid gland are former
The conjugate of threonine derivative, wherein trilute derivative are n-hydroxysuccinimide (NHS) and triiodo first
The sulfonated HOSu NHS of chemical synthesis substance or N- (Sulfo-NHS) and triiodo thyroid gland original ammonia of shape gland original ammonia acid
The chemical synthesis substance of acid.
The trilute antibody of the biotin labeling is biotin and trilute antibody
Conjugate, wherein trilute antibody are mouse monoclonal antibody.
Further, the coated magnetic particle suspension of the Streptavidin is grain of the surface package with Streptavidin
The ferriferrous oxide particles that diameter is 1um~1.5um are suspended in magnetic particle and are coated with suspension in object buffer solution, the Streptavidin
A concentration of 0.5mg/mL of coated magnetic particle suspension；The magnetic particle coating object buffer solution is 100mM Tris（Three hydroxyl first
Base aminomethane）Buffer solution, PH 7.0.
Further, the trilute derivative of the alkali phosphatase enzyme mark is to be diluted in enzyme marker to delay
The conjugate of alkaline phosphatase and trilute derivative in fliud flushing, the alkaline phosphatase and triiodo thyroid gland
The conjugate of former threonine derivative is 1 with the dilution ratio of enzyme marker buffer solution:400~1:2000, preferred dilution ratio
It is 1:800；Enzyme marker buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0 is preferred a concentration of
20mM, preferred PH are 7.4.
Further, the trilute antibody of the biotin labeling is to be diluted in biotinylated derivative buffering
The conjugate of biotin and trilute antibody in liquid, wherein trilute antibody are mouse Dan Ke
Grand antibody；The dilution ratio of the conjugate of the biotin and trilute antibody and biotinylated derivative buffer solution
It is 1:200~1:1000, preferred dilution ratio is 1:500；The biotinylated derivative buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 8.0.
Further, the test dilution is the buffer solution containing dissociation agent, wherein dissociation agent is 8- aniline -1- naphthalene sulphurs
Acid（ANS）, the concentration range of the test dilution is 0.01%~1%, preferred a concentration of 0.1%；The buffer solution is 20mM
Tris buffer solutions, PH 7.4；
The trilute calibration object is the calibration object buffer solution containing 20% fetal calf serum, by triiodo thyroid gland original ammonia
Sour sterling is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；Calibration object buffer solution is
20mM Tris buffer solutions, PH 7.4；
The trilute quality-control product is the calibration object buffer solution containing 20% fetal calf serum, by triiodo thyroid gland original ammonia
Sour sterling is diluted to working concentration, respectively 1.00,4.00ng/mL；The quality-control product buffer solution is 20mM Tris buffer solutions,
PH value is 7.4.
Further, the trilute derivative is n-hydroxysuccinimide (NHS) and triiodo first shape
The chemical synthesis substance of gland original ammonia acid, chemical structural formula are as follows：
Or the sulfonated HOSu NHSs of the trilute derivative N- (Sulfo-NHS) and triiodo thyroid gland
The chemical synthesis substance of former propylhomoserin, chemical structural formula are as follows：
Further, the trilute derivative is prepared by following processing step：
S1. the preparation of trilute-succinate：It takes trilute to be added in distilled water, then adds
Enter copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate
Ice bath, which cools down 2 hours, has crystallization to be precipitated, and with ethyl alcohol recrystallization, obtains white flaky crystals trilute-succinic acid
Ester；
The preparation of S2.N- HOSu NHSs (NHS) and trilute synthetic：Take triiodo thyroid gland original ammonia
Acid-succinate is dissolved in 50mM citric acid solutions（PH4.0）In, n-hydroxysuccinimide (NHS) and 1- (3- bis- is added
Methylaminopropyl) -3- ethyl-carbodiimide hydrochlorides (EDC), react 8h for 2~8 DEG C under the conditions of being protected from light, cross chromatographic column and collect N- hydroxyls
The eluent of base succinimide (NHS) and trilute synthetic, after concentration, are recrystallized with absolute ethyl alcohol, are obtained
It is former to get triiodo thyroid gland to white, needle-shaped crystals n-hydroxysuccinimide (NHS) and trilute synthetic
Threonine derivative.
A kind of preparation method of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, packet
It includes：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, it is 4 DEG C~37 DEG C reactions 0.5 in temperature
After~24 hours, then ProteinG affinity columns (GE companies) is used to purify enzyme labelled antibody, obtain trilute enzyme knot
Object is closed, trilute enzyme conjugates is diluted in enzyme marker buffer solution, the trilute enzyme
The dilution ratio of conjugate and enzyme marker buffer solution is 1:400~1:2000, preferred dilution ratio is 1:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）Be added in sodium carbonate buffer (pH8.0), biotin derivative be added, temperature be 4 DEG C~37 DEG C reactions 0.5~
After 24 hours, ProteinG affinity columns (GE companies) purifying biological element labelled antibody is then used, trilute is obtained
Trilute biotin conjugate is diluted in biotinylated derivative buffer solution by biotin conjugate, and described three
The dilution ratio of iodine thyronine biotin conjugate and biotinylated derivative buffer solution is 1:200~1:1000, preferably
Dilution ratio be 1:500；
（3）Test the preparation method of dilution：With 20mM Tris buffer solutions（PH7.4）ANS, which is diluted to working concentration, is
0.1%；
（4）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；
（5）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 1.00,4.00ng/mL；
（6）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, test dilution, trilute calibration object, trilute quality-control product are assembled into
Box preserves under the conditions of 2~8 DEG C.
Further, a kind of preparation of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box
Method, including：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, after 37 DEG C are reacted 4 hours, use
ProteinG affinity columns (GE companies) purify enzyme labelled antibody, trilute enzyme conjugates are obtained, by triiodo thyroid gland
Former propylhomoserin enzyme conjugates is diluted in enzyme marker buffer solution, and the trilute enzyme conjugates is slow with enzyme marker
The dilution ratio of fliud flushing is 1:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）It is added in sodium carbonate buffer (pH8.0), biotin derivative is added and uses ProteinG after 37 DEG C are reacted 4 hours
Affinity column (GE companies) purifying biological element labelled antibody, obtains trilute biotin conjugate, by triiodo first shape
Gland original ammonia acid biotin conjugate is diluted in biotinylated derivative buffer solution, and the trilute biotin combines
The dilution ratio of object and biotinylated derivative buffer solution is 1:500；
（3）Test the preparation method of dilution：With 20mM Tris buffer solutions（PH7.4）ANS, which is diluted to working concentration, is
0.1%；
（4）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；
（5）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 1.00,4.00ng/mL；
（6）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, test dilution, trilute calibration object, trilute quality-control product are assembled into
Box preserves under the conditions of 2~8 DEG C.
Kit Performance Evaluating Indexes of the present invention：Accuracy, line are carried out to the kit prepared using this method
Property, precision, specificity and stability are measured.
Kit reaction principle of the present invention：Using magnetic microparticles as the solid phase of immune response, chemiluminescence is utilized
Immunoassay method coordinates with chemiluminescence class analyzer, for measuring total triiodo thyroid gland original ammonia in human serum/blood plasma
Acid content.The technical principle of reaction is：Trilute in sample to be tested, calibration object or quality-control product and alkaline phosphatase
The trilute monoclonal antibody of the trilute derivative competitive binding biotin labeling of enzyme label,
The magnetic particle of coating Streptavidin is then added, being combined with biotin by Streptavidin makes antigen antibody complex connect
On magnetic particle, the Direct precipitation in externally-applied magnetic field detaches the compound that immune response is formed with unbonded other materials.
The compound of precipitation is cleaned, is added enzyme-catalyzed chemical luminescence substrate, substrate by catalytic pyrolysis, forms and unstable swashs under enzyme effect
State intermediate is sent out, photon is just sent out when excitation state intermediate returns to ground state, forms luminescence-producing reaction, is measured and is reacted using light-emitting appearance
Luminous intensity.Within the measurement range, luminous intensity is inversely proportional with the trilute concentration in sample, uses improvement
Four parameter Logistic equation models can quantify calculate trilute concentration in sample to be tested.
The total triiodothyronine magnetic microparticle chemiluminescence immunological quantitative determining kit of the present invention, preparation process letter
It is single, of low cost, stability is good, performance reaches the peer-level of famous foreign brand reagent.
The novelty of the present invention is：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark is using trilute
Derivative（T3-NHS）Coupling phosphatase directly is carried out, other patents that compare or producer are trilutes-
BSA derivatives（T3-CMO-BSA）Or trilute-OVA derivatives（T3-CMO-OVA）, this method cost is lower,
Technique is simpler, more controllably.There may be the interference of certain steric hindrance, shadows for the high molecular weight protein derivative of trilute
The accuracy of sound test result, and trilute derivative of the present invention（T3-NHS）It is and triiodo thyroid gland
The similar small-molecule substance of former propylhomoserin structure, the steric hindrance interference effectively avoided the problem that.
（2）The preparation method of biotin labelled antibodies：It is directly coupled with antibody using the biotin through overactivation, nothing
Need to add EDC the coupling agents such as NHS, technique is simpler, and cost is lower.
Specific implementation mode
It is further illustrated the present invention with reference to example, the advantages and features of the present invention becomes apparent from what is be described.But
It is to be understood that this example is only a kind of example of the present invention, any restrictions can't be done to the scope of the present invention.
A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, including Streptavidin
Coated magnetic particle suspension, the trilute derivative of alkali phosphatase enzyme mark, the triiodo first of biotin labeling
Shape gland original ammonia acid antibody tests dilution, total triiodothyronine calibration object, total triiodothyronine quality-control product.
The coated magnetic particle suspension of Streptavidin of the present invention is four of surface package with Streptavidin
Fe 3 O, grain size are 1um~1.5um, are suspended in magnetic particle coating object buffer solution, a concentration of 0.1mg/mL~1.0mg/
ML, preferred a concentration of 0.5mg/mL；It is 20mM~200mM Tris that magnetic particle, which is coated with object buffer solution,（Trihydroxy methyl amino first
Alkane）Buffer solution, PH ranging from 6.5~8.0, preferred a concentration of 100mM, preferred PH are 7.0.
The trilute derivative of alkali phosphatase enzyme mark of the present invention is alkaline phosphatase and triiodo
The conjugate of thyronine derivative, wherein trilute derivative are n-hydroxysuccinimide (NHS)
With the sulfonated HOSu NHS of chemical synthesis substance or N- (Sulfo-NHS) and triiodo first of trilute
The chemical synthesis substance of shape gland original ammonia acid.It is diluted in enzyme marker buffer solution, dilution ratio 1:400~1:2000, preferably
Dilution ratio be 1:800；Enzyme marker buffer solution is 20mM~200mM Tris buffer solutions, and PH ranging from 6.5~8.0 is excellent
A concentration of 20mM of choosing, preferred PH are 7.4.
The trilute derivative of the present invention is n-hydroxysuccinimide (NHS) and triiodo
The chemical synthesis substance of thyronine, chemical structural formula are as follows：
Or the sulfonated HOSu NHSs of the trilute derivative N- (Sulfo-NHS) and triiodo thyroid gland
The chemical synthesis substance of former propylhomoserin, chemical structural formula are as follows：
The preparation process of trilute derivative of the present invention：
（1）The preparation of trilute-succinate：Take trilute（It purchases from Sigma companies）
3.5g is in 200mL distilled water, addition copper sulphate 800mg, back flow reaction 5 hours in 100 DEG C of water-baths, after being cooled to room temperature,
It is filtered to remove solid impurity, then has within 2 hours crystallization to be precipitated the cooling of filtrate ice bath, with ethyl alcohol recrystallization, obtains white plates
Crystallize trilute-succinate 2.6g.
（2）The preparation of n-hydroxysuccinimide (NHS) and trilute synthetic：Take the triiodo first of 1.2g
Shape gland original ammonia acid-succinate is dissolved in 50mM citric acid solutions（PH4.0）In, n-hydroxysuccinimide (NHS) is added
600mg and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) 200mg, 2~8 DEG C of reactions under the conditions of being protected from light
8h crosses the eluent that chromatographic column collects n-hydroxysuccinimide (NHS) and trilute synthetic, after concentration,
It is recrystallized with absolute ethyl alcohol, obtains white, needle-shaped crystals n-hydroxysuccinimide (NHS) and synthesized with trilute
Object 160mg is to get T3-NHS.
It is biotin and trilute the present invention relates to the trilute antibody of biotin labeling
The conjugate of antibody, wherein trilute antibody are mouse monoclonal antibody.It is diluted in biotinylated derivative buffering
In liquid, dilution ratio 1:200~1:1000, preferred dilution ratio is 1:500；Biotinylated derivative buffer solution be 20mM~
200mM Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 8.0.
Test dilution of the present invention is the buffer solution containing dissociation agent, wherein dissociation agent main component is 8- benzene
Amine -1-naphthalene sulfonic aicd（ANS）, concentration range is 0.01%~1%, preferred a concentration of 0.1%；Buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 7.4.
Trilute calibration object of the present invention is the calibration object buffer solution containing 20% fetal calf serum, by three
Iodine thyronine sterling is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；Calibration
It is 20mM~200mM Tris buffer solutions to savor buffer solution, and PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are
7.4。
Trilute quality-control product of the present invention is the calibration object buffer solution containing 20% fetal calf serum, by three
Iodine thyronine sterling is diluted to working concentration, respectively 1.00,4.00ng/mL；Quality-control product buffer solution be 20mM~
200mM Tris buffer solutions, pH range are 6.5~8.0, and preferred a concentration of 20mM, preferred pH value is 7.4.
One, the preparation of kit：
（1）Magnetic particle is coated with object buffer solution and prepares：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 0.50g |
| Bovine serum albumin(BSA) | 50.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.00 ± 0.10.
（2）It is prepared by enzyme marker buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 50.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（3）It is prepared by biotinylated derivative buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 4.50g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 10.00g |
| Proclin300 | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 8.00 ± 0.10.
（4）Dilution is tested to prepare：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 4.50g |
| Tween-20 | 1.00g |
| Bovine serum albumin(BSA) | 10.00g |
| Proclin300 | 1.00g |
| 8- aniline -1-naphthalene sulfonic aicd | 1.00g |
Above-mentioned material is added in 1000mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（5）It is prepared by calibration object buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 1.00g |
| Fetal calf serum | 200mL |
| Proclin300 | 1.00g |
Above-mentioned material is added in 800mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（6）It is prepared by quality-control product buffer solution：
| Material | Dosage |
| Trishydroxymethylaminomethane | 2.42g |
| Sodium chloride | 18.00g |
| Tween-20 | 2.00g |
| Fetal calf serum | 200mL |
| Proclin300 | 1.00g |
Above-mentioned material is added in 800mL deionized waters, dissolving is sufficiently stirred, adjusts PH to 7.40 ± 0.10.
（1）The coated magnetic particle suspension manufacturing methods of Streptavidin：
By the coated magnetic particle mother liquor of the Streptavidin of commercialization（It purchases in Nanjing Pan Gu's gene nano Science and Technology Ltd.）
It is 0.5mg/mL with magnetic bead coating object buffer solution diluted concentration.
（2）The trilute derivative preparation method of alkali phosphatase enzyme mark：
100ug alkaline phosphatases are added in 1mL10mM sodium carbonate buffers (pH8.0), 1ug triiodo thyroid gland original ammonia is added
Acid derivative（T3-NHS, it is company that buying is refined in Shenzhen）, after 37 DEG C are reacted 4 hours, with ProteinG affinity columns, (GE is public
Department) purifying enzyme labelled antibody, obtain trilute enzyme conjugates.It is diluted in enzyme marker buffer solution, dilution ratio
It is 1:1000.
（3）The trilute preparation method for antibody of biotin labeling：
By 20ug trilute antibody（Mouse, monoclonal are purchased in Meridian companies of the U.S.）1mL10mM is added
In sodium carbonate buffer (pH8.0), 50ug biotin derivatives are added, after 37 DEG C are reacted 4 hours, with ProteinG affinity columns
(GE companies) purifying biological element labelled antibody, obtains trilute biotin conjugate.It is diluted in biotin labeling
In object buffer solution, dilution ratio 1:500.
（4）The preparation method of trilute calibration object：
Trilute sterling is diluted to working concentration, respectively 0 with calibration object buffer solution, 0.50,1.00,
2.00,4.00,8.00 ng/mL.
（5）The preparation method of trilute quality-control product：
Trilute sterling is diluted to working concentration, respectively 1.00,4.00ng/mL with quality-control product buffer solution.
（6）Assembling：Mentioned reagent component is assembled into box, is preserved under the conditions of 2~8 DEG C.
Two, the test method of kit：
（1）Sample-adding and incubation process：Total triiodothyronine calibration object, quality-control product or fresh patient's sample 50uL is drawn to add
Enter in reaction tube, then be added alkali phosphatase enzyme mark trilute derivative 50uL and biotin labeling three
Iodine thyronine antibody 50uL, 37 DEG C of incubation reactions 10 minutes；Then the coated magnetic particle of Streptavidin is added to suspend
Liquid 50uL, 37 DEG C of incubation reactions 10 minutes；
（2）Magneto separate cleaning process：Reaction tube after the completion of incubation reaction is placed on Magneto separate frame and stands 1 minute, in removing
Clear liquid；Magnetic bead is added for the first time and is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, remove supernatant；Second
Secondary addition magnetic bead is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；Magnetic bead is added in third time
It is coated with object buffer solution 300uL, is placed on Magneto separate frame and stands 1 minute, removes supernatant；
（3）Luminescence process：530 substrate solutions of Lumi-Phos are added（It purchases in Lumigen companies of the U.S.）200uL, 37 DEG C are protected from light
It is incubated after five minutes, luminous value is measured with 9507 semi-automatic Chemiluminescence Apparatus of shore pine.
Three, the performance test results of kit：
（1）The range of linearity is 0~8.00ng/mL, linear coefficient：r≥0.9900；
（2）Imprecision is no more than 6% in batch；
（3）Accuracy：The rate of recovery is between 90%~110%；
（4）Minimum detectability：Test result is not higher than 0.1ng/mL；
（5）Specificity：The reverse triiodothyronine of 50ng/mL（rT3）, 500ng/mL total thyroxin（TT4）, test
As a result it is not higher than 0.1ng/mL：
| Chaff interferent | Concentration | Test result | Conclusion |
| Reverse triiodothyronine | 50ng/mL | 0.08ng/mL | < 0.1ng/mL |
| Total thyroxin | 500ng/mL | 0.06ng/mL | < 0.1ng/mL |
（6）Stability：37 DEG C accelerate 7 days in 2~8 DEG C of ± 10% ranges of reagent test result error；
| Sample | Concentration | 37 DEG C of acceleration, 7 days relative deviations |
| Low value Quality Control | 1.0ng/mL | -2.73% |
| High level Quality Control | 4.0ng/mL | -3.16% |
It can be seen that by above result：Kit of the present invention is compared with foreign same type kit, performance test knot
Fruit is very close to reaching good results, trilute magnetic microparticle chemiluminescence immune quantitative detection reagent of the invention
Box has good applicability and advance.
Embodiment described above is only the preferred embodiment of the present invention.It should be pointed out that dilution ratio of the present invention
Refer to that ratio diluted in mass ratio is not departing from the technology of the present invention side for those skilled in the art
Under the premise of case, some improvements and modifications can also be made, these improvement and modification also should be regarded as protection scope of the present invention, this
The available prior art in the part being not known in embodiment is realized.
Claims (10)
1. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, it is characterised in that：It is wrapped
Include the coated magnetic particle suspension of Streptavidin, the trilute derivative of alkali phosphatase enzyme mark, biotin
The trilute antibody of label tests dilution, total triiodothyronine calibration object, total triiodo thyroid gland original
Propylhomoserin quality-control product.
2. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent according to claim 1
Box, it is characterised in that：The coated magnetic particle suspension of Streptavidin is four oxygen of the surface package with Streptavidin
Change three-iron is suspended in the suspension formed in magnetic particle coating object buffer solution, and the coated magnetic particle of Streptavidin suspends
A concentration of 0.1mg/mL~1.0mg/mL of liquid, the magnetic particle coating object buffer solution is 20mM~200mM Tris（Three hydroxyl first
Base aminomethane）Buffer solution, PH ranging from 6.5~8.0；
The trilute derivative of the alkali phosphatase enzyme mark is alkaline phosphatase and trilute
The conjugate of derivative, wherein trilute derivative are n-hydroxysuccinimide (NHS) and triiodo thyroid gland
The sulfonated HOSu NHS of chemical synthesis substance or N- (Sulfo-NHS) of former propylhomoserin and trilute
Chemical synthesis substance；
The trilute antibody of the biotin labeling is the coupling of biotin and trilute antibody
Object, wherein trilute antibody are mouse monoclonal antibody.
3. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The coated magnetic particle suspension of Streptavidin is surface package with Streptavidin
The ferriferrous oxide particles that grain size is 1um~1.5um are suspended in magnetic particle and are coated with suspension in object buffer solution, and the strepto- is affine
A concentration of 0.5mg/mL of the coated magnetic particle suspension of element；The magnetic particle coating object buffer solution is 100mM Tris（Three hydroxyls
Aminomethane）Buffer solution, PH 7.0.
4. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The trilute derivative of the alkali phosphatase enzyme mark is to be diluted in enzyme marker
The conjugate of alkaline phosphatase and trilute derivative in buffer solution, the alkaline phosphatase and triiodo first shape
The conjugate of gland original ammonia acid derivative is 1 with the dilution ratio of enzyme marker buffer solution:400~1:2000, preferred thinner ratio
Example is 1:800；Enzyme marker buffer solution is 20mM~200mM Tris buffer solutions, PH ranging from 6.5~8.0, preferred concentration
It is 7.4 for 20mM, preferred PH.
5. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The trilute antibody of the biotin labeling is to be diluted in biotinylated derivative to delay
The conjugate of biotin and trilute antibody in fliud flushing, wherein trilute antibody are that mouse is single
Clonal antibody；The thinner ratio of the conjugate of the biotin and trilute antibody and biotinylated derivative buffer solution
Example is 1:200~1:1000, preferred dilution ratio is 1:500；The biotinylated derivative buffer solution is 20mM~200mM
Tris buffer solutions, PH ranging from 6.5~8.0, preferred a concentration of 20mM, preferred PH are 8.0.
6. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The test dilution is the buffer solution containing dissociation agent, wherein dissociation agent is 8- aniline -1- naphthalenes
Sulfonic acid（ANS）, the concentration range of the test dilution is 0.01%~1%, preferred a concentration of 0.1%；The buffer solution is
20mM Tris buffer solutions, PH 7.4；
The trilute calibration object is the calibration object buffer solution containing 20% fetal calf serum, by triiodo thyroid gland original ammonia
Sour sterling is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；Calibration object buffer solution is
20mM Tris buffer solutions, PH 7.4；
The trilute quality-control product is the calibration object buffer solution containing 20% fetal calf serum, by triiodo thyroid gland original ammonia
Sour sterling is diluted to working concentration, respectively 1.00,4.00ng/mL；The quality-control product buffer solution is 20mM Tris buffer solutions,
PH value is 7.4.
7. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The trilute derivative is n-hydroxysuccinimide (NHS) and triiodo first
The chemical synthesis substance of shape gland original ammonia acid, chemical structural formula are as follows：
Or the sulfonated HOSu NHSs of the trilute derivative N- (Sulfo-NHS) and triiodo thyroid gland
The chemical synthesis substance of former propylhomoserin, chemical structural formula are as follows：
。
8. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection according to claim 1 or 2
Kit, it is characterised in that：The trilute derivative is prepared by following processing step：
S1. the preparation of trilute-succinate：It takes trilute to be added in distilled water, then adds
Enter copper sulphate, back flow reaction 5 hours in 100 DEG C of water-baths after being cooled to room temperature, are filtered to remove solid impurity, then by filtrate
Ice bath, which cools down 2 hours, has crystallization to be precipitated, and with ethyl alcohol recrystallization, obtains white flaky crystals trilute-succinic acid
Ester；
The preparation of S2.N- HOSu NHSs (NHS) and trilute synthetic：Take triiodo thyroid gland original ammonia
Acid-succinate is dissolved in 50mM citric acid solutions（PH4.0）In, n-hydroxysuccinimide (NHS) and 1- (3- bis- is added
Methylaminopropyl) -3- ethyl-carbodiimide hydrochlorides (EDC), react 8h for 2~8 DEG C under the conditions of being protected from light, cross chromatographic column and collect N- hydroxyls
The eluent of base succinimide (NHS) and trilute synthetic, after concentration, are recrystallized with absolute ethyl alcohol, are obtained
It is former to get triiodo thyroid gland to white, needle-shaped crystals n-hydroxysuccinimide (NHS) and trilute synthetic
Threonine derivative.
9. a kind of preparation method of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box, feature
It is, it includes：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, it is 4 DEG C~37 DEG C reactions 0.5 in temperature
After~24 hours, then ProteinG affinity columns (GE companies) is used to purify enzyme labelled antibody, obtain trilute enzyme knot
Object is closed, trilute enzyme conjugates is diluted in enzyme marker buffer solution, the trilute enzyme
The dilution ratio of conjugate and enzyme marker buffer solution is 1:400~1:2000, preferred dilution ratio is 1:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）Be added in sodium carbonate buffer (pH8.0), biotin derivative be added, temperature be 4 DEG C~37 DEG C reactions 0.5~
After 24 hours, ProteinG affinity columns (GE companies) purifying biological element labelled antibody is then used, trilute is obtained
Trilute biotin conjugate is diluted in biotinylated derivative buffer solution by biotin conjugate, and described three
The dilution ratio of iodine thyronine biotin conjugate and biotinylated derivative buffer solution is 1:200~1:1000, preferably
Dilution ratio be 1:500；
（3）Test the preparation method of dilution：With 20mM Tris buffer solutions（PH7.4）ANS, which is diluted to working concentration, is
0.1%；
（4）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；
（5）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 1.00,4.00ng/mL；
（6）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, test dilution, trilute calibration object, trilute quality-control product are assembled into
Box preserves under the conditions of 2~8 DEG C.
10. a kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection examination according to claim 9
The preparation method of agent box, which is characterized in that it includes：
（1）The trilute derivative preparation method of alkali phosphatase enzyme mark：Sodium carbonate is added in alkaline phosphatase
In buffer solution (pH8.0), trilute derivative is added（T3-NHS）, after 37 DEG C are reacted 4 hours, use
ProteinG is affine column purification enzyme labelled antibody, obtains trilute enzyme conjugates, by trilute enzyme
Conjugate is diluted in enzyme marker buffer solution, and the trilute enzyme conjugates is dilute with enzyme marker buffer solution
It is 1 to release ratio:1000；
（2）The trilute preparation method for antibody of biotin labeling：By trilute antibody（Mouse,
Monoclonal）It is added in sodium carbonate buffer (pH8.0), biotin derivative is added and uses ProteinG after 37 DEG C are reacted 4 hours
Affinity column purifying biological element labelled antibody, obtains trilute biotin conjugate, by trilute
Biotin conjugate is diluted in biotinylated derivative buffer solution, the trilute biotin conjugate and biology
The dilution ratio of plain marker buffer solution is 1:500；
（3）Test the preparation method of dilution：With 20mM Tris buffer solutions（PH7.4）ANS, which is diluted to working concentration, is
0.1%；
（4）The preparation method of trilute calibration object：With calibration object buffer solution by trilute sterling
It is diluted to working concentration, respectively 0,0.50,1.00,2.00,4.00,8.00 ng/mL；
（5）The preparation method of trilute quality-control product：With quality-control product buffer solution by trilute sterling
It is diluted to working concentration, respectively 1.00,4.00ng/mL；
（6）Assembling：By the trilute derivative of above-mentioned alkali phosphatase enzyme mark, the triiodo first shape of biotin labeling
Gland original ammonia acid antibody, test dilution, trilute calibration object, trilute quality-control product are assembled into
Box preserves under the conditions of 2~8 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810550557.0A CN108776218A (en) | 2018-05-31 | 2018-05-31 | A kind of total triiodothyronine magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
Applications Claiming Priority (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113493514A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate of anti-triiodothyronine monoclonal antibody, total triiodothyronine quantitative detection kit and use method thereof |
| CN115043898A (en) * | 2021-03-08 | 2022-09-13 | 迈克生物股份有限公司 | Biotinylated antigen derivatives, related kit and use |
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