CN109444402A - Norepinephrine electrochemiluminescent immunoassay detection kit - Google Patents
Norepinephrine electrochemiluminescent immunoassay detection kit Download PDFInfo
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- G—PHYSICS
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- 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
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- 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
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Abstract
The invention discloses a kind of norepinephrine electrochemiluminescent immunoassay detection kits, including detection system and system for pretreating sample；Wherein detection system includes the solid phase carrier for being directly or indirectly coated with norepinephrine antibody, norepinephrine antibody-solutions, Avidin connection tracer solution and calibration object；System for pretreating sample includes enrichment material, acylating agent, methylase.The present invention is first to norepinephrine enrichment, acylated and methylation pre-treatment in urine or blood plasma; utilize c/s-diol specificity affinity media adsorbing separation norepinephrine; take the acylated scheme of acylating agent that one end is biotin; after norepinephrine after acidylating and methylating is identified and is reacted by antibody; in conjunction with the method for the Avidin that trace labelling object connects, the Noradrenaline Contents in sample are accurately measured.Kit sensitivity of the present invention and precision are high, and the automation of detection, power-assisted pheochromocytoma clinical diagnosis are realized using luminescence technology.
Description
Technical field
The present invention relates to Measurements for Biotechnique, more particularly, to a kind of norepinephrine electrochemiluminescent immunoassay detection kit.
Background technique
Norepinephrine, scientific name 1- (3,4- dihydroxyphenyl) -2- ethylaminoethanol, is after adrenaline removes N- methyl
The substance of formation, also belongs to catecholamine in chemical structure.It is both a kind of neurotransmitter, mainly by Sympathetic Postganglionic Cell Bodies
It synthesizes and secretes with intracerebral adrenergic nerve tip, be the primary transmitter and a kind of hormone of the latter's release, by adrenal gland
Medullary substance synthesis and secretion, but content is less.Norepinephrine in blood circulation is mainly from adrenal medella.
Certain tumours will lead to the abnormal metabolism of Catecholamine matter, such as pheochromocytoma, Chromaffionoma.Thermophilic chromium
Oncocyte is lasting or discontinuously largely discharges Catecholamine matters, therefore the clinical symptom for mainly having catecholamine excessively high, causes
The function and metabolic disorder of the hypertension and multiple organs of duration or paroxysmal, patient may occur in which headache, palpitaition, hidrosis three
Symptoms, the especially severe person such as sign and hypertension can Complicated with Acute Left Heart Insufficiency or cerebrovascular accidents.Low blood can also occur for this disease
Pressure, or even shock, or there is the performance that hypertension and low blood pressure alternate.Currently, the catecholamines in detection blood plasma are to examine
The laboratory techniques of disconnected pheochromocytoma routine.Clinically, the coherence check index more often carried out includes adrenaline, Yi Jiduo
Bar amine and the metabolite vanillylmandelic acid (VMA) of catecholamine etc..
The market mainstream detection method of norepinephrine mainly includes high performance liquid chromatography (HPLC), enzyme-linked exempts from present
Epidemic disease determining adsorption (ELISA), radiommunoassay, but HPLC time-consuming is more long, the measurement of at high cost and batch relatively difficult to achieve, and enzyme
Linked immunoassay method lacks accuracy and reproducibility.
Summary of the invention
It is shone using luminescence method to what norepinephrine in urine was detected the purpose of the present invention is to provide a kind of
Immunity detection reagent.
To achieve the above object, the present invention can take following technical proposals:
Norepinephrine electrochemiluminescent immunoassay detection kit of the present invention, including detection system and system for pretreating sample；
Wherein:
The detection system includes the solid phase carrier for being directly or indirectly coated with norepinephrine antibody, norepinephrine
Antibody-solutions, Avidin connect tracer solution and calibration object；
The system for pretreating sample includes enrichment material, acylating agent, methylase.
The tracer is adamantane, acridinium ester, luminol and its derivative, different luminol and its derivative, alkaline phosphorus
At least one of sour enzyme (ALP) or horseradish peroxidase (HRP).
The Avidin is recombination or native purified Avidin, the use of concentration is 1/1000~1/20000, buffering
It include the BSA or other albumen such as Casein of 1~50 g/L, isinglass etc. in liquid.
The norepinephrine antibody is that anti-norepinephrine monoclonal antibody or anti-norepinephrine are polyclonal
Antibody, the carrier material of the solid-phase coating are magnetic microsphere or microwell plate.
The diameter of the magnetic microsphere is 0.1-8 μm, and surface is connected with one or more activity functional groups, including but not
It is limited to-COOH ,-NH2,-OH, have can be magnetized rapidly under the action of an external magnetic field, the category that remanent magnetism is zero after withdrawing magnetic field
Property；The norepinephrine antibody can be coated on magnetic microsphere by direct or indirect coated method, can also be with
The form of antibody-solutions is connected on magnetic microsphere in the reaction.
The indirect coating magnetic microsphere is to be coated with indirectly by FITC or anti-FITC antibody or by secondary antibody.
The calibration object be norepinephrine antigen concentration from low to high range between the ng/mL of 0ng/mL~500
The series of calibration product of multiple and different concentration points include the bovine serum albumin(BSA) and preservative of 1~50 g/L in buffer.
Enrichment material in its system for pretreating sample is c/s-diol specificity affinity media, including boric acid affinity gel
Magnetic bead, boric acid affinity gel plate, boric acid affinity gel pipe.
The acylating agent is the biotinylated acylating agent in one end, including but not limited to 6- (dimaleoyl imino) caproic acid amber
Imide ester, N- [6- (biotin amino) caproyl] -6-aminocaprolc acid N- succinimide ester or N- succinimide base 6-
Biotin lpsilon etc.；It the use of concentration is 0.3~10 mg/mL, buffer is phosphoric acid, boric acid, carbonate buffer system.
The methylase includes but is not limited to catechol O-methyltransferase (COMT), the transfer of benzyl carbinol amine-n-methyl
Enzyme (PNMT) etc.；It the use of concentration is the mg/mL of 0.5 mg/mL~1.Methyl donor used in it includes but is not limited to S- adenosine
Methionine (SAM), methionine (MET), front three ethylethanolamine, dimethylamino caprolactone etc.；Use 0.05~1 mg/ of concentration
mL；Buffer used is any one of PBS, Tris-NaCL, Tris-HCL, pH=5.0~10.0.
The application method of kit of the present invention are as follows:
1, carry out pre-treatment to sample first: urine or plasma sample are added in c/s-diol specificity affinity media, adsorbed,
After extraction, acylating reagent is added, mixes 10~50 min of reaction, then washes away unreacted acylating reagent, cis- two will be adsorbed in
Norepinephrine elution 0.5~2 h of release on alcohol affinity media, is added 10~100 μ l of methylase solution, instead later
0.5~2 h is answered, sample is transferred to reaction cup by treated, is detected using the full-automatic detection analysis instrument of AutoLumo.
2, it detects
2.1 norepinephrine antibody or FITC- norepinephrine antibody exist in the form of antibody-solutions, connect on magnetic particle
Being connected to can be with the secondary antibody or FITC antibody of norepinephrine antibody response, specific load procedure are as follows: school after a, processing
Quasi- product and 10~100 μ l of sample are separately added into different reacting holes；B, 10~50 μ l magnetic particle suspension, 10~100 μ are added
The antibody-solutions of l；C, 37 DEG C of 15~30 min of incubation are placed under magnetic environment and clean 5 times；D, 50~200 μ l are added and are marked with and show
The avidin solution of track object；E, 37 DEG C of 15~30 min of incubation are placed under magnetic environment and clean 5 times；F, luminous substrate, detection is added
Light signal strength；G, by calibration object response curve, the noradrenaline of sample to be tested is calculated according to pattern detection light intensity meter
Cellulose content.
2.2 norepinephrines are connected to magnetic particle, specific load procedure in a manner of direct or indirect are as follows: a, processing
Calibration object and 10~200 μ l of sample are separately added into different reacting holes afterwards；B, 10~50 μ l magnetic particle suspension are added； c,
37 DEG C of 15~30 min of incubation, are placed under magnetic environment and clean 5 times；D, be added 50~200 μ l be marked with tracer Avidin it is molten
Liquid；E, 37 DEG C of 15~30 min of incubation are placed under magnetic environment and clean 5 times；F, luminous substrate is added, detects light signal strength；g,
By calibration object response curve, the Noradrenaline Contents of sample to be tested are calculated according to pattern detection light intensity meter.
The present invention uses 6 scaling methods, takes the norepinephrine calibration object of 6 various concentration gradients first, and multiple holes are surveyed
The luminous value for determining the calibration object of various concentration gradient, averages, and respectively using concentration value as X-axis, the Log value of luminous value is Y-axis, root
It carries out curve fitting according to (Lin-Log) four parametric technique.
Method using mentioned reagent box detection norepinephrine concentration includes using immune point of Full-automatic chemiluminescence
Analyzer, chemical illumination immunity analysis instrument used are preferably the full-automatic detection analysis instrument of AutoLumo (Zhengzhou Antu bioengineering
Limited liability company).
The advantage of the invention is that the shortcomings that overcoming existing norepinephrine detection method and insufficient, first to urine or
Norepinephrine enrichment, acylated and methylation pre-treatment, utilize the absorption point of c/s-diol specificity affinity media in blood plasma
It leaves away methylepinephrine, takes the acylated scheme of acylating agent that one end is biotin, gone on first kidney after acidylating and methylating
After parathyrine is identified and reacted by antibody, in conjunction with the method for the Avidin that trace labelling object connects, accurately measures and gone in sample
Methylepinephrine content.Kit sensitivity of the present invention and precision are high, and the automation of detection, power-assisted are realized using luminescence technology
Pheochromocytoma clinical diagnosis.
Detailed description of the invention
Fig. 1 is that the correlation of kit of the present invention and HPLC illustrates.
Specific embodiment
More detailed explanation is done to the present invention below by specific embodiment, in order to the reason of those skilled in the art
Solution.
Norepinephrine antibody used in the embodiment of the present invention, secondary antibody, Avidin label tracer (HRP,
ALP, luminol and its derivative, different luminol and its derivative, adamantane) to derive from Zhengzhou Yi Meinuo biotechnology limited
Company.
The reagent component (such as some necessary buffers such as cleaning solution etc.) that is not referred in detail in kit of the invention,
Reagent box package and the independent packaging container of each reagent component etc. can be carried out according to the routine operation of fields, symbol
Close relevant industries regulation.The operating procedure not referred in detail in the method for kit of the present invention can also refer to fields
Routine operation carries out.
Embodiment 1 prepares norepinephrine electrochemiluminescent immunoassay detection kit
1, solid support material is prepared
The preparation of magnetic microsphere suspension: the PBS buffer solution first by the magnetic microsphere stoste of selection Jing Guo 10 times of stoste volumes
It is activated after rinsing 2~5 times using EDC, NHS or glutaraldehyde, the magnetic microsphere after activation and concentration are 5~40 μ g/mL's
Antibody is coated with by being connected chemically either method, after the magnetic microsphere after coating is closed after over cleaning using envelope guarantor's fluid-tight,
Constant volume simultaneously dispenses, and 2~8 DEG C of environment save backup.
A, magnetic particle connection secondary antibody can be prepared using the method, b, magnetic particle connect anti-EITC antibody, c, magnetic particle
Connect the magnetic particle suspension of anti-norepinephrine antibody.
2, Avidin connection tracer solution is prepared
First, in accordance with formula Tris-NaCL(0.05-0.2M), bovine serum albumin(BSA) (BSA) (0.5%-4%), aminopyrine (ADP)
(0.5%-2%), iodine propine n-butyl amine formic acid esters (IPBC-II) (0.5%-2%), polyethylene glycol-4000 (PEG-4000) (0.1%-
2%) enzyme solutions dilution is prepared, Avidin is added according still further to 1:5000~1:50000 and connects tracer, obtains a, Avidin-
HRP solution, b, Avidin-ALP solution, c, Avidin-luminol (derivative) solution, d, Avidin-different luminol are (derivative
Object) solution, e, Avidin-adamantane solution, f, Avidin-acridine ester solution after mixing 2~8 DEG C save it is stand-by.
3, norepinephrine antibody-solutions are prepared
First, in accordance with formula Tris-NaCL(0.05-0.2M), bovine serum albumin(BSA) (BSA) (0.5%-4%), aminopyrine (ADP)
(0.5%-2%), iodine propine n-butyl amine formic acid esters (IPBC-II) (0.5%-2%), polyethylene glycol-4000 (PEG-4000) (0.1%-
2%) antibody-solutions dilution is prepared, norepinephrine antibody is added according still further to 0.5~10 μ g/mL, prepares antibody-solutions, mixes
It is saved for use for 2~8 DEG C after closing uniformly.
4, FITC connection norepinephrine antibody-solutions are prepared
Using Chadwick method, the norepinephrine antibody of 2 mg/mL is added in the phosphate buffer of pH8.0, is placed in
In ice bank, FITC is dissolved with 3% aqueous sodium carbonate and 200 μ g/mL are added, the two mixed in equal amounts, in 0~4 DEG C of refrigerator,
Persistently stir 18 on magnetic stirrer~it mixes well for 24 hours, in conjunction with after, by the norepinephrine antibody-solutions of label
Centrifugation, 2500 r/min, 20 min remove wherein a small amount of sediment, are fitted into bag filter, then be placed in a beaker, use pH8.0
0~4 DEG C of refrigerator dialysed overnight of buffered saline.
FITC- norepinephrine antibody after purification is added in the above solution according to 0.5~10 μ g/mL and prepares FITC-
Norepinephrine antibody-solutions save for use for 2~8 DEG C after mixing.
5, sample enrichment material is prepared
1.0~5.0 g c/s-diol specificity affinity medias are weighed in 250 mL beakers, 50~250 mL purified waters are added,
Oscillation mixes, and takes carrier material, and every part of 100~500 μ l of addition are uniformly distributed it by physical absorption, and nitrogen is blown or 37 DEG C
Drying.
6, acylating agent is prepared
The n,N-Dimethylformamide (DMF) for measuring certain volume weighs appropriate acylating agent, made it completely dissolved in addition DMF,
It is proportionally added into dehydrated alcohol (C again2H5OH), phosphate buffer (PBS, pH are 5.0~7.4), the concentration of acylating agent is at this time
1~10 mg/mL is saved for use for 2~8 DEG C after mixing.
7, methylase is prepared
Take methylase freeze-dried powder respectively, the purified water for being added 1.25 mL dissolved, prepare methyl donor solution (containing 0.1~
0.5 mg/mL methyl donor), COMT enzyme buffer liquid (1 moL/L Tris-HCL) it is spare, using it is preceding by volume 1:1:1 mix
(being used in 15 min after mixing).
The application method of the kit of the present invention of embodiment 2
1, Sample pretreatment: taking 10~50 μ l of urine detection sample in c/s-diol specificity affinity media, and addition 1000~
The Extraction buffer of 5000 μ l shakes 0.5~2 h on oscillator, removes liquid and pats dry, the purified water of 2 mL, oscillator is added
2~10 min of upper concussion remove liquid, add the Extraction buffer of 100~500 μ l, and the acyl of 10~100 μ l is added in every hole
Change reagent, mix immediately, 10~50 min are shaken on oscillator, removing liquid pats dry, the purified water of 2 mL of addition, on oscillator
2~10 min are shaken, 100~600 μ l is drawn and discharges buffer, shake 0.5~2 h on oscillator, take 10~100 μ l of supernatant
It is transferred in 96 reaction cups, 10~100 μ l of methylase solution is added, shakes 0.5~2 h on oscillator, uses AutoLumo
Full-automatic detection analysis instrument is detected.
2, it detects: by Avidin connection tracer, norepinephrine antibody-solutions and general conventional substrate, cleaning
For liquid composition kit: being separately added into treated calibration object and sample in reaction cup, sample-adding amount is 50 holes μ l/.
Every hole is separately added into 20 μ l of magnetic particle suspension, 50 μ l of sample, 50 μ l of antibody-solutions, 37 DEG C of 15 min of incubation after mixing,
Washing lotion is washed 6 times.100 μ l of enzyme conjugates, 37 DEG C of 17 min of incubation after mixing is added in every hole, and washing lotion is washed 6 times, every hole difference
Substrate A liquid and each 50 μ l of substrate B liquid is added, detects luminous value after mixing in 1~5 min, sample is back-calculated out by calibration object curve
This concentration value.
The performance evaluation of the kit of the present invention of embodiment 3
1, sensitivity technique
Blank limits (LOB): 5 parts of blank clinical samples close to 0 value, each sample are repeated 3 times, and are done in total 4 days, obtain 60 knots
The data of fruit nonnegative number；
Detection line (LOD): after LOB is determined, collecting 5 parts of low value clinical samples for being in 1~4 times of LOB, and each sample is repeated 3 times,
It does in total 4 days, obtains 60 data；
Functional Sensitivity (FS): using LOD test in data, 5 concentration samples survey 3 times daily, survey 4 days in total, each sample
Originally 12 are obtained as a result, calculating the mean value of each sample, SD and CV%, the concentration closest to 20% is Functional Sensitivity；Such as 1 institute of table
Show.
The kit sensitivity technique of the present invention of table 1
Table 1 is shown: first concentration that can accurately detect is 0.601 ng/mL, the concentration that second batch can be detected accurately
It is 0.631 ng/mL, is all satisfied requirement.
2, precision detects
Two batches reagent is taken to carry out precision test respectively, respectively with quality-control product and clinical high/medium/low value sample, replication 20
It is secondary, the variation of measurement concentration is calculated, measurement result is as shown in chart 2, table 3.
The detection of the kit accuracy of the present invention of table 2
The detection of the kit accuracy of the present invention of table 3
Table 2, table 3 the result shows that: the coefficient of variation is respectively less than 8%.
3, kit cross reaction examination of the present invention
Examination goes dopamine, adrenaline, metanephrine, methoxyepinephrine and levodopa to the present invention
The interference of kit, as a result as shown in table 4 below.
Table 4
4 result surface of table: kit and primary cross substance crossing-over rate of the present invention is respectively less than 0.05%, to kit of the present invention without
Interference.
4, kit clinical performance examination of the present invention
Measured simultaneously with kit of the present invention and HPLC 40 clinical diagnosis congenital adrenal hyperplasias, hypertension, pheochromocytoma,
The urine specimens such as primary hyperparathyroidism are shown in Fig. 1: y=1.0423x+0.471, r with HPLC correlation2=0.9799,
Illustrate that this kit can be used to provide help to the timely diagnosing and treating of pheochromocytoma.
Claims (10)
1. a kind of norepinephrine electrochemiluminescent immunoassay detection kit, it is characterised in that: including detection system and Sample pretreatment
System；Wherein:
The detection system includes the solid phase carrier for being directly or indirectly coated with norepinephrine antibody, norepinephrine
Antibody-solutions, Avidin connect tracer solution and calibration object；
The system for pretreating sample includes enrichment material, acylating agent, methylase.
2. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that: the tracer
For adamantane, acridinium ester, luminol and its derivative, different luminol and its derivative, alkaline phosphatase or horseradish peroxidase
At least one of enzyme.
3. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that: the Avidin
The use of concentration is 1/1000~1/20000 for recombination or native purified Avidin, includes 1~50 g/L in buffer
BSA or other albumen.
4. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that: described to remove first kidney
Upper parathyrine antibody is monoclonal or polyclonal antibody, and the carrier material of the solid-phase coating is magnetic microsphere or microwell plate.
5. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 4, it is characterised in that: the magnetism is micro-
The diameter of ball is 0.1-8 μm, and surface is connected with one or more activity functional groups；The norepinephrine antibody can lead to
It crosses direct or indirect coated method to be coated on magnetic microsphere, magnetic can also be connected in the reaction in the form of antibody-solutions
On property microballoon.
6. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 5, it is characterised in that: the indirect packet
It is to be coated with indirectly by FITC or anti-FITC antibody or by secondary antibody by magnetic microsphere.
7. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that: the calibration object
For norepinephrine antigen concentration, multiple and different concentration points of the range between the ng/mL of 0ng/mL~500 are from low to high
Column calibration object includes the bovine serum albumin(BSA) and preservative of 1~50 g/L in buffer.
8. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that: the enrichment material
Material is c/s-diol specificity affinity media, including boric acid affinity gel magnetic bead, boric acid affinity gel plate, boric acid affinity gel
Pipe.
9. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that:
The acylating agent is the biotinylated acylating agent in one end, including 6- (dimaleoyl imino) caproic acid succinimide ester, N-
[6- (biotin amino) caproyl] -6-aminocaprolc acid N- succinimide ester or N- succinimide base 6- biotin ammonia oneself
Acid.
10. norepinephrine electrochemiluminescent immunoassay detection kit according to claim 1, it is characterised in that:
The methylase includes catechol O-methyltransferase, phenylethanolamine-N-methyl transferase；It the use of concentration is 0.5
The mg/mL of mg/mL~1.
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| CN109917144A (en) * | 2019-04-15 | 2019-06-21 | 郑州伊美诺生物技术有限公司 | A kind of preparation method and applications of methoxyepinephrine specific antibody |
| CN113495157A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate diluent, total thyroxine quantitative detection kit and use method thereof |
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