CN109187971A - Neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
Neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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- CN109187971A CN109187971A CN201810939822.4A CN201810939822A CN109187971A CN 109187971 A CN109187971 A CN 109187971A CN 201810939822 A CN201810939822 A CN 201810939822A CN 109187971 A CN109187971 A CN 109187971A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
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- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The present invention provides a kind of neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof, belongs to vitro detection technical field.The kit includes: the neuronspecific enolase monoclonal antibody of Streptavidin magnetic particle suspension liquid, the neuronspecific enolase monoclonal antibody of chemiluminescent labels label and coupling label substance markers.The present invention provides a kind of preparation method of neuronspecific enolase chemiluminescence immune detection reagent kit.Neuronspecific enolase chemiluminescence immune detection reagent kit of the invention is full automatic measurement sample, and directly give numerical value, reduce manual operation error, and it realizes unattended, time needed for shortening clinical detection, detection accuracy is higher simultaneously, and reagent and instrument form closed system, and systematic error is small.
Description
Technical field
The invention belongs to vitro detection technical fields, are related to a kind of neuronspecific enolase (NSE) chemiluminescence
Immunity detection reagent and preparation method thereof.
Background technique
NSE is that catalysis 2-phosphoglyceric acid becomes phosphoric acid alkene in the process for neuron and neuroendocrine cell tricarboxylic acid cycle
The key enzyme of alcohol of formula pyruvic acid.Gene nucleotide series overall length 2423bp encodes 434 amino acid residues, and biological half-life can
20h can be greater than, molecular weight 78kD, isoelectric pH 4.7 is a kind of acid protease.NSE is enolase gene family member
One of, the dimer that enolase is made of tri- subunits of α, β, γ, isodynamic enzyme is divided into α α, β β, γ γ, α γ and α β five
Kind.α subunit is primarily present in liver, kidney etc. and organizes, therefore the enolase (NNE) of referred to as Non nervous system；β subunit is primarily present in
Skeletal muscle and cardiac muscle, the referred to as enolase (MSE) of muscle specific；γ subunit is primarily present in nerve fiber, γ γ, α γ
The isodynamic enzyme of composition is that neuron and neuroendocrine cell are peculiar, therefore are named as neuronspecific enolase (neuro-
Specificenolase, NSE).
In terms of clinical application, for NSE mainly as the marker of central nervous system damage, the dynamic change of secretion can
Indicate the sensibility and specificity of nervous system injury.It is usually used in diagnosing tumor, monitoring therapeuticing effect and prognosis evaluation etc..It is a variety of
Central nervous system disease is in the cerebral injury as caused by cerebral injury, brain tumor, retinal damage and other reasons, NSE in blood
Different degrees of raising can all occur in concentration, and NSE can be used as the sensibility and specificity index of damage.
NSE is for diagnosis of small cell lung cancer, the tumor marker with high specific and high sensitivity, specificity
Up to 80%~90%.It can be used for antidiastole, the therapeutic effect after monitoring Small Cell Lung Cancer radiotherapy, chemotherapy.With neuron spy
Specific enolase monitors the recurrence of Small Cell Lung Cancer, determines early 4~12 weeks of recurrence than clinic；Neuron specific enolase
Enzyme can also be used in the antidiastole of neuroblastoma and the nephroblastoma, and monitor the change of illness state of neuroblastoma,
Evaluate curative effect and report recurrence；
NSE shows very high immunocompetence in endocrine tumors, and can directly detect in serum, therefore can
To be used to monitor the disease progression of tumor patient, therapeutic effect is evaluated.Serologic detection is than radiographic test, anatomical examination side
Just rapidly, therefore NSE as tumor marker in clinic using very extensive.
The domestic and import reagent box testing principle that China is approved listing at present mainly has enzyme-linked immunization (ELISA), electricity
Chemiluminescence immunoassay (ECLI), Time-resolved Fluoimmunoassay (TRFIA) and Chemiluminescence immunoassay (CLIA).Traditional enzyme
Linked immunosorbent assay is not only cumbersome, and the degree of automation is low, is affected by human factors greatly, and specificity and sensitivity all need to be mentioned
It is high.Electrochemiluminescence immunoassay (ECLI), Time-resolved Fluoimmunoassay (TRFIA) and Chemiluminescence immunoassay (CLIA) have
The advantages of high sensitivity, the range of linearity wide, easy to operate, easy to accomplish automation is that ideal clinical micro biochemical is examined point
Analysis means.But neuronspecific enolase (NSE) is clinically detected at present mainly based on external import reagent, it is external
Import reagent is expensive, and delivery date is long, and heavy financial burden and inconvenience are brought to patient, is unfavorable for pushing away in basic hospital
And.
Chemiluminescence immunoassay technology is a kind of highly sensitive microdetermination technology, and it is high, specific to have sensibility
By force, the features such as measurement range is wide, reagent stability is good, easy to operate.Currently based on the detection of chemiluminescence immune assay principle
Instrument is successfully commercialized, as Abbott Laboratories' diagnostic companies (Abbott) ARCHITECT (i) type chemical illumination immunity analysis instrument and its
Matched reagent box, using acridinium ester label antibody as detection probe, it is reaction carriers that antibody, which is coated with magnetic bead, and combines automation
Detection system, can quickly detect a variety of disease markers.But based on Chemiluminescence Immunoassay for detecting the domestic of NSE
The research of kit is relatively fewer, is not able to satisfy domestic demand.
Summary of the invention
The purpose of the present invention is to solve it is existing detection NSE method detection time it is long, sensitivity is low, poor repeatability
The problem of, and a kind of neuronspecific enolase chemiluminescence immune detection reagent kit is provided and preparation method thereof.
Present invention firstly provides a kind of neuronspecific enolase chemiluminescence immune detection reagent kit, the kits
Include:
Streptavidin magnetic particle suspension liquid
The neuronspecific enolase monoclonal antibody of chemiluminescent labels label
The neuronspecific enolase monoclonal antibody of coupling label substance markers.
Preferably, in the kit, Streptavidin magnetic particle suspension liquid mass percent be 0.01%~
1%.
Preferably, in the Streptavidin magnetic particle suspension liquid, the partial size of Streptavidin magnetic particle is 0.05~3
μm。
Preferably, in the neuronspecific enolase monoclonal antibody of the chemiluminescent labels label, mind
It is 1:(1~20 through first specificity olefinic alcohol enzyme monoclonal antibody and chemiluminescent labels molar ratio), chemiluminescent labels
The concentration of the neuronspecific enolase monoclonal antibody of label is >=0.1 μ g/mL.
Preferably, the chemiluminescent labels are acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium.
Preferably, in the neuronspecific enolase monoclonal antibody of the coupling label substance markers, neuron
Specificity olefinic alcohol enzyme monoclonal antibody and the molar ratio of coupling marker are 1:(1~20), the nerve of coupling label substance markers
The concentration of first specificity olefinic alcohol enzyme monoclonal antibody is >=0.7 μ g/mL.
Preferably, the coupling marker is biotin.
Preferably, the kit further includes Chemoluminescent substrate, the Chemoluminescent substrate include A liquid and
B liquid, the A liquid are nitric acid solution, and B liquid is sodium hydroxide solution.
Preferably, the kit further includes neuronspecific enolase calibration object,
The neuronspecific enolase calibration object be concentration be respectively 0.00ng/mL, 5ng/mL, 20ng/mL,
The neuronspecific enolase solution of 50ng/mL, 200ng/mL and 500ng/mL.
The present invention also provides the preparations of the chemiluminescence immune detection reagent kit of above-mentioned neuronspecific enolase
Method includes the following steps:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer；
Step 2: the preparation of the neuronspecific enolase monoclonal antibody of chemiluminescent labels label
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, carbonic acid buffer is then added,
The centrifugation of chemiluminescent labels solution is added after mixing, is protected from light being put into after the centrifugation seal of tube in magazine, is then put into magazine
It is mixed in gas bath constant temperature oscillator, confining liquid is added, is put into gas bath constant temperature oscillator and mixes, by the antibody closed by pure
Change, collect, be then placed in buffer and dilute, saves；
Step 3: the preparation of the neuronspecific enolase monoclonal antibody of coupling label substance markers
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, TRIS buffer is then added,
Coupling marker solution centrifugation is added after mixing, lysine is added after label reaction, and the reaction was continued, finally by reaction solution desalination
It is put into buffer and dilutes after column purification, collection, save.
Beneficial effects of the present invention
Present invention firstly provides a kind of neuronspecific enolase chemiluminescence immune detection reagent kit, the kits
Using Streptavidin magnetic particle suspension liquid as solid phase carrier, double-antibody sandwich principle is detected, in conjunction with luminous intensity and spirit
The higher chemiluminescent substance confrontation NSE of sensitivity is marked, using hydrogen peroxide chemistry luminescence system, with double antibody sandwich method
Realize the quantitative detection to NSE, which selects acridinium ester for the marker material of chemiluminescence immunoassay system, the material
The energy jump generated when having excitation state to return to ground state is direct chemiluminescence, does not need the participation of enzyme, saves time and cost；
It can be firmly combined together using the analog of Streptavidin MagneSphere and biotin labeling, reduce non-specific adsorption,
Improve the accuracy of test sample, strong antijamming capability.Neuronspecific enolase chemiluminescence immunoassay inspection of the invention
Test agent box is full automatic measurement sample, and directly gives numerical value, reduces manual operation error, and realize it is unattended,
Time needed for shortening clinical detection, while detection accuracy is higher, reagent and instrument form closed system, and systematic error is small.
Detailed description of the invention
Fig. 1 is the NSE standard curve that embodiment 5 obtains.
Specific embodiment
It is right in the following with reference to the drawings and specific embodiments in order to keep advantages of the present invention, purpose and method more full and accurate understandable
A specific embodiment of the invention is described in detail.Many details are explained in the following description in order to understand, but this
Invention can be implemented with different with other modes that are describing.
Present invention firstly provides a kind of neuronspecific enolase chemiluminescence immune detection reagent kit, the kits
Include:
Streptavidin magnetic particle suspension liquid
The neuronspecific enolase monoclonal antibody of chemiluminescent labels label
The neuronspecific enolase monoclonal antibody of coupling label substance markers.
According to the present invention, in the kit, Streptavidin magnetic particle suspension liquid mass percent is preferably
0.01%~1%, more preferably 0.072%.
According to the present invention, in the chain Streptavidin magnetic particle suspension liquid, the partial size of mould affine biscuit porcelain particle is preferably
0.05~3 μm, more preferably 3 μm.When the partial size of Streptavidin magnetic particle is lower than 0.05 μm, antigen or antibody and magnetic bead
Percentage bound is low, and it is relatively low to may cause whole light quantity subnumber；It is non-specific when the partial size of Streptavidin magnetic particle is higher than 3 μm
It is obvious in conjunction with effect, it may cause kit poor sensitivity etc..
According to the present invention, in the neuronspecific enolase monoclonal antibody of the chemiluminescent labels label,
Neuronspecific enolase monoclonal antibody and chemiluminescent labels molar ratio are preferably 1:(1~20), more preferably
1:3~20；The concentration of the neuronspecific enolase monoclonal antibody of chemiluminescent labels label is >=0.1 μ g/mL.
The chemiluminescent labels are preferably acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium, more preferably acridinium ester.
According to the present invention, in the neuronspecific enolase monoclonal antibody of the coupling label substance markers, nerve
First specificity olefinic alcohol enzyme monoclonal antibody and the molar ratio of coupling marker are preferably 1:(1~20), more preferably 1:5~
20；The concentration of the neuronspecific enolase monoclonal antibody of coupling label substance markers is >=0.7 μ g/mL, the coupling
Marker is preferably biotin.
According to the present invention, the kit further includes Chemoluminescent substrate, and the Chemoluminescent substrate includes A liquid
With B liquid；The A liquid is nitric acid solution, and B liquid is sodium hydroxide solution.
According to the present invention, the kit further includes neuronspecific enolase calibration object.
It is respectively 0.00ng/mL, 5ng/mL, 20ng/ that the neuronspecific enolase calibration object, which is preferably concentration,
The neuronspecific enolase solution of mL, 50ng/mL, 200ng/mL and 500ng/mL.
The present invention also provides the preparations of the chemiluminescence immune detection reagent kit of above-mentioned neuronspecific enolase
Method includes the following steps:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer；
The concentration of the Streptavidin magnetic particle solution is preferably 50~100mg/ml；Streptavidin magnetic particle is molten
The volume ratio of liquid and TBST solution is preferably (0.5~1): (5~15)；The mixing time is preferably 10-15 minutes, described
Buffer be 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5 or 100mM PBS, 0.1% tween-
20,0.1%Proclin300, pH7.2；The concentration of the solid-phase reagent is preferably 0.01%~1%, more preferably
0.05%；The source of the Streptavidin magnetic particle solution be it is commercially available, be selected from agilent company, article No. PL6827-1006.
Step 2: the preparation of the neuronspecific enolase monoclonal antibody of chemiluminescent labels label
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, is preferably centrifuged 10s at room temperature
~30s guarantees that antibody is located at centrifuge tube bottom position, phosphate buffer is then added, chemiluminescent labels are added after mixing
Solution centrifugation, the centrifuging temperature is preferably room temperature, and centrifugation time is preferably 0.5min~3min；It will be put after the centrifugation seal of tube
Enter to be protected from light in magazine, then magazine is put into gas bath constant temperature oscillator (25 DEG C) and is mixed, the mixing time is preferably 2-
4h is added confining liquid, is put into gas bath constant temperature oscillator and mixes, and the off-period is preferably 1-2h, anti-by what is closed
Body is then placed in buffer and dilutes by purifying, collection, saves；
The quality (μ g) of the neuronspecific enolase monoclonal antibody: the body of chemiluminescent labels solution
Product (μ l) is preferably (250-500): (5-15)；The concentration of the chemiluminescent labels solution is preferably 2-2.5mg/mL；
The confining liquid is preferably lysine, and mass fraction is preferably 20-25%；The buffer is 50mM MES, 0.05%
Tween-20,0.05%Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20,0.1%Proclin300, pH6.0；
Step 3: the preparation of the neuronspecific enolase monoclonal antibody of coupling label substance markers
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, it is ensured that antibody is located at centrifuge tube bottom
Portion position is preferably centrifuged 10s~30s at room temperature, and TRIS buffer is added after centrifugation and mixes well, and coupling label is then added
Object is centrifuged 30-45s with centrifuge under room temperature, and 2-8 DEG C of mixing, the mixing time is preferably 2-4h, and closing is added
Liquid is put into mixing (25 DEG C) in gas bath constant temperature oscillator, and the off-period is preferably 1-2h, and the antibody closed is passed through
Purifying is collected, and is then placed in buffer and is diluted, and is saved；
The quality (μ g) of the neuronspecific enolase monoclonal antibody: the volume of coupling marker solution
(mL) preferably (500-750): (2-5)；The concentration of the coupling marker solution is preferably 2-3mg/mL；The closing
Liquid is preferably lysine, the source of the coupling marker be it is commercially available, be selected from ACROBiosystems company.The buffering
Liquid be 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5 or 100mM PBS, 0.1% Tween-20,
0.1%Proclin300, pH6.0.
Neuronspecific enolase chemiluminescence immune detection reagent kit of the invention is for detecting neuron-specific
When property enolase, using Full-automatic chemiluminescence immunoassay analysis meter (CM180) to neuronspecific enolase calibration object
It is detected, draws standard curve, be built in computer software；Then clinical sample is tested according to demand, according to the light quantity of sample
The concentration of subnumber calculating neuronspecific enolase；Finally neuronspecific enolase Full-automatic chemiluminescence is exempted from
Epidemic disease analysis system carries out the evaluation of performance (sensitivity, linear, anti-interference/specificity).Combined with specific embodiments below to this hair
It is bright to do further detailed description.
Embodiment 1: the preparation of neuronspecific enolase chemiluminescence immune detection reagent kit:
(1) preparation of Streptavidin magnetic particle suspension liquid:
The Streptavidin magnetic particle solution 0.5mL (50mg) of concentration 100mg/mL is taken, the TBST solution that 10mL is added fills
Divide and mix 10min, be placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Make after repeated washing 3 times
With 50mM MES, 0.05% tween, 0.05%Proclin300, it is 0.05% that magnetic bead concentration is made into the buffer of pH6.5
Solid-phase reagent, 2~8 DEG C of preservations.
(2) acridinium ester label technique:
250ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
PBS buffer solution is added afterwards, mixes well, 5 μ l 2mg/mL acridinium ester DMF solutions is added after mixing, with centrifuge room temperature condition
Lower centrifugation 0.5min.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature oscillation later
Device (25 DEG C) mixes 4h.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed mixes, envelope
Closing the time is 1h.By the antibody closed using (25 prepacked column of sephadex G) is purified on AKTA purifying instrument, delayed with PB
Fliud flushing elution, Fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.It is when use that neuron after purification is special
Specific enolase enzyme antibody concentrated solution 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 are dilute
It releases to final concentration of 0.1 μ g/ml, 2~8 DEG C of preservations.
(3) the neuronspecific enolase monoclonal antibody preparation process of coupling label substance markers:
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
TRIS buffer solution is added afterwards, mixes well, the DMF solution of 2ml 2mg/mL biotin is added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 30s.2-8 DEG C mixes 4 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25
DEG C), middling speed mixes, off-period 1h.The antibody closed is purified using AKTA purifying instrument (250 column of sephadex G),
It is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By mind after purification when use
Through first specificity olefinic alcohol enzyme antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5's
Buffer is diluted to final concentration of 1.0 μ g/mL, 2~8 DEG C of preservations.
The preparation of 2 neuronspecific enolase chemiluminescence immune detection reagent kit of embodiment:
(1) preparation of Streptavidin magnetic particle suspension liquid:
Taking concentration is 0.72 milliliter of the Streptavidin magnetic particle solution (72mg) of 100mg/ml, and the TBST that 15mL is added is molten
It after liquid mixes well 15 minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 100mM PBS, 0.1% Tween-20,0.1%Proclin300, the buffer of pH7.2 after 3 times is
0.072% solid-phase reagent, 2~8 DEG C of preservations.
(2) acridinium ester label technique:
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 30s)
TRIS is added afterwards and rushes solution, mixes well, 15 μ l 2.5mg/mL acridinium ester DMF solutions is added after mixing, with centrifuge room temperature item
45s is centrifuged under part.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature oscillator later
(23 DEG C) mix 3.5h.25% lysine confining liquid of 2mL is added, is put into gas bath constant temperature oscillator (23 DEG C), middling speed mixes, envelope
Closing the time is 1.5h.By the antibody closed using (25 prepacked column of sephadex G) is purified on AKTA purifying instrument, PB is used
Buffer elution, Fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By neuron after purification when use
Specificity olefinic alcohol enzyme antibody concentrated solution 100mM PBS, 0.1% Tween-20,0.1%Proclin300, the buffering of pH6.0
Liquid is diluted to final concentration of 0.2 μ g/ml, 2~8 DEG C of preservations.
(3) the neuronspecific enolase monoclonal antibody preparation process of coupling label substance markers:
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
TRIS buffer solution is added afterwards, mixes well, the DMF solution of 2ml 2mg/mL biotin is added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 30s.2-8 DEG C mixes 4 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25
DEG C), middling speed mixes, off-period 1h.The antibody closed is purified using AKTA purifying instrument (250 column of sephadex G),
It is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By mind after purification when use
Through first specificity olefinic alcohol enzyme antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5's
Buffer is diluted to final concentration of 1.0 μ g/mL, 2~8 DEG C of preservations.
750ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
After phosphate buffer solution is added, mix well, after mixing be added 5ml 3mg/mL biotin DMF solution, with centrifuge room temperature
Under the conditions of be centrifuged 45s.4 DEG C mix 6 hours.25% lysine confining liquid of 3mL is added, is put into gas bath constant temperature oscillator (23 DEG C),
Middling speed mixes, off-period 2h.The antibody closed is purified using AKTA purifying instrument (250 column of sephadex G), uses PB
Buffer elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By neuron after purification when use
Specificity olefinic alcohol enzyme antibody concentrated solution 100mM PBS, 0.1% Tween-20,0.1%Proclin300, the buffering of pH6.0
Liquid is diluted to final concentration of 1.2 μ g/ml, 2~8 DEG C of preservations.
The preparation of 3 neuronspecific enolase chemiluminescence immune detection reagent kit of embodiment:
(1) preparation of Streptavidin magnetic particle suspension liquid:
The Streptavidin magnetic particle solution 0.5mL (50mg) of concentration 100mg/mL is taken, the TBST solution that 10mL is added fills
Divide and mix 10min, be placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Make after repeated washing 3 times
With 50mM MES, 0.05% tween, 0.05%Proclin300, it is 0.05% that magnetic bead concentration is made into the buffer of pH6.5
Solid-phase reagent, 2~8 DEG C of preservations.
(2) acridinium ester label technique:
250ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
PBS buffer solution is added afterwards, mixes well, 5 μ l 2mg/mL acridinium ester DMF solutions is added after mixing, with centrifuge room temperature condition
Lower centrifugation 0.5min.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature oscillation later
Device (25 DEG C) mixes 2h.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed mixes, envelope
Closing the time is 1h.By the antibody closed using (25 prepacked column of sephadex G) is purified on AKTA purifying instrument, delayed with PB
Fliud flushing elution, Fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.It is when use that neuron after purification is special
Specific enolase enzyme antibody concentrated solution 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 are dilute
It releases to final concentration of 0.1 μ g/ml, 2~8 DEG C of preservations.
(3) the neuronspecific enolase monoclonal antibody preparation process of coupling label substance markers:
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
TRIS buffer solution is added afterwards, mixes well, the DMF solution of 2ml 2mg/mL biotin is added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 30s.2-8 DEG C mixes 3 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25
DEG C), middling speed mixes, off-period 2h.The antibody closed is purified using AKTA purifying instrument (250 column of sephadex G),
It is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By mind after purification when use
Through first specificity olefinic alcohol enzyme antibody concentrated solution 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5's
Buffer is diluted to final concentration of 1.0 μ g/mL, 2~8 DEG C of preservations.
Embodiment 4: neuronspecific enolase chemiluminescence immune detection reagent kit detection method:
With Full-automatic chemiluminescence immunoassay analysis meter (CM180) for detection instrument, methodology is double antibody sandwich method, instrument
Sequentially add the serum sample of 10 μ L, the neuronspecific enolase monoclonal antibody of 50 μ L acridinium ester labels, 50 μ L life
The neuronspecific enolase monoclonal antibody and 40 μ L Streptavidin magnetic particles of object element label.After being incubated for 20min, into
Row Magneto separate.Reactant is sent into darkroom by instrument, primary that luminous substrate liquid A liquid (HNO is added3Solution) and B liquid (NaOH solution)
It is reacted, last recording light quantum number.
Embodiment 5: neuronspecific enolase chemiluminescence immune detection reagent kit performance evaluation
(1) accuracy (rate of recovery)
The NSE sample (A) that concentration is about 150ng/mL (its concentration deviation is allowed to be ± 20%) is added to 2~5ng/mL
In sample (B), the volume ratio between the sample A being added and sample B is 1:9, calculates rate of recovery R, the rate of recovery by formula (1)
It should be in (85%~115%) range.
In formula: the R-rate of recovery；
V-sample A volume；
V0The volume of-sample B；
The detectable concentration after sample A is added in c-sample B；
c0The detectable concentration of-sample B；
csThe concentration of-sample A.
Table 1NSE accuracy test data
(2) blank limits
Zero primary calibration method product or Sample dilution 20 times are measured in parallel, its signal value is recorded, calculate mean M and standard deviation
SD, and the value of M+2SD is calculated, it measures adjacent concentration primary calibration object 3 times, records its signal value, be averaged.According to zero one
It is primary out that concentration-signal value result between grade calibration object and adjacent concentration primary calibration object carries out two o'clock linear regression fit
Equation, signal value corresponding to M+2SD bring equation into, and gained concentration is blank limit, as a result should be less than 2.5ng/mL.
Table 2NSE blank limits test data
(3) linear
At least five kinds of concentration will be diluted to by a certain percentage close to the high level sample of the range of linearity upper limit, wherein low value concentration
Sample must be close to the lower limit of the range of linearity.The sample standard deviation of each concentration is repeated to detect 3 times, average value is calculated, result is averaged
Value and dilution ratio carry out straight line fitting with least square method, calculate linearly dependent coefficient r, as a result should meet the requirements (linear model
It encloses for 2.5ng/mL~500ng/mL, linearly dependent coefficient r >=0.9900).
It can be seen from the linear test data of table 3NSE and Fig. 1 NSE invention reagent and import reagent standard curve comparison diagram
Invention reagent has the broader range of linearity and higher linear related coefficient compared with import reagent, and then can be with
Guarantee to provide more accurate inspection result in detection range.
The linear test data of table 3NSE
(4) repeated
Respectively repeat detection 10 times with the samples of low, high two kinds of various concentrations, calculate 10 measurement results average value (M) and
Standard deviation (SD) calculates the coefficient of variation (CV) by formula (2), and the coefficient of variation (CV) answers≤8.0%.
CV=SD/M × 100% ... ... ... ... ... ... ... (2)
In formula: the CV-coefficient of variation；
SD-measurement result standard deviation；
M-measurement result average value.
Table 4NSE reperformance test data
(5) difference between batch
Same a sample is detected respectively with 3 lot number kits, then the interassay coefficient of variation between 3 lot number kits
(CV)≤15.0%.
Table 5NSE difference between batch test data
(6) thermal stability
Kit is placed 14 days under the conditions of 37 DEG C, and calibration object and clinical light quantity subnumber decaying answer≤± 15%.
6 heat stability testing data of table
(7) anti-interference
Testing result is not by jaundice (bilirubin < 72mg/dL), hyperlipemia (disease) (fat emulsion < 1800mg/dL, class wind
The influence of the wet factor (1500U/mL) and biotin (< 72ng/mL).
Standard: the rate of recovery is in ± 10% range of initial value.
(8) specific
Measure the non-specific neuronal enolase (NNE) that concentration is 500 μ g/L and the cell angle that concentration is 100 μ g/L
Protein 19 segment (CYFRA21-1) sample, measurement result should be not higher than 0.5 μ g/L.
Claims (10)
1. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit, which is characterized in that the kit includes:
Streptavidin magnetic particle suspension liquid
The neuronspecific enolase monoclonal antibody of chemiluminescent labels label
The neuronspecific enolase monoclonal antibody of coupling label substance markers.
2. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is, in the kit, Streptavidin magnetic particle suspension liquid mass percent is 0.01%~1%.
3. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is, in the Streptavidin magnetic particle suspension liquid, the partial size of Streptavidin magnetic particle is 0.05~3 μm.
4. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is, in the neuronspecific enolase monoclonal antibody of the chemiluminescent labels label, neuronal specificity
Enolase monoclonal antibody and chemiluminescent labels molar ratio are 1:(1~20), the nerve of chemiluminescent labels label
The concentration of first specificity olefinic alcohol enzyme monoclonal antibody is >=0.1 μ g/mL.
5. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1 or 4,
It is characterized in that, the chemiluminescent labels are acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium.
6. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is, in the neuronspecific enolase monoclonal antibody of the coupling label substance markers, neuron specific enolase
Changing enzyme monoclonal antibody and being coupled the molar ratio of marker is 1:(1~20), the neuronal specificity alkene of coupling label substance markers
The concentration of Enolase monoclonal antibody is >=0.7 μ g/mL.
7. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1 or 6,
It is characterized in that, the coupling marker is biotin.
8. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is that the kit further includes Chemoluminescent substrate, and the Chemoluminescent substrate includes A liquid and B liquid, the A
Liquid is nitric acid solution, and B liquid is sodium hydroxide solution.
9. a kind of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1, special
Sign is that the kit further includes neuronspecific enolase calibration object,
The neuronspecific enolase calibration object is that concentration is respectively 0.00ng/mL, 5ng/mL, 20ng/mL, 50ng/
The neuronspecific enolase solution of mL, 200ng/mL and 500ng/mL.
10. a kind of system of neuronspecific enolase chemiluminescence immune detection reagent kit according to claim 1
Preparation Method, which comprises the steps of:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, it is placed on magnetic separator, until supernatant is without muddiness,
Supernatant is abandoned, magnetic particle is left and taken, is made into solid-phase reagent after cleaning in buffer；
Step 2: the preparation of the neuronspecific enolase monoclonal antibody of chemiluminescent labels label
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, carbonic acid buffer is then added, is mixed
The centrifugation of chemiluminescent labels solution is added afterwards, is protected from light being put into after the centrifugation seal of tube in magazine, magazine is then put into gas bath
It is mixed in constant temperature oscillator, confining liquid is added, is put into gas bath constant temperature oscillator and mixes, the antibody closed process is purified,
It collects, is then placed in buffer and dilutes, save；
Step 3: the preparation of the neuronspecific enolase monoclonal antibody of coupling label substance markers
Neuronspecific enolase monoclonal antibody is put into centrifuge tube and is centrifuged, TRIS buffer is then added, is mixed
Coupling marker solution centrifugation is added afterwards, lysine is added after label reaction, and the reaction was continued, finally that reaction solution desalting column is pure
It is put into buffer and dilutes after change, collection, save.
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| CN110196336A (en) * | 2019-06-04 | 2019-09-03 | 迪瑞医疗科技股份有限公司 | Hematopoietin chemiluminescence immune detection reagent kit and preparation method thereof |
| CN110736837A (en) * | 2019-09-17 | 2020-01-31 | 北京九强生物技术股份有限公司 | Latex immunoturbidimetry detection kit for neuron-specific enolase |
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| CN111381046A (en) * | 2020-03-12 | 2020-07-07 | 迪瑞医疗科技股份有限公司 | Calprotectin chemiluminescence immunoassay kit and preparation method thereof |
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