CN109239369A - Corticotrophin assay kit and preparation method thereof - Google Patents
Corticotrophin assay kit and preparation method thereof Download PDFInfo
- Publication number
- CN109239369A CN109239369A CN201811241180.7A CN201811241180A CN109239369A CN 109239369 A CN109239369 A CN 109239369A CN 201811241180 A CN201811241180 A CN 201811241180A CN 109239369 A CN109239369 A CN 109239369A
- Authority
- CN
- China
- Prior art keywords
- buffer
- corticotropin
- concentration
- antibody
- corticotrophin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Inorganic Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of corticotrophin assay kits and preparation method thereof, belong to technical field of immunoassay, solve the problems such as prior art test accuracy is bad.The kit includes following reagent: R1: the suspension containing the coated magnetic particle of Streptavidin；R2: the suspension of the corticotropin antibody containing chemiluminescent labels label；R3: the suspension and calibration object and quality-control product of the corticotropin antibody containing biotin labeling.The corticotrophin assay kit prepared using method of the invention, detection sensitivity with higher, it is easy to operate, quantitative detection is accurate, quick the advantages that, can be widely used for the clinical detection of corticotropin.
Description
Technical field
The present invention relates to the technical field of immunoassay more particularly to a kind of corticotrophin assay kit and
Preparation method.
Background technique
Corticotropin (ACTH) is 30 nonapeptides, can promote adrenocortical hyperplasia and adrenal gland skin
The generation and secretion of matter hormone.The ACTH produced from shark, the frog, ostrich, mammal hypophysis is 30 nonapeptides, different genera
1~24 and 34~39 amino acids residues of the ACTH of animal are essentially identical, it will be apparent that architectural difference is reflected in molecule
Carboxylic petiolarea (25~33).The sequence of amino acid of its ammonia end part (1~24) is more consistent, and is during biology is active
Heart district domain, and can artificial synthesized ACTH and its ammonia petiolarea two tetradecapeptides, physiological activity is identical as natural A CTH's.25th
~33 sequences are then the part of species variation and the region of decision immunologic specificity.ACTH is still found in addition to being distributed in hypophysis
The tissue such as hypothalamus, adrenal medella, enteron aisle and placenta.It is from opiod peptide-melanotropin-in its biosynthetic process
Corticotropin original (abbreviation POMC) transformation, ACTH itself but also as α-melanotropin (α-MSH) precursor, or even
Also there is the similar peptide of ACTH in low invertebrate.ACTH can be clinically used for treating certain collagenosis, gout, branch gas
Pipe asthma etc..
ACTH (1-39) (human) amino acid sequence is as follows:
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-
Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-
Leu-Glu-Phe
ACTH can promote adrenocortical hyperblastosis and the generation of cortin and secretion, the generation of ACTH and
Secretion was secreted the cortin contained in turn by the direct regulation and control of hypothalamus cortico-trophin-releasing factor (CRF) (CRF)
It will affect hypophysis and hypothalamus, form the activity that negative-feedback regu- lation weakens them.Daily rhythm fluctuation is presented in the secretion of ACTH, falls asleep
ACTH secretion afterwards can gradually decrease, and midnight is minimum, then gradually increase again, until awakening, which is got up, advances into secretion peak, daytime is tieed up
It holds and is reduced again in reduced levels, sleep.
ACTH increase be found in primary adrenal cortical hypofunction, ectopic ACTH syndrome, Nelson syndrome,
Cushing disease, heredity adrenal cortex to ACTH not response syndrome, congenital adrenal cortical hyper plasia, periodicity ACTH,
ADH secretion increases syndrome, other (such as operation, wound, shock, hypoglycemia).ACTH reduction is found in anterior pituitary function
Decline disease, adrenal cortical adenoma, pure ACTH shortage syndrome etc..
Immunoassay method currently used for detecting ACTH mainly has enzyme-linked immunosorbent assay and chemiluminescence immune assay
Method.Enzyme-linked immunosorbent assay uses horseradish peroxidase or alkali phosphatase enzyme mark antibody, generates for catalytic luminescence substrate
Color change, it is easy to operate, but marker easy in inactivation, luminous substrate needs are protected from light, the detection sensitivity of reagent is lower, causes
Test result inaccuracy.Chemiluminescence immunoassay is formed among a kind of excitation state using catalyst chemiluminescent substance
Body issues photon when excitation state intermediate returns to stable ground state, has easy to operate, high degree of automation, detects speed
The advantages that fast.But antibody direct coated magnetic bead is be easy to cause to the agglutination phenomenon of magnetic bead, cause reagent stability poor, influences to examine
The accuracy of the result of survey.
Summary of the invention
The present invention in view of the above technical problems, provides a kind of corticotrophin assay kit and its preparation side
Method, the kit have high sensitivity, accurately for the adrenocorticotropic hormone content in quantitative detection human serum/urine
The advantages that property is good, easy to operate, at low cost.
To achieve the goals above, the invention provides the following technical scheme:
Corticotrophin assay kit, including following reagent:
R1: the suspension containing the coated magnetic particle of Streptavidin；
R2: the suspension of the corticotropin antibody containing chemiluminescent labels label；
R3: the suspension of the corticotropin antibody containing biotin labeling；
Wherein, the concentration of the coated magnetic bead of Streptavidin is 0.03%-0.1% in R1；
The concentration for the corticotropin antibody that chemiluminescent labels mark in R2 is 0.1-2 μ g/ml；
The concentration of the corticotropin antibody of biotin labeling is 0.5-2 μ g/ml in R3.
Preferably, the partial size of the magnetic particle in R1 is 1-3 μm.
Preferably, the molar ratio of corticotropin antibody and chemiluminescent labels is 1:1-1:10 in R2.
Preferably, chemiluminescent labels are acridinium ester or acridine ester derivant.
Preferably, the molar ratio of corticotropin antibody and biotin is 1:1-1:10 in R3.
Preferably, above-mentioned corticotrophin assay kit, further includes calibration object, the preparation of the calibration object
Method are as follows: the corticotropin stoste for taking 0.4 μ g/mL is added in calibration object Matrix buffer, is diluted to
The calibration object mother liquor of 4000pg/mL；Again by calibration object mother liquor be successively diluted to concentration be 2000pg/mL, 1000pg/mL,
Each concentration point of 200pg/mL, 50pg/mL, 10pg/mL, 1pg/mL, using calibration object Matrix buffer as 0pg/mL concentration
Point；Calibration object Matrix buffer by 0.1mol/L PB, 0.15mol/L sodium chloride, 1% bovine serum albumin bletilla 0.05%
Tween-20 composition, pH value 6.5.
Preferably, above-mentioned corticotrophin assay kit, further includes quality-control product, the preparation of the quality-control product
Method are as follows: calibration object mother liquor is added in calibration object Matrix buffer, being configured to concentration is the Quality Control of 1000pg/mL high concentration
Product；Calibration object mother liquor is added in calibration object Matrix buffer, being configured to concentration is 50pg/mL low concentration quality-control product.
The present invention also provides the preparation methods of above-mentioned corticotrophin assay kit, which is characterized in that
The following steps are included:
Taking concentration is the Streptavidin magnetic particle solution of 50-100mg/ml, and the TBST solution that 5-20 times of volume is added fills
Divide after mixing 10-15 minutes, be placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness；Repeated washing 3
With the 50mM MES buffer containing 0.05% Tween-20 and 0.05%Proclin300, pH6.5 at magnetic bead concentration after secondary
For the solid-phase reagent R1 of 0.03%-0.1%；
S2, reagent preparation R2, R2's the preparation method comprises the following steps:
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Raw material buffer is to contain 0.01%
The 20mM PB of SDS, pH 7.4；Label buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with acridinium ester by a mole volume ratio 1:1-1:10,37 DEG C of labels 4
After hour, Block buffer, off-period 1h is added；Block buffer is to contain the bad ammonia of 150mmol/L NaCl and 10%
The 100mmol/L P BS of acid, pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the acridine ester molecule not being coupled, centrifuge speed
For 9000rpm, centrifugation time 30min, diluted with the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0
To the reagent R2 of final concentration of 0.1-2 μ g/ml；
S3, reagent preparation R3, R3's the preparation method comprises the following steps:
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Label buffer is 100mmol/L PBS,
150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with biotin by a mole volume ratio 1:1-1:10,37 DEG C of labels 4
After hour, Block buffer, off-period 1h is added；Raw material buffer is the 20mM containing 0.01%SDS, pH 7.4
PB；Label buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the biotin molecule not being coupled, centrifuge speed
It, will be from the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0 for 9000rpm, centrifugation time 30min
Antibody after the heart is configured to the reagent R3 of final concentration of 0.5-2 μ g/ml.
The present invention also provides the detection methods of above-mentioned corticotrophin assay kit, including following step
It is rapid:
1) by sample to be tested and R2 and R3 reagent according to volume ratio 3:1:1 hybrid reaction 10min, promote adrenal gland skin in sample
The corticotropin the of matter hormone antigen and biotin labeling corticotropin first antibody, acridinium ester label
Two antibody are immunoreacted, and form biotin-corticotropin antibody-corticotropin-rush adrenal gland
Cortin antibody-acridine ester solvent；
2) R1 reagent is added with R1 reagent volume ratio 2.5:1 according still further to sample to be tested and reacts 10min, Streptavidin MagneSphere
In conjunction with biotin, magnetic bead-Streptavidin-biotin-corticotropin antibody-corticotrophins are formed
Element-corticotropin antibody-acridine ester complexes；
3) cleaning buffer solution washing is added, unreacted solution is cleaned and is removed；
4) acid exciting liquid is added, keeps acridinium ester label free；Alkaline excitation liquid is added, acridinium ester is made to emit photon；
Optical signal is acquired, photomultiplier tube reception light quantity subnumber is directly proportional to the content of corticotropin, records luminous value.
Wherein, cleaning buffer solution includes 50g/L disodium hydrogen phosphate, 10g/L sodium dihydrogen phosphate, 100g/L NaCl, 2%
The Proclin-300 of Triton-100 and 2%；
Acid exciting liquid is hydrogen peroxide and aqueous solution of nitric acid, and the mass concentration of hydrogen peroxide is 1~2%, and nitric acid rubs
Your concentration is 6~8mM；
Alkaline excitation liquid is sodium hydrate aqueous solution, and the molar concentration of sodium hydroxide is 0.3~0.4M.
Kit of the invention is using corticotropin (ACTH) in sandwich method principle quantitative detection human plasma
Biotin-corticotropin antibody complex is added in Streptavidin-magnetic particle suspension, passes through life for content
The compatible reaction of object element and Streptavidin forms magnetic microsphere-Avidin-Biotin-corticotropin compound, leads to
Antigen-antibody reaction is crossed, antigen antibody complex is formed and forms magnetic particle-biotin-corticotropin antibody-rush kidney
Compound is adsorbed on reaction cup bottom with magnetic field, washed off by upper gland cortin-corticotropin antibody-acridinium ester
Soda acid exciting liquid is added, according to the content of corticotropin in luminous intensity quantitative measurment sample in free ingredient.
Compared with prior art, the invention has the benefit that
Detection corticotrophin assay kit provided by the invention, using biotin-avidin system, effectively
Magnetic bead agglutination phenomenon caused by antibody direct coated magnetic bead is avoided, solves asking for the difference of accuracy caused by reagent stability
Topic.And biotin-avidin system is by the special role between streptavidin-biotin, by the rush of biotin labeling
Cortex hormone of aadrenaline antibody is connected on magnetic particle, while corticotropin antibody, the biotin of acridinium ester label
The corticotropin antibody of label is immunoreacted with the corticotropin in sample, forms antigen-antibody
Compound, and pass through in the reaction bonded to magnetic particle between biotin and streptavidin, using paramagnetic particle conduct
Solid phase carrier, large specific surface area improve the sensitivity of detection.In addition, this cohesive process is the generation when detecting sample
, before the use, the antibody of the coated magnetic bead of Streptavidin and biotin labeling is separated storage to kit, therefore can
To avoid the agglutination of magnetic bead caused by antibody direct coated magnetic bead.
The present invention is directly shone using acridinium ester chemiluminescent system, reduces background blank, high sensitivity, stability
Good, high specificity, good linearity, detection time is short, for clinical diagnosis provide it is more special, stable, quick, reliably detect hand
Section solves the disadvantages of blank testing height of reagent in the prior art, stability is poor, sensitivity is low, accuracy is poor.In addition, this
The detection corticotrophin assay kit provided is invented, the calibration object and quality-control product used is traceable to international mark
Quasi- substance further ensures the accuracy of test result.
The preparation method and detection method of detection corticotrophin assay kit provided by the invention, this method
Detection corticotrophin assay kit simple to operation, being prepared, detection sensitivity is high, stability is good, surveys
Test result is accurate and reliable.
Detailed description of the invention
In order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only one recorded in the present invention
A little embodiments are also possible to obtain other drawings based on these drawings for those of ordinary skill in the art.
Fig. 1 is the standard song that corticotrophin assay of embodiment of the present invention kit tests international reference materials
Line；
Fig. 2 is the standard curve of corticotrophin assay kit detection calibration product provided by the invention.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, below in conjunction with attached drawing and implementation
Example is further detailed the present invention.The source of the Streptavidin magnetic particle solution be it is commercially available, it is prompt selected from peace
Lun Science & technology Co., Ltd, article No. PL6727-1001.
The preparation of embodiment 1 calibration object, quality-control product
The corticotropin stoste 1ml for taking 0.4 μ g/mL, is added in 99ml calibration object buffer, is diluted to
The calibration object mother liquor of 4000pg/mL.Again by calibration object mother liquor be successively diluted to concentration be 2000pg/mL, 1000pg/mL,
Each concentration point of 200pg/mL, 50pg/mL, 10pg/mL, 1pg/mL, using calibration object Matrix buffer as 0pg/mL concentration
Point.The composition of calibration object buffer: by the sodium chloride of PB, 0.15mol/L of 0.1mol/L, 1% bovine serum albumin bletilla
0.05% tween-20 composition, pH value 6.5,2-8 DEG C of storage are spare.
Calibration object mother liquor 10ml is added in 30ml calibration object Matrix buffer, being configured to 40ml concentration is 1000pg/
ML high concentration quality-control product.
Calibration object mother liquor 0.5ml is added in 39.5ml calibration object Matrix buffer, being configured to 40ml concentration is
50pg/mL low concentration quality-control product.
The preparation of 2 R1 of embodiment
Taking concentration is the Streptavidin magnetic particle solution of 100mg/ml, and the TBST solution that 20 times of volumes are added mixes well
After ten minutes, it is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness；With containing after repeated washing 3 times
Have the 50mM MES buffer of 0.05% Tween-20 and 0.05%Proclin300, pH6.5 at magnetic bead concentration be 0.03%
Solid-phase reagent R1.
The preparation of 3 R1 of embodiment
Taking concentration is the Streptavidin magnetic particle solution of 50mg/ml, and the TBST solution that 5 times of volumes are added mixes well 15
It after minute, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness；With containing after repeated washing 3 times
The 50mM MES buffer of 0.05% Tween-20 and 0.05%Proclin300, pH6.5 are 0.1% at magnetic bead concentration
Solid-phase reagent R1.
The preparation of 4 R2 of embodiment
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Raw material buffer is to contain 0.01%
The 20mM PB of SDS, pH 7.4；Label buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with acridinium ester by a mole volume ratio 1:1,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Block buffer be containing 150mmol/L NaCl and 10% lysine,
The 100mmol/L P BS of pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the acridine ester molecule not being coupled, centrifuge speed
For 9000rpm, centrifugation time 30min, diluted with the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0
To the reagent R2 of final concentration of 0.1 μ g/ml.
The preparation of 5 R2 of embodiment
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Raw material buffer is to contain 0.01%
The 20mM PB of SDS, pH 7.4；Label buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with acridinium ester by a mole volume ratio 1:10,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Block buffer be containing 150mmol/L NaCl and 10% lysine,
The 100mmol/L P BS of pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the acridine ester molecule not being coupled, centrifuge speed
For 9000rpm, centrifugation time 30min, diluted with the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0
To the reagent R2 of final concentration of 2 μ g/ml
The preparation of 6 R3 of embodiment
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Label buffer is 100mmol/L PBS,
150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with biotin by a mole volume ratio 1:1,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Raw material buffer is the 20mM PB containing 0.01%SDS, pH 7.4；Mark
Note buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the biotin molecule not being coupled, centrifuge speed
It, will be from the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0 for 9000rpm, centrifugation time 30min
Antibody after the heart is configured to the reagent R3 of final concentration of 0.5 μ g/ml.
The preparation of 7 R3 of embodiment
Corticotropin antibody is taken, is label buffer by raw material buffer exchange, is centrifuged with 30KDa ultrafiltration
Pipe carries out ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Label buffer is 100mmol/L PBS,
150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with biotin by a mole volume ratio 1:10,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Raw material buffer is the 20mM PB containing 0.01%SDS, pH 7.4；Mark
Note buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the biotin molecule not being coupled, centrifuge speed
It, will be from the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0 for 9000rpm, centrifugation time 30min
Antibody after the heart is configured to the reagent R3 of final concentration of 2 μ g/ml.
The standard curve of 8 corticotropin chemical luminescence reagent kit of embodiment
The international reference materials for taking corticotropin, being configured to theoretical concentration is respectively 0pg/mL, 1pg/
It is prepared by mL, 10pg/mL, 50pg/mL, 200pg/mL, 1000pg/mL, 2000pg/mL, R1 embodiment 4 prepared by Example 2
R2, R3 prepared by embodiment 6 tests the international reference materials of above-mentioned concentration, test it is bright as shown in table 1, draw standard mark
Directrix curve is as shown in Figure 1.
The test light result of 1 international reference materials of table
| International reference materials theoretical concentration (pg/mL) | Test bright (RLU) |
| 0 | 205 |
| 1 | 647 |
| 10 | 1730 |
| 50 | 7548 |
| 200 | 27949 |
| 1000 | 120830 |
| 2000 | 223782 |
Example 1 prepare theoretical concentration be respectively 0pg/mL, 1pg/mL, 10pg/mL, 50pg/mL, 200pg/mL,
The calibration object and theoretical concentration of 1000pg/mL, 2000pg/mL are respectively 50pg/mL, 1000pg/mL quality-control product, Example 2
R2 prepared by the R1 embodiment 4 of preparation, R3 prepared by embodiment 6, test the calibration object and quality-control product of above-mentioned concentration, and test is bright
As shown in table 2, standard curve is drawn as shown in Fig. 2, the light quantity subnumber of calibration object, quality-control product is brought into international standard object
The standard curve (Fig. 1) of matter calculates calibration object, quality-control product concentration, as shown in table 2.
The assigned result of the auspicious calibration object of 2 enlightening of table, quality-control product
By above-mentioned Tables 1 and 2 data it is found that the line of corticotropin chemical luminescence reagent kit of the present invention
Property good (R=0.999), accuracy is good, and calibration object can be traceable to international reference materials.
The detection method of corticotrophin assay kit specifically:
With Full-automatic chemiluminescence immunoassay analysis meter (CM-180) for detection instrument, methodology mode is double-antibody sandwich
Method, i.e. instrument sequentially add the R3 of the sample (doubtful containing corticotropin) of 150 μ l, 50 μ l (containing biotin mark
The corticotropin antibody of note), (corticotropin containing chemiluminescent substance label is anti-by the R2 of 50 μ l
Body), react 10min after, then plus 40 μ l R1 (contain the coated magnetic particle of Streptavidin), react 10min after, clean, into
Row Magneto separate.Reactant is sent into darkroom by instrument, sequentially adds luminous substrate liquid acid exciting liquid and alkaline excitation liquid carries out instead
It answers, last recording light quantum number.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Example documented by technical solution modify perhaps equivalent replacement of some of the technical features but these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (9)
1. corticotrophin assay kit, which is characterized in that including following reagent:
R1: the suspension containing the coated magnetic particle of Streptavidin；
R2: the suspension of the corticotropin antibody containing chemiluminescent labels label；
R3: the suspension of the corticotropin antibody containing biotin labeling；
Wherein, the concentration of the coated magnetic bead of Streptavidin is 0.03%-0.1% in R1；
The concentration for the corticotropin antibody that chemiluminescent labels mark in R2 is 0.1-2 μ g/ml；
The concentration of the corticotropin antibody of biotin labeling is 0.5-2 μ g/ml in R3.
2. corticotrophin assay kit according to claim 1, which is characterized in that magnetic particle in R1
Partial size is 1-3 μm.
3. corticotrophin assay kit according to claim 1, which is characterized in that promote adrenal gland skin in R2
The molar ratio of matter hormone antibody and chemiluminescent labels is 1:1-1:10.
4. corticotrophin assay kit according to claim 3, which is characterized in that chemiluminescent labels
For acridinium ester or acridine ester derivant.
5. corticotrophin assay kit according to claim 1, which is characterized in that promote adrenal gland skin in R3
The molar ratio of matter hormone antibody and biotin is 1:1-1:10.
6. corticotrophin assay kit according to claim 1, which is characterized in that it further include calibration object,
The calibration object the preparation method comprises the following steps: take the corticotropin stoste of 0.4 μ g/mL, be added to calibration object substrate buffer
In liquid, it is diluted to the calibration object mother liquor of 4000pg/mL；Again by calibration object mother liquor be successively diluted to concentration be 2000pg/mL,
Each concentration point of 1000pg/mL, 200pg/mL, 50pg/mL, 10pg/mL, 1pg/mL, using calibration object Matrix buffer as
The point of 0pg/mL concentration；Calibration object Matrix buffer by 0.1mol/L PB, 0.15mol/L sodium chloride, 1% ox blood it is pure
Albumen and 0.05% tween-20 composition, pH value 6.5.
7. corticotrophin assay kit according to claim 6, which is characterized in that it further include quality-control product,
The quality-control product the preparation method comprises the following steps: calibration object mother liquor is added in calibration object Matrix buffer, being configured to concentration is
1000pg/mL high concentration quality-control product；Calibration object mother liquor is added in calibration object Matrix buffer, being configured to concentration is 50pg/
ML low concentration quality-control product.
8. the preparation method of corticotrophin assay kit according to claim 1-5, feature
It is, comprising the following steps:
S1, reagent preparation R1, R1's the preparation method comprises the following steps:
Taking concentration is the Streptavidin magnetic particle solution of 50-100mg/ml, and the TBST solution that 5-20 times of volume is added is sufficiently mixed
It after 10-15 minutes even, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness；After repeated washing 3 times
It is at magnetic bead concentration with the 50mM MES buffer containing 0.05% Tween-20 and 0.05%Proclin300, pH6.5
The solid-phase reagent R1 of 0.03%-0.1%；
S2, reagent preparation R2, R2's the preparation method comprises the following steps:
Take corticotropin antibody, by raw material buffer exchange be label buffer, with 30KDa ultra-filtration centrifuge tube into
Row ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Raw material buffer is to contain 0.01%SDS, pH
7.4 20mM PB；Label buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with acridinium ester by a mole volume ratio 1:1-1:10,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Block buffer be containing 150mmol/L NaCl and 10% lysine,
The 100mmol/L P BS of pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the acridine ester molecule not being coupled, centrifuge speed is
9000rpm, centrifugation time 30min are diluted to the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0
The reagent R2 of final concentration of 0.1-2 μ g/ml；
S3, reagent preparation R3, R3's the preparation method comprises the following steps:
Take corticotropin antibody, by raw material buffer exchange be label buffer, with 30KDa ultra-filtration centrifuge tube into
Row ultrafiltration centrifugation, centrifuge speed 9000rpm, centrifugation time 30min；Label buffer is 100mmol/L PBS,
150mmol/L NaCl, pH6.0；
Corticotropin antibody is uniformly mixed with biotin by a mole volume ratio 1:1-1:10,37 DEG C label 4 hours
Afterwards, Block buffer, off-period 1h is added；Raw material buffer is the 20mM PB containing 0.01%SDS, pH 7.4；Mark
Note buffer is the 100mmol/L PB containing 150mmol/L NaCl, pH6.0；
Ultrafiltration centrifugation is carried out with 30KDa ultra-filtration centrifuge tube after closing, removes the biotin molecule not being coupled, centrifuge speed is
9000rpm, centrifugation time 30min will be centrifuged with the 100mmol/L PB buffer containing 150mmol/L NaCl, pH6.0
Antibody afterwards is configured to the reagent R3 of final concentration of 0.5-2 μ g/ml.
9. the detection method of corticotrophin assay kit according to claim 1, which is characterized in that including
Following steps: by sample to be tested and R2 and R3 reagent according to volume ratio 2:1:1 hybrid reaction 10min, according still further to sample to be tested with
R1 reagent volume ratio 2.5:1 is added R1 reagent and reacts 10min, and washing is added exciting liquid and carries out luminescence-producing reaction, acquires optical signal,
Record luminous value.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811241180.7A CN109239369A (en) | 2018-10-24 | 2018-10-24 | Corticotrophin assay kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811241180.7A CN109239369A (en) | 2018-10-24 | 2018-10-24 | Corticotrophin assay kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109239369A true CN109239369A (en) | 2019-01-18 |
Family
ID=65081543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811241180.7A Pending CN109239369A (en) | 2018-10-24 | 2018-10-24 | Corticotrophin assay kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109239369A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112540171A (en) * | 2020-12-04 | 2021-03-23 | 郑州标源生物科技有限公司 | Adrenocorticotropic hormone quality control product and preparation method thereof |
| CN117417446A (en) * | 2022-07-18 | 2024-01-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548930A (en) * | 2018-03-30 | 2018-09-18 | 迈克生物股份有限公司 | Corticotropin chemiluminescence detection kit |
-
2018
- 2018-10-24 CN CN201811241180.7A patent/CN109239369A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548930A (en) * | 2018-03-30 | 2018-09-18 | 迈克生物股份有限公司 | Corticotropin chemiluminescence detection kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112540171A (en) * | 2020-12-04 | 2021-03-23 | 郑州标源生物科技有限公司 | Adrenocorticotropic hormone quality control product and preparation method thereof |
| CN112540171B (en) * | 2020-12-04 | 2023-12-22 | 郑州标源生物科技有限公司 | Corticotropin quality control product and preparation method thereof |
| CN117417446A (en) * | 2022-07-18 | 2024-01-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
| CN117417446B (en) * | 2022-07-18 | 2024-04-19 | 东莞市朋志生物科技有限公司 | Anti-corticotropin antibodies or functional fragments thereof, reagents and kits for detecting corticotropin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107656071B (en) | NT-proBNP detection kit and use method thereof | |
| EP1016867B1 (en) | Immunoassay method for bnp | |
| US4578361A (en) | Creatinine antibody | |
| CN111323577B (en) | Anti-cardiolipin antibody IgG magnetic particle chemiluminescence immunoassay kit | |
| JPH0926423A (en) | Synthetic calibrator for use in immunoassay containing body under test formed as composite for inactive carrier moleculeor partial alignment thereof | |
| CN111175491A (en) | sBCMA magnetic particle chemiluminescence immunoassay kit and preparation method and application thereof | |
| CN111781385A (en) | NT-proBNP detection kit and preparation method thereof | |
| JP2000508075A (en) | Luminescence-specific binding assay | |
| CN104697829B (en) | Acidic treatment agent, sample preprocessing method, kit and detection method for the detection of I chemiluminescence immunoassays of IGF- | |
| WO2006088624A2 (en) | Stabilized compositions containing natriuretic peptides | |
| CN108444988A (en) | Thyroglobulin chemiluminescence detection kit | |
| CN109239369A (en) | Corticotrophin assay kit and preparation method thereof | |
| CN101949945B (en) | Kit for detecting free thyroxin by using magnetic particles as solid-phase carriers and preparation method thereof | |
| CN111007269A (en) | Total I type collagen amino-terminal extension peptide chemiluminescence immunoassay kit and preparation method thereof | |
| JPH0667960B2 (en) | Method for separating analyte bound to binding protein and sample preparation reagent for measuring vitamin B12 in serum | |
| CN108333370A (en) | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects cTnI kits | |
| CN110031635A (en) | Flash type homogeneous chemistry luminescence technology detects cardiac muscle troponin I/T method | |
| CN110398589B (en) | Kit for quantitatively detecting content of anti-mullerian hormone and detection method thereof for non-disease diagnosis purpose | |
| CN113238055A (en) | Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof | |
| JP3819612B2 (en) | Method for immunological measurement of β-hCG | |
| JP5997446B2 (en) | Liquid reagent for immobilizing thyroid hormone and use thereof | |
| CN111381046A (en) | Calprotectin chemiluminescence immunoassay kit and preparation method thereof | |
| JPH07198721A (en) | Buffer solution for immunological measurement | |
| CN108414736B (en) | Method for detecting target object by using chemiluminescence enhancer modified antibody | |
| JPH11311624A (en) | Assay surface enabling analysis material liberation stage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190118 |
|
| RJ01 | Rejection of invention patent application after publication |