CN105131105A - Cortisol immunogen, derivative, antibody, detection reagent and preparation method - Google Patents
Cortisol immunogen, derivative, antibody, detection reagent and preparation method Download PDFInfo
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- CN105131105A CN105131105A CN201510444151.0A CN201510444151A CN105131105A CN 105131105 A CN105131105 A CN 105131105A CN 201510444151 A CN201510444151 A CN 201510444151A CN 105131105 A CN105131105 A CN 105131105A
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- hydrocortisone
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 title claims abstract description 175
- 229960000890 hydrocortisone Drugs 0.000 title claims abstract description 92
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 230000002163 immunogen Effects 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000000243 solution Substances 0.000 claims description 42
- 102000004190 Enzymes Human genes 0.000 claims description 38
- 108090000790 Enzymes Proteins 0.000 claims description 38
- 102100031126 6-phosphogluconolactonase Human genes 0.000 claims description 32
- 108010029731 6-phosphogluconolactonase Proteins 0.000 claims description 32
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 claims description 32
- 150000001885 cortisol derivatives Chemical class 0.000 claims description 21
- 239000000758 substrate Substances 0.000 claims description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims description 14
- 241001465754 Metazoa Species 0.000 claims description 13
- 238000010171 animal model Methods 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 11
- 239000007983 Tris buffer Substances 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000000890 antigenic effect Effects 0.000 claims description 9
- 238000013016 damping Methods 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 7
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 230000021615 conjugation Effects 0.000 claims description 5
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 108010078791 Carrier Proteins Proteins 0.000 claims description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 4
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims description 4
- 238000002649 immunization Methods 0.000 claims description 4
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine group Chemical class C(CCC)N(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 4
- 108010008038 Synthetic Vaccines Proteins 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 3
- 238000005227 gel permeation chromatography Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 239000012264 purified product Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 108010017384 Blood Proteins Proteins 0.000 claims description 2
- 102000004506 Blood Proteins Human genes 0.000 claims description 2
- 241000283707 Capra Species 0.000 claims description 2
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 108010034949 Thyroglobulin Proteins 0.000 claims description 2
- 102000009843 Thyroglobulin Human genes 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 230000036039 immunity Effects 0.000 claims description 2
- 238000004020 luminiscence type Methods 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 230000000392 somatic effect Effects 0.000 claims description 2
- 229960002175 thyroglobulin Drugs 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000005847 immunogenicity Effects 0.000 abstract description 5
- 210000002966 serum Anatomy 0.000 abstract description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract 1
- 230000001837 anti-cortisol effect Effects 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000004202 carbamide Substances 0.000 abstract 1
- 238000002965 ELISA Methods 0.000 description 12
- 238000003018 immunoassay Methods 0.000 description 12
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- 238000000034 method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000007689 inspection Methods 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000010219 correlation analysis Methods 0.000 description 3
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
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- 238000003786 synthesis reaction Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000003270 steroid hormone Substances 0.000 description 2
- 102000014702 Haptoglobin Human genes 0.000 description 1
- 108050005077 Haptoglobin Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000003163 gonadal steroid hormone Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
- C07J5/0046—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa
- C07J5/0053—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa not substituted in position 16
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/534—Production of labelled immunochemicals with radioactive label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a cortisol immunogen, a derivative, an antibody, a detection reagent and a preparation method. The cortisol immunogen has a high immunogenicity and can be used for preparing a high-potency anti-cortisol specific antibody through introduction without any cross reaction with common 62 medicines. A cortisol immunogen detection reagent prepared from the antibody can accurately and quickly determine the content of cortisol in bio-samples, such as urea, serum, plasma and the like. Compared with detection reagents on market at present, the detection reagent is simple in operation, is high in specificity and is accurate in result. The detection reagent also can reduce detection cost of the cortisol effectively and is beneficial to clinical large-scale popularization and application.
Description
Technical field
The invention belongs to biological technical field, relate to hydrocortisone immunogen, derivative, antibody, detection reagent and preparation method.
Background technology
Hydrocortisone (Hydrocortisone), its structural formula is as shown in formula III:
Hydrocortisone, also known as hydrocortisone, is a kind of steroid hormone that suprarenal gland produces in stress reaction.Hydrocortisone is main glucocorticosteroid, participates in sex hormone metabolism in human body, and can affect body sugar, fat, protein metabolism and regulate a series of vital movement such as water, electrolyte balance in body.Human body is secreted hydrocortisone total amount and be about 200mg every day, and secretory volume presents obvious periodical change according to division of day and night every day (24h).After cortisol secretion enters blood, the overwhelming majority and steroid hormone haptoglobin (CBG) combine.Hydrocortisone free in blood only accounts for the 1%-3% of hydrocortisone total amount, but only has free hydrocortisone just to have biological activity.Usual free cortisol with keep relative equilibrium in conjunction with hydrocortisone, but its content all has remarkable rising in body wound, infection, motion, the situation such as excited, acth secretion hydrocortisone superfunction or lowly also can produce distinctive clinical manifestation.Therefore, in Present clinical medical science and pharmacology research and practice, measuring the level of hydrocortisone has become an important index.
At present, the detection method of hydrocortisone mainly comprises: high performance liquid chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry coupling method (LC/MS/MS), radio immunoassay (RIA), chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) etc.These detection methods respectively have its advantage and disadvantage, but have certain limitation in clinical large-scale application.The hydrocortisone detection reagent of good, highly sensitive, the high specificity of deficient in stability in the market, especially the measured Automated inspection reagent of matter, therefore, research and develop that a kind of quality reaches clinical requirement, practical, cost performance is high, the hydrocortisone that can be applicable to automatic clinical chemistry analyzer measures the focus that reagent has become domestic and international external diagnosis reagent industry.
Summary of the invention
The defect that the present invention exists to overcome prior art, adopt unique hydrocortisone derivative to prepare the strong hydrocortisone immunogen of immunogenicity and antibody thereof, the hydrocortisone homogeneous enzyme immunoassay detection reagent prepared with this antibody can be implemented on automatic clinical chemistry analyzer hydrocortisone high-throughput, rapid detection.This detection reagent has the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, effectively can also reduce hydrocortisone testing cost, is conducive to clinical expansion and uses.
One object of the present invention is the hydrocortisone immunogen providing a kind of immunogenicity strong.
Another object of the present invention is to provide a kind of for the formation of the immunogenic hydrocortisone derivative of hydrocortisone.
Another object of the present invention is to provide a kind of hydrocortisone immunogenic preparation method.
Another object of the present invention is to provide the anti-cortisol-specif antibody of the high specificity using hydrocortisone immunogen of the present invention to prepare.
Another object of the present invention is to provide a kind of hydrocortisone detection reagent and preparation method thereof.
Hydrocortisone immunogen of the present invention, immunogenicity is high, can induce the anti-cortisol-specif antibody obtaining high-titer.This antibodies specific is high, strong with the bonding force of hydrocortisone.The hydrocortisone detection reagent prepared by this antibody, can determine the Determination of cortisol in sample quickly and accurately.The present invention is achieved by the following technical solutions:
A kind of hydrocortisone immunogen, its structural formula is as shown in formula I:
Carrier, for having immunogenic protein or polypeptide, is selected from the one of serum protein, hemocyanin and thyroglobulin.Be more preferably serum albumin, more preferably bovine serum albumin.
Described hydrocortisone immunogen is formed by connecting by hydrocortisone derivative and above-mentioned carrier.A kind of hydrocortisone derivative, its chemical structure is as shown in formula II:
The immunogenic preparation method of hydrocortisone, comprises the following steps:
(1) carrier proteins 100 ~ 300mg is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirring and dissolving in small beaker: 100 ~ 300mg hydrocortisone derivative, 1.75 ~ 5.25ml dimethyl formamide, 1.75 ~ 5.25ml ethanol, 3.5 ~ 10.5ml10mM, the potassium phosphate buffer of pH5.0,100 ~ 300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25 ~ 75mgN-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30 ~ 60min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains hydrocortisone immunogen.
A kind of anti-cortisol-specif antibody, for by the complete antibody molecule produced after hydrocortisone immunogen immune laboratory animal, or for retaining and the antibody fragment of cortisol-specif binding ability or antibody derivatives.
Described complete antibody molecule, antibody fragment or antibody derivatives are the polyclonal antibody adopting single hydrocortisone immunogen to obtain animal booster immunization, or are the monoclonal antibody obtained through somatic hybridization after immunity; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse.Be preferably rabbit.
Described anti-cortisol-specif antibody adopts ordinary method inoculation experiments animal by above-mentioned obtained hydrocortisone immunogen, gets antiserum(antisera) after booster immunization.The preparation method of anti-cortisol-specif antibody, comprises the following steps:
(1) with PBS, the BSA-hydrocortisone immunogen of above-mentioned synthesis is diluted to 0.1 ~ 3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5 ~ 5.0ml antigenic solution, laboratory animal is injected;
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 0.5 ~ 5.0ml and equivalent Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times;
(3) get blood to above-mentioned laboratory animal, separation and purification obtains the anti-cortisol-specif antibody of tiring as 1:30000 ~ 1:50000.
A kind of hydrocortisone detection reagent, containing described anti-cortisol-specif antibody and indicator, described indicator is selected from the one in enzyme reagent, radio isotope reagent, fluorescent reagent or luminescence reagent; Described enzyme reagent is made up of the substrate of cortex alcoholase mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and the substrate of enzyme is G-6-P.
A preparation method for hydrocortisone detection reagent, is characterized in that comprising following steps:
(1) reagent A: the Tris buffer solution of the Reduced nicotinamide-adenine dinucleotide of 2.018 ~ 8.072g, 5.625 ~ 22.50mM oxidation state and 0.856 ~ 3.422g, 5.625 ~ 22.50mM G-6-P, 0.5 ~ 2L55mM, pH=8.0 is made homogeneous phase enzyme substrates; Be added in above-mentioned homogeneous phase enzyme substrates by described anti-cortisol-specif antibody, the volume ratio of anti-cortisol-specif antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by cortex alcoholase mark conjugate, the volume ratio of cortex alcoholase mark conjugate and Tris damping fluid is 1:100 ~ 1:10000.
Described anti-cortisol-specif antibody and the volume ratio of homogeneous phase enzyme substrates are preferably 1:500;
Described cortex alcoholase mark conjugate and the volume ratio of Tris damping fluid are preferably 1:2500.
The preparation method of described cortex alcoholase mark conjugate comprises following steps:
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: take the glucose-6-phosphate dehydrogenase (G6PD) that 7.5 ~ 22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6 ~ 18mL
2with in the solution of 100mgNaCl, pH=9.0; Add the Reduced nicotinamide-adenine dinucleotide of 112.5 ~ 337.5mg reduction-state, 67.5 ~ 202.5mg G-6-P and 0.375 ~ 1.125mL Trivalin SF in the solution; Dropwise add 1 ~ 3mL dimethyl sulfoxide (DMSO) again;
(2) activation of hydrocortisone derivative: take 5 ~ 15mg hydrocortisone derivative under anhydrous conditions, is dissolved in 300 ~ 900 μ L dimethyl formamides; Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C; Add 1.5 ~ 4.5 μ L Tributylamines; Add 0.75 ~ 2.25 μ L isobutyl chlorocarbonate;-2 ~-8 DEG C are stirred 30 ~ 60 minutes;
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and hydrocortisone derivative: the hydrocortisone derivative solution that step (2) activates dropwise is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) dissolves; 2-8 DEG C of stirring is spent the night;
(4) purified product: connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
Hydrocortisone homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is unmixed and separated, so the substrate of enzyme and above-mentioned anti-cortisol-specif antibody are mixed.
Hydrocortisone immunogens of the present invention is strong, immunogenicity is high, and the anti-cortisol-specif antibodies specific prepared is strong, height of tiring, and with common 62 kinds of medicines without any cross reaction; Homogeneous enzyme immunoassay detection reagent containing above-mentioned anti-cortisol-specif antibody can determine the Determination of cortisol in the biological samples such as urine, serum, blood plasma easily and fast, exactly, and can on automatic clinical chemistry analyzer the multiple sample of Simultaneously test, realize the rapid mensuration of high-throughput of hydrocortisone, accuracy is high, high specificity, tolerance range is all enhanced before comparing with detection efficiency, achieve the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is the ELISA detection reaction curve of hydrocortisone;
Fig. 2 is the homogeneous enzyme immunoassay response curve of hydrocortisone;
Fig. 3 is hydrocortisone homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The immunogenic synthesis of embodiment one hydrocortisone
Hydrocortisone immunogen is by the hydrocortisone derivative shown in bovine serum albumin (BovineSerumAlbumin, BSA) Yu formula II
group is formed by connecting, and concrete steps are as follows:
1. bovine serum albumin 200mg is dissolved in 50ml0.2M, in the phosphoric acid buffer of pH8.5;
2. following chemical is joined stirring and dissolving in small beaker: 200mg hydrocortisone derivative, 3.5ml dimethyl formamide, 3.5ml ethanol, 7.0ml10mM, the potassium phosphate buffer of pH5.0,200mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mgN-hydroxy thiosuccinimide, by these chemical at room temperature stirring and dissolving reaction 30min;
3. the solution dissolved is dropped in BSA solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains hydrocortisone immunogen.
Embodiment two: the preparation of anti-cortisol-specif antibody
Hydrocortisone immunogen embodiment one prepared adopts ordinary method inoculation experiments animal rabbit, and get antiserum(antisera) after booster immunization, concrete steps are as follows:
1. with PBS, the hydrocortisone immunogen of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2.2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and equivalent Freund's incomplete adjuvant, above-mentioned experimental animal rabbit is injected once, afterwards every surrounding injection once, amount to injection 4 times.
3. the experimental animal rabbit of pair step 2 gets blood, and separation and purification obtains the anti-cortisol-specif antibody of tiring as 1:30000 ~ 1:50000.
Embodiment three: the ELISA inspection of hydrocortisone
1. the foundation of the ELISA examination criteria curve of hydrocortisone
(1) preparation of standard substance
Hydrocortisone powder (being purchased from Sigma company) is dissolved in methanol solution, is prepared into the storage liquid of 1mmol/L.Storage liquid being diluted successively with ELISA damping fluid is the standardized solution of 500.00nmol/L, 250.00nmol/L, 125.00nmol/L, 62.50nmol/L, 31.25nmol/L and 0.00nmol/L.Wherein, ELISA damping fluid contains 50.0mMTris, the BSA of 145mMNaCl and 0.25%.
(2) the ELISA method of inspection preparation standard curve of hydrocortisone is utilized
With PBS, anti-cortisol-specif antibody dilution prepared in embodiment two is become the final concentration solution of 1:8000,100 μ L/ holes are coated on 96 hole elisa plates, place 12-24h for 4 DEG C; After the above-mentioned 96 hole elisa plates being coated with anti-hydrocortisone antibody being washed 3 times with PBS, add the BSA solution of 0.5% of 200 μ L/ holes, close for 4 DEG C and place 8-16h.Then wash 3 times with PBS, add the standard substance in 20 μ L/ holes.Add the HRP-hydrocortisone conjugate of 100 μ L/ hole working concentrations again; After incubated at room temperature 30min, PBS washes plate 5 times; Then every hole adds 100 μ LTMB substrates, incubated at room 30min.Every hole adds 100 μ L stop buffers (2M sulfuric acid) again.Measure the light absorption value of 450nm.The light absorption value calibration of the 450nm corresponding to each standard substance, production standard curve, result as shown in Figure 2.
2. the detection of Determination of cortisol in testing sample
(1) testing sample is made
Preparation method: hydrocortisone powder (being purchased from Sigma company) is dissolved in the storage liquid that methanol solution makes 1mmol/L, and this storage liquid is diluted in blank diaper, 0.00 is respectively to final concentration, 50.00,250.00,450.00nmol/L, forms urine specimen that is blank, basic, normal, high concentration.This blank diaper is not containing the Healthy People urine of hydrocortisone.
(2) testing method
Utilize the ELISA method of inspection of above-mentioned hydrocortisone, the urine specimen of above-mentioned blank, basic, normal, high concentration replaced standard substance, test above-mentioned blank, basic, normal, high concentration urine specimen at the light absorption value of 450nm.
(3) test result
The typical curve of the ELISA inspection of the hydrocortisone of contrast shown in Fig. 1, calculates Determination of cortisol in each sample, and carries out 3 multiple holes mensuration to each sample, and the actual content according to hydrocortisone in above-mentioned sample calculates the rate of recovery, and result is as shown in table 1.
The ELISA of table 1 hydrocortisone detects recovery experiment
From result in table 1: the hydrocortisone rate of recovery adopting the ELISA detection reagent of hydrocortisone of the present invention to measure in different concns sample is all higher, equal > 90%, illustrate that anti-cortisol-specif antibody of the present invention may be used for the detection of hydrocortisone in sample, and result precision is high.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1. the preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
(1) accurately take the G6PDH that 15mg specification is 100KU, room-temperature dissolution contains 72.6mg (0.05M) Tris, 8mgMgCl in 12mL
2(3.3mM) with in the solution of 100mgNaCl, these pH value of solution=9.0, this step is carried out in beaker C.
(2) in above-mentioned beaker C, the Reduced nicotinamide-adenine dinucleotide (NADH) of 225mg reduction-state is added, 135mg G-6-P (G-6-P) and 0.75mL Trivalin SF (Carbitol).
(3) in above-mentioned beaker C, 2mL dimethyl sulfoxide (DMSO) (dimethysulfoxide, DMSO) is dropwise added again.
2. the activation of hydrocortisone derivative:
(1) take the above-mentioned hydrocortisone derivative of 10mg under anhydrous conditions, be dissolved in 600 μ LDMF.
(2) above-mentioned solution temperature is made to drop to-2 ~-8 DEG C.
(3) 3 μ L Tributylamines (tributylamine) are added.
(4) 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate) are added.
(5)-2 ~-8 DEG C are stirred 30 minutes.
The connection of 3.G6PDH and hydrocortisone derivative:
(1) the hydrocortisone derivative solution of above-mentioned activation is dropwise joined in the G6PDH solution of above-mentioned dissolving.
(2) 2-8 DEG C of stirring is spent the night.
4. purified product:
By the solution in G-25 gel chromatography column purification step 3, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
Embodiment five: the preparation of hydrocortisone homogeneous enzyme immunoassay detection reagent
1. the preparation of reagent A: the Reduced nicotinamide-adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g (11.25mM) G-6-P (G-6-P) are placed in beaker D, make homogeneous phase enzyme substrates with the Tris buffer solution of 1L55mM, pH=8.0; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-cortisol-specif antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase enzyme substrates is 1:500.
2. the preparation of reagent B: glucose-6-phosphate dehydrogenase (G6PD) embodiment four prepared-hapten conjugation thing is added in the Tris damping fluid of 120mM, pH=8.2, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:2500.
Embodiment six: the inspection of hydrocortisone homogeneous enzyme immunoassay and result
1. obtain typical curve:
(1) auspicious BS-480 automatic clinical chemistry analyzer reaction parameter (see table 2) advanced in years is set.
(2) operation steps is: first reagent adding A, then adds standard substance, finally adds reagent B.After adding reagent B, measure the OD of different time points
340light absorption value, calculates speed of reaction during different standards product concentration, needs the volume ratio constantly adjusting reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal graphic representation, as shown in Figure 3.
Table 2 steps auspicious BS-480 automatic clinical chemistry analyzer reaction parameter
2. pattern detection: the typical curve obtained by homogeneous enzyme immunoassay detection reagent of the present invention, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in by cortisol standards in human urine, be respectively 50.00 to concentration, 250.00,450.00nmol/L.Detection data and data analysis are in table 3.
Table 3 sample determination and precision and rate of recovery assessment
Detected result: the accuracy that homogeneous enzyme immunoassay detection reagent of the present invention measures is high, and the rate of recovery reaches 95%-105%, and precision is high, and CV is all lower than 5%.
Embodiment seven: interfering effects of drug is tested
Choose 62 kinds of Common drugs and carry out Interference Detection, adjustment concentration, to 1.00nmol/L, adopts the homogeneous enzyme immunoassay method of embodiment six to measure:
1. by reagent A contact reacts prepared by interference medicament to be measured and embodiment five, then add reagent B;
2. detect the OD of above-mentioned mixing solutions
340light absorption value, obtains the concentration of respective substance according to the typical curve of embodiment six.
62 kinds of common medicine names and measurement result are specifically see table 4.
Table 4 common interference drug determination result
Measurement result shows: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to hydrocortisone is all less than 0.01nmol/L.As can be seen here, antibody of the present invention is the specific antibody of anti-hydrocortisone, with other medicines no cross reaction.
Embodiment eight: correlation analysis
Use Beckman (chemoluminescence method) reagent and homogeneous enzyme immunoassay reagent of the present invention to carry out correlation analysis respectively to 100 routine clinical samples, the data of mensuration are see table 5.
Table 5 clinical sample measured value
Map to above-mentioned data, see Fig. 3, the linear equation obtained is: y=0.9835x+3.2603, coefficient R
2=0.9799, show that the accuracy of detection reagent of the present invention mensuration hydrocortisone clinical samples is high.It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. a hydrocortisone immunogen, its structural formula is as shown in formula I:
Carrier, for having immunogenic protein or polypeptide, is selected from the one in serum protein, hemocyanin or thyroglobulin.
2. a hydrocortisone derivative, is characterized in that, its chemical structural formula is as shown in formula II:
3. the immunogenic preparation method of hydrocortisone as claimed in claim 1, is characterized in that comprising following steps:
(1) carrier proteins 100 ~ 300g is dissolved in 25 ~ 75ml0.2M, in the phosphoric acid buffer of pH8.5;
(2) following chemical is joined stirring and dissolving in small beaker: 100 ~ 300mg hydrocortisone derivative according to claim 2,1.75 ~ 5.25ml dimethyl formamide, 1.75 ~ 5.25ml ethanol, 3.5 ~ 10.5ml10mM, the potassium phosphate buffer of pH5.0,100 ~ 300mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 25 ~ 75mgN-hydroxy thiosuccinimide, by above-mentioned chemical at room temperature stirring and dissolving reaction 30 ~ 60min;
(3) solution dissolved is dropped in carrier protein solution, and stir at 2 ~ 8 DEG C and spend the night, obtain antigen; Synthetic antigen is carried out purifying through dialysis, obtains hydrocortisone immunogen.
4. an anti-cortisol-specif antibody, for by the complete antibody molecule produced after hydrocortisone immunogen immune laboratory animal according to claim 1, or for retaining and the antibody fragment of cortisol-specif binding ability or antibody derivatives.
5. the anti-cortisol-specif antibody of one according to claim 4, it is characterized in that described complete antibody molecule, antibody fragment or antibody derivatives, for the polyclonal antibody adopting single hydrocortisone immunogen to obtain animal booster immunization, or it is the monoclonal antibody obtained through somatic hybridization after immunity; Described laboratory animal is the one of rabbit, goat, mouse, sheep, cavy or horse.
6. a preparation method for the anti-cortisol-specif antibody according to any one of claim 4-5, is characterized in that comprising following steps:
(1) with PBS, hydrocortisone immunogen is diluted to 0.1 ~ 3.0mg/ml, obtains antigenic solution, then mix with equivalent Freund's complete adjuvant with 0.5 ~ 5.0ml antigenic solution, laboratory animal is injected;
After (2) 2 ~ 3 weeks, then with the identical antigenic solution of 0.5 ~ 5.0ml and equivalent Freund's incomplete adjuvant, above-mentioned laboratory animal is injected once, afterwards every surrounding injection once, amount to injection 3 ~ 6 times;
(3) get blood to the laboratory animal of step (2), separation and purification obtains the anti-cortisol-specif antibody of tiring as 1:30000 ~ 1:50000.
7. a hydrocortisone detection reagent, containing the anti-cortisol-specif antibody described in claim 4 or 5 and indicator, described indicator is selected from the one in enzyme reagent, radio isotope reagent, fluorescent reagent or luminescence reagent; Described enzyme reagent is made up of the substrate of cortex alcoholase mark conjugate and enzyme, and enzyme mark conjugate is glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and the substrate of enzyme is G-6-P.
8. a preparation method for hydrocortisone detection reagent as claimed in claim 7, is characterized in that comprising following steps:
(1) reagent A: the Tris buffer solution of the Reduced nicotinamide-adenine dinucleotide of 2.018 ~ 8.072g, 5.625 ~ 22.50mM oxidation state and 0.856 ~ 3.422g, 5.625 ~ 22.50mM G-6-P, 0.5 ~ 2L55mM, pH=8.0 is made homogeneous phase enzyme substrates; Be added in above-mentioned homogeneous phase enzyme substrates by the anti-cortisol-specif antibody described in claim 4 or 5, the volume ratio of anti-cortisol-specif antibody and homogeneous phase enzyme substrates is 1:100 ~ 1:10000;
(2) reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by cortex alcoholase mark conjugate, the volume ratio of cortex alcoholase mark conjugate and Tris damping fluid is 1:100 ~ 1:10000.
9. the preparation method of hydrocortisone detection reagent according to claim 8, is characterized in that the preparation method of described cortex alcoholase mark conjugate comprises following steps:
(1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: take the glucose-6-phosphate dehydrogenase (G6PD) that 7.5 ~ 22.5mg specification is 100KU, room-temperature dissolution contains 72.6mg0.05MTris, 8mg3.3mMMgCl in 6 ~ 18mL
2with in the solution of 100mgNaCl, pH=9.0; Add the Reduced nicotinamide-adenine dinucleotide of 112.5 ~ 337.5mg reduction-state, 67.5 ~ 202.5mg G-6-P and 0.375 ~ 1.125mL Trivalin SF in the solution; Dropwise add 1 ~ 3mL dimethyl sulfoxide (DMSO) again;
(2) activation of hydrocortisone derivative: take 5 ~ 15mg hydrocortisone derivative under anhydrous conditions, is dissolved in 300 ~ 900 μ L dimethyl formamides; Above-mentioned solution temperature is made to drop to-2 ~-8 DEG C; Add 1.5 ~ 4.5 μ L Tributylamines; Add 0.75 ~ 2.25 μ L isobutyl chlorocarbonate;-2 ~-8 DEG C are stirred 30 ~ 60 minutes;
(3) connection of glucose-6-phosphate dehydrogenase (G6PD) and hydrocortisone derivative: the hydrocortisone derivative solution that step (2) activates dropwise is joined in the glucose-6-phosphate dehydrogenase (G6PD) solution that step (1) dissolves; 2-8 DEG C of stirring is spent the night;
(4) purified product: connect product by G-25 gel chromatography column purification, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores at 2-8 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365342A (en) * | 2017-07-17 | 2017-11-21 | 苏州博源医疗科技有限公司 | Aldosterone derivative, immunogene and synthetic method, specific antibody and detection reagent and preparation method, kit |
| CN108586562A (en) * | 2018-05-08 | 2018-09-28 | 苏州博源医疗科技有限公司 | A kind of cortex 01 derivatives and the preparation method and application thereof |
| CN111504920A (en) * | 2019-01-09 | 2020-08-07 | 北京九强生物技术股份有限公司 | 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent |
| CN114560939A (en) * | 2022-03-30 | 2022-05-31 | 广东工业大学 | Single-chain antibody for detecting cortisol and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447984A (en) * | 2014-12-20 | 2015-03-25 | 苏州博源医疗科技有限公司 | Docetaxel immunogen, anti-docetaxel specific antibody and docetaxel detection reagent |
-
2015
- 2015-07-27 CN CN201510444151.0A patent/CN105131105A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447984A (en) * | 2014-12-20 | 2015-03-25 | 苏州博源医疗科技有限公司 | Docetaxel immunogen, anti-docetaxel specific antibody and docetaxel detection reagent |
Non-Patent Citations (2)
| Title |
|---|
| F.K. ZEUGSWETTER等: "Configuration of antibodies for assay of urinary cortisol in dogs influences analytic specificity", 《DOMESTIC ANIMAL ENDOCRINOLOGY》 * |
| RONG LIU等: "One-Step Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for Direct Determination of Cortisol in Serum", 《HYBRIDOMA》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107365342A (en) * | 2017-07-17 | 2017-11-21 | 苏州博源医疗科技有限公司 | Aldosterone derivative, immunogene and synthetic method, specific antibody and detection reagent and preparation method, kit |
| CN107365342B (en) * | 2017-07-17 | 2019-08-06 | 苏州博源医疗科技有限公司 | Aldosterone derivative, immunogene and synthetic method, specific antibody and detection reagent and preparation method, kit |
| CN108586562A (en) * | 2018-05-08 | 2018-09-28 | 苏州博源医疗科技有限公司 | A kind of cortex 01 derivatives and the preparation method and application thereof |
| CN111504920A (en) * | 2019-01-09 | 2020-08-07 | 北京九强生物技术股份有限公司 | 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent |
| CN111504920B (en) * | 2019-01-09 | 2023-06-30 | 北京九强生物技术股份有限公司 | Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent |
| CN114560939A (en) * | 2022-03-30 | 2022-05-31 | 广东工业大学 | Single-chain antibody for detecting cortisol and preparation method and application thereof |
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