CN109917144A - A kind of preparation method and applications of methoxyepinephrine specific antibody - Google Patents
A kind of preparation method and applications of methoxyepinephrine specific antibody Download PDFInfo
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- CN109917144A CN109917144A CN201910297156.3A CN201910297156A CN109917144A CN 109917144 A CN109917144 A CN 109917144A CN 201910297156 A CN201910297156 A CN 201910297156A CN 109917144 A CN109917144 A CN 109917144A
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Abstract
The invention discloses a kind of preparation methods of methoxyepinephrine specific antibody, the methoxyepinephrine specific antibody is using methoxyepinephrine coupling carrier albumen preparation artificial antigen as immunogene, and immunization experiment animal obtains.The present invention uses methoxyepinephrine coupling carrier albumen as immunogene, it is prepared for the antibody of the anti-methoxyepinephrine of specificity, the ELISA kit of methoxyepinephrine in detection sample is established with specific antibody prepared by the present invention, with adrenaline, norepinephrine, dopamine, metanephrine no cross reaction, the methoxyepinephrine content in sample can be rapidly and accurately detected, has higher sensitivity and specificity.
Description
Technical field
The present invention relates to vitro diagnostic techniques, more particularly, to a kind of system of methoxyepinephrine specific antibody
Preparation Method, the invention further relates to the specific antibody prepared detection sample in methoxyepinephrine content kit
In application.
Background technique
Chromaffin cell tumor (Pheochromocytoma, PHEO) be mainly derived from adrenal medella, sympathetic ganglion or
The thermophilic network tissue at other positions.Diagnosis chromaffin cell tumor outstanding feature object include catecholamine (Catecholamine, CA) and
Its metabolin metanephrine (Metanephrine, MN), methoxyepinephrine (Normetanephrine,
NMN).
Detection blood plasma or catecholamine in urine and its metabolin such as MN and NMN are that clinically to diagnose thermophilic network thin at present
The main method of born of the same parents' tumor.Common detection method includes high performance liquid chromatography (HPLC), capillary electrophoresis (CE), mass spectrography
(MS), gas chromatography (GC), biochemical method and enzyme linked immunosorbent assay (ELISA) etc..When clinical use, these methods respectively have
Advantage and disadvantage, HPLC method result precision is high, but takes a long time, higher cost；Biochemical method and enzyme linked immunosorbent assay are easy to operate,
But specificity and sensitivity still need to further increase.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of methoxyepinephrine specific antibody, You Benfa
The methoxyepinephrine detection kit that the antibody of bright preparation is established, can fast and accurately detect the methoxy in sample
Base Noradrenaline Contents.
To achieve the above object, the present invention can take following technical proposals:
The preparation method of methoxyepinephrine specific antibody of the present invention, the methoxyepinephrine
Specific antibody is that artificial antigen is prepared using methoxyepinephrine coupling carrier albumen as immunogene, and immunization experiment is dynamic
Object obtains；The immunogene specific the preparation method comprises the following steps:
Carrier protein is dissolved as 4mg/ml with the MES buffer of 0.05mol/L, pH=4.7 by the first step；With 0.05mol/L, pH
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt is 20mg/ml by=4.7 MES buffer；With
Methoxyepinephrine is dissolved as 2mg/ml by the MES buffer of 0.05mol/L, pH=4.7；
Second step, by methoxyepinephrine and carrier protein according to 200:1(W/W) ratio be added reaction cup in, room temperature
It mixes, according to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: methoxyepinephrine=1:50(W/W)
Ratio calculate the dosage of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, room temperature is slowly added in reaction cup,
Room temperature is protected from light overnight；
Third step, 12h that reaction solution dialysed in 4 DEG C of environment with the PBS buffer solution of 0.01mol/L, pH=7.2 is to get to immune
It is former.
The carrier protein is bovine serum albumin(BSA) (BSA), keyhole limpet hemocyanin (KLH), oralbumin (OVA) etc..
Conventional animal model, such as mouse, cavy, rabbit, goat, sheep or horse can be used in experimental animal used.
Methoxyepinephrine specific antibody of the present invention is preparing methoxyepinephrine detection
Application in kit: the antibody of preparation is coated on solid phase carrier, is handled sample with biotinylated acylating agent,
The methoxyepinephrine after being acylated in sample is set to be identified and be combined by the antibody on solid phase carrier；Wash away unbonded acyl
After agent, the Avidin for using horseradish peroxidase-labeled is added；After washing again, substrate solution, quantitative detection sample is added
In methoxyepinephrine content.
The solid phase carrier can be magnetic particle or microwell plate；The acylating agent can be 6-(dimaleoyl imino)
Caproic acid succinimide ester, N- [6-(biotin amino) caproyl] -6-aminocaprolc acid N- succinimide ester, N- succinyl are sub-
Amino 6- biotin lpsilon etc..
The sample is serum, blood plasma or urine.
The advantage of the invention is that using methoxyepinephrine coupling carrier albumen as immunogene, it is prepared for spy
The antibody of anisotropic anti-methoxyepinephrine establishes methoxyl group in detection sample with specific antibody prepared by the present invention
The ELISA kit of norepinephrine, with adrenaline, norepinephrine, dopamine, metanephrine without intersecting
Reaction, can rapidly and accurately detect the methoxyepinephrine content in sample, have higher sensitivity and special
Property.
Detailed description of the invention
Fig. 1 is that the detection kit established with antibody of the present invention is illustrated with HPLC detection method correlation.
Specific embodiment
More detailed explanation is made to the method for the present invention below by specific embodiment, in order to those skilled in the art couple
The understanding of the application.Unless otherwise specified, reagent use herein and instrument are this field conventional products, in the application
Test method used is also conventional method in this field.
Embodiment 1 prepares methoxyepinephrine immunogene (carrier protein selects KLH)
KLH is dissolved as 4mg/ml with the MES buffer of 0.05mol/L, pH=4.7 by the first step；With 0.05mol/L, pH=4.7
MES buffer by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt be 20mg/ml；Use 0.05mol/
L, methoxyepinephrine is dissolved as 2mg/ml by the MES buffer of pH=4.7；
Second step, by methoxyepinephrine and KLH according to 200:1(W/W) ratio be added reaction cup in, room temperature mix,
According to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: methoxyepinephrine=1:50(W/W) ratio
Example calculates the dosage of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and room temperature is slowly added in reaction cup, room temperature
It is protected from light overnight；
Third step, 12h that reaction solution dialysed in 4 DEG C of environment with the PBS buffer solution of 0.01mol/L, pH=7.2 is to get to immune
It is former.
Embodiment 2 prepares methoxyepinephrine specific antibody (experimental animal model selection New Zealand great Bai
Rabbit)
By the immunogene Freund's complete adjuvant prepared in embodiment 1 it is fully emulsified after, subcutaneous multiple spot is 2.5kg or so to weight
Male New Zealand White Rabbit be immunized, first immunisation dosage be 2mg/ only；28 days, 56 days difference are spaced after immune for the first time
It carries out for the second time and third time is immune, immunogene and incomplete Freund's adjuvant are mixed after emulsifying in equal volume and be immunized, and immunizing dose is
1mg/ is only.After 3rd time 10 days immune, rabbit ear vein blood sampling, polyvalent antibody Protein A column is purified, and it is anti-to obtain specificity
Methoxyepinephrine polyclonal antibody.
Embodiment 3 establishes methoxyepinephrine ELISA detection kit, and (selection of its solid phase carrier is micro- using magnetic
Grain)
1, magnetic particle is coated with
Magnetic particle stoste after taking 30 μ l to mix is washed 5 times with the PBS buffer solution of 300 μ l, then with 10% glutaraldehyde to magnetic
Activating microparticles 1 hour, then the magnetic particle after activation is washed 2 times with 7~8 PBS buffer solution of pH；Methoxyl group is gone on first kidney
Parathyrine antibody is added in magnetic bead according to the amount of 0.3 μ g/ person-portion, and 4 DEG C are coated with 2 hours.It is finally small with the confining liquid closing 2 containing BSA
When, 2-8 DEG C of magnetic bead after closing saves backup.
2, the pre-treatment of sample
It takes urine specimen 50ul to be added in reaction cup, the hydrochloric acid 300ul of 0.1mol/L is added, after sample mixes at 60 DEG C of acidifications
2h is managed, the biotinylated 6-(dimaleoyl imino of 100ul is added after being cooled to room temperature) caproic acid succinimide ester, room temperature acyl
It is detected after changing processing 30min.
3, pattern detection
The chromaffin cell tumor clinical samples 40 with HPLC method definite value from hospital are collected, take 50ul after acylated processing
It is transferred in another reaction cup, the magnetic particle that addition is coated with anti-methoxyepinephrine antibody in each reaction cup hangs
Liquid 20ul, 37 DEG C of reaction 15min after mixing；It is washed 5 times after reaction, the Avidin of HRP label is added in each reaction cup
100ul, 37 DEG C of reaction 17min after concussion mixes；It is washed 5 times after reaction, luminous substrate A and luminous bottom is added in each reaction cup
Object B mixed liquor 100ul, is protected from light 5min after mixing and is detected.
The ELISA detection kit and HPLC method established with anti-methoxyepinephrine antibody prepared by the present invention
It is simultaneously congenital adrenal hyperplasia, hypertension, chromaffin cell tumor, Graves' disease etc. to 40 clinical diagnosises
Urine specimen is detected, testing result and HPLC method testing result correlation y=1.1016x+6.6628 as shown in Figure 1:,
R2=0.9832。
Claims (5)
1. a kind of preparation method of methoxyepinephrine specific antibody, it is characterised in that: the methoxyl group removes first kidney
Upper parathyrine specific antibody is that artificial antigen is prepared using methoxyepinephrine coupling carrier albumen as immunogene, is immunized
Experimental animal obtains.
2. the preparation method of methoxyepinephrine specific antibody according to claim 1, it is characterised in that: institute
Stating carrier protein is bovine serum albumin(BSA), keyhole limpet hemocyanin or oralbumin.
3. the methoxyepinephrine specific antibody of claim 1 is preparing methoxyepinephrine detection reagent
Application in box, it is characterised in that: the antibody of preparation is coated on solid phase carrier, with biotinylated acylating agent by sample into
Row processing makes the methoxyepinephrine after being acylated in sample be identified and be combined by the antibody on solid phase carrier；It washes away not
In conjunction with acylating agent after, be added use horseradish peroxidase-labeled Avidin；After washing again, substrate solution is added, it is quantitative
Detect the content of the methoxyepinephrine in sample.
4. methoxyepinephrine specific antibody as claimed in claim 3 is preparing methoxyepinephrine inspection
Application in test agent box, it is characterised in that: the solid phase carrier is magnetic particle or microwell plate；The acylating agent is 6-
(dimaleoyl imino) caproic acid succinimide ester, N- [6-(biotin amino) caproyl] -6-aminocaprolc acid N- succinyl are sub-
Amine ester or N- succinimide base 6- biotin lpsilon.
5. methoxyepinephrine specific antibody as claimed in claim 3 is preparing methoxyepinephrine inspection
Application in test agent box, it is characterised in that: the sample is serum, blood plasma or urine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112225672A (en) * | 2020-10-16 | 2021-01-15 | 湖南苏阳医疗科技有限公司 | Methoxy norepinephrine derivative, immunogen, specific antibody, preparation method and application thereof |
| CN112266330A (en) * | 2020-10-15 | 2021-01-26 | 湖南苏阳医疗科技有限公司 | Methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, preparation method and application thereof |
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| US20120191434A1 (en) * | 2010-08-25 | 2012-07-26 | Gt Life Sciences, Inc. | Articles of manufacture and methods for modeling chinese hamster ovary (cho) cell metabolism |
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| CN109444402A (en) * | 2018-12-18 | 2019-03-08 | 郑州安图生物工程股份有限公司 | Norepinephrine electrochemiluminescent immunoassay detection kit |
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| CN112266330A (en) * | 2020-10-15 | 2021-01-26 | 湖南苏阳医疗科技有限公司 | Methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, preparation method and application thereof |
| CN112266330B (en) * | 2020-10-15 | 2023-02-24 | 长沙博源医疗科技有限公司 | Methoxyarenol derivative, immunogen, anti-methoxyadrol specific antibody, preparation method and application thereof |
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Application publication date: 20190621 |