CN104140464B

CN104140464B - People's GFAP epitope peptide, antigen, antibody, purposes and test kit

Info

Publication number: CN104140464B
Application number: CN201310173533.5A
Authority: CN
Inventors: 朱建安
Original assignee: ANQUN BIOENGINEERING Co Ltd SHENZHEN
Current assignee: ANQUN BIOENGINEERING Co Ltd SHENZHEN
Priority date: 2013-05-10
Filing date: 2013-05-10
Publication date: 2016-12-28
Anticipated expiration: 2033-05-10
Also published as: CN104140464A

Abstract

The present invention relates to people's GFAP epitope peptide, antigen, antibody, purposes and test kit.The aminoacid sequence of people's GFAP epitope peptide of the present invention, for one of sequence shown in sequence table SEQ ID NO.1 and SEQ ID NO.2.The GFAP antigen of the present invention is prepared with protein carrier coupling by people's GFAP epitope peptide.GFAP monoclonal antibody or the polyclonal antibody of the present invention are prepared by the GFAP antigen of the present invention.The GFAP monoclonal antibody of the present invention or polyclonal antibody are used for preparing GFAP external diagnosis reagent case.People's GFAP epitope peptide of the present invention has good antigenicity, can produce monoclonal antibody and the polyclonal antibody of high degree of specificity with its antigen prepared (immunogen) immune animal, thus can be applicable to the vitro detection of people GFAP.

Description

People's GFAP epitope peptide, antigen, antibody, purposes and test kit

Technical field

The invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to people's glial fibrillary acidic protein (GFAP) antigen table Position peptide, the GFAP specific antigen prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, described anti- Body purposes on preparation people's GFAP external diagnosis reagent case, people's GFAP external diagnosis reagent case, and a kind of for quantitatively inspection Survey fluorescence immune chromatography test paper of people's GFAP albumen and preparation method thereof in determinand.

Background technology

In recent years, the research of neural biochemical marker has been caused the attention of many researcheres, has found a kind of reliable, non- Invasive, can reflect that the biochemical marker of central nervous system damage scope and prognosis is the common aspiration of numerous researcher. By to blood and the research of cerebrospinal fluid (CSF), it has now been found that some biochemical markers can be used to judge nervous system Damage range and prognosis is predicted, this is better than the imaging examinations such as CT and MRI disease early stage.Wherein, Glial fibrillary acidic protein (GFAP) is exactly such a biochemical marker.

Glial fibrillary acidic protein (GFAP) be a kind of molecular weight be the acidic protein of 50-52KDa, it belongs to cytoskeleton Albumen, is Astrocytic marker protein, have in spider cell abundant, uniquely express.Glial fibrillary acidic protein is Ripe astrocyte distinctive intermediate filament composition, the evolution of nervous physiology function and various pathology is had important by it Effect.

Under normal circumstances, GFAP is by the power adjustment of astrocyte.The GFAP positive star glue of hypertrophy after damage Cell plastid can promote the mitosis of astrocyte, and makes primitive progenitors differentiation become the astrocyte of maturation. Various central nervous system injury all can cause astrocyte to react.The cell number that is masked as of astrocyte reaction increases Adding, cyton is loose, and astrocyte branch increases, and the expression of GFAP strengthens.Therefore, GFAP can be as maincenter god Through system injury scope and the biochemical marker of prognosis.Additionally, the expression that GFAP is in glioma also there will be exception, because of This its can also as reflect gliomatous grade malignancy Specific marker.

Research finds, GFAP and nervous system pathology process (such as cerebral trauma, cerebral infarction, apoplexy, neurodegenerative disease) There is obvious dependency.When the central nervous system of patient occurs damage, GFAP overflows from the neurogliocyte of damaged Going out, enter intercellular fluid, the GFAP of solubility is entered cerebrospinal fluid by intercellular fluid, enters blood through the blood brain barrier destroyed and follows Ring.Therefore, it can by the GFAP level in patients serum to judge the state of an illness and the prognosis of nervous system injury.

The optimal method of the GFAP level in detection serum is immune detection.Therefore, searching suitably has immunity The GFAP epitope peptide of originality, prepare specific GFAP antigen and antibody for emphasis.

Summary of the invention

For the problem in the presence of the above-mentioned prior art of solution, the invention provides a kind of people's GFAP epitope peptide, use GFAP specific antigen prepared by this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, it is at preparation people GFAP Purposes on test kit, and people's GFAP external diagnosis reagent case.

Specifically, the invention provides:

A kind of people's GFAP epitope peptide, the aminoacid sequence of wherein said GFAP epitope peptide be both it One:

(1)Thr-Val-Arg-Gln-Lys-Leu-Gln-Asp-Glu-Thr-Asn-Leu-Arg-Leu-Glu-Ala- Glu-Asn-Asn-Leu-Ala-Ala-Tyr；

(2)Tyr-Gln-Glu-Ala-Leu-Ala-Arg-Leu-Glu-Glu-Glu-Gly-Gln-Ser-Leu-Lys- Asp。

Present invention also offers a kind of GFAP antigen, it is by making described people's GFAP epitope peptide (1) and carrier Albumen coupling is prepared from.

Present invention also offers a kind of GFAP antigen, it is by making described people's GFAP epitope peptide (2) and carrier Albumen coupling is prepared from.

Present invention also offers a kind of people's GFAP antibody, it is the monoclonal antibody being prepared from by described GFAP antigen Or polyclonal antibody, wherein said GFAP antigen is by making described people's GFAP epitope peptide (1) and carrier protein couplet It is prepared from.

Present invention also offers a kind of people's GFAP antibody, it is the monoclonal antibody being prepared from by described GFAP antigen Or polyclonal antibody, wherein said GFAP antigen is by making described people's GFAP epitope peptide (2) and carrier protein couplet It is prepared from.

Present invention also offers according to described people's GFAP antibody in the purposes prepared on people's GFAP external diagnosis reagent case.

Present invention also offers a kind of people's GFAP external diagnosis reagent case, it comprises described people's GFAP antibody as being coated Antibody, wherein said coated antibody is preferably monoclonal antibody.

Preferably, described test kit also comprises binding antibody, and described binding antibody is described people's GFAP antibody, and should Binding antibody is preferably polyclonal antibody, and when described binding antibody derive from described people's GFAP epitope peptide (1) and (2), during one in, described coated antibody derives from the another one in described people's GFAP epitope peptide (1) and (2).

Preferably, described test kit also comprises the second antibody of enzyme labelling.

The present invention compared with prior art has the advantages that:

1. people's GFAP epitope peptide of the present invention has good antigenicity, with its antigen prepared (immunogen) immunity Animal can produce monoclonal antibody and the polyclonal antibody of high degree of specificity.

2. the GFAP monoclonal antibody prepared by the present invention and polyclonal antibody can high special with in blood sample GFAP combine.

3. people's GFAP external diagnosis reagent case of the present invention can detect the glial fibrillary acidic protein in serum effectively The level of GFAP, can be used to judge neural damage range and be predicted prognosis.

4. fluorescence analysis is combined by the present invention with flash chromatography immunological technique, it is provided that a kind of for detection by quantitative The fluorescence immune chromatography test paper of people GFAP albumen in determinand, with the people's GFAP albumen in this detection paper determinand, operation letter Just, quickly, only need just can complete for 10 minutes sample detection, and detection range width, specificity are high, sensitivity is good, it is possible to rapidly The assisted diagnosis state of an illness in time, monitors prognosis.

5. the present invention is during the fluorescence immune chromatography test paper preparing described people's GFAP albumen, by substantial amounts of test Grope, optimize the preparation condition of each side so that when detecting with the fluorescence immune chromatography test paper of the present invention, the fluorescence letter back of the body Ratio is greatly improved, thus improves detection sensitivity and credible result degree；Additionally, the present invention is also by the detection zone of reagent paper and matter The change of the fluorescence intensity ratio in control district carrys out the content of GFAP in response sample, and this only examines or check detection zone with traditional chromatographic technique Absolute fluorescence intensity compare, decrease the impact of external condition and background etc. to the full extent, further increase detection knot Really credibility.

Detailed description of the invention

Below by way of the description of detailed description of the invention, the invention will be further described, but this is not the limit to the present invention System, those skilled in the art are according to the basic thought of the present invention, and various modifications may be made or improves, but without departing from this The basic thought of invention, the most within the scope of the present invention.

One, people GFAP epitope peptide

People's GFAP albumen specifically described herein be it known in the art, its aminoacid sequence be it known in the art, permissible The specialized databases such as NCBI are found.

The invention provides a kind of people's GFAP epitope peptide (1) and (2), its aminoacid sequence is respectively such as sequence table SEQ Shown in ID No.1 and SEQ ID No.2, for:

(1)T-V-R-Q-K-L-Q-D-E-T-N-L-R-L-E-A-E-N-N-L-A-A-Y；With

(2)Y-Q-E-A-L-A-R-L-E-E-E-G-Q-S-L-K-D。

The present inventor gropes through substantial amounts of theoretical research and experiment, and final screening obtains two kinds and has good Antigenic epitope peptide.

GFAP epitope peptide (1) is one section of people's GFAP albumen n end the 150th to 172 and contains 23 amino acid whose peptide fragments.

GFAP epitope peptide (2) is one section of the 324th to 340, people's GFAP PROTEIN C end and contains 17 amino acid whose peptide fragments.

The two peptide fragment is respectively provided with the feature that hydrophilic, antigenicity are strong and are readily synthesized.

At present, the present invention studies discovery, and the GFAP epitope peptide of the present invention has a following function:

1. there is antigenicity；2. after being connected with carrier protein, produce specific antibody as immunogen stimulating animal；3. The antibody prepared with epitope peptide can specifically be combined with people GFAP.

The preparation method of the GFAP epitope peptide of the present invention can be by chemical synthesis: utilize American AB I431A type polypeptide Automatic synthesizer, by Solid phase synthesis epitope peptide.The epitope peptide (1) of the present invention and the molecular weight of (2) are respectively 2690.27,1979.35, available mass spectrum is determined, and measures the epitope peptide sequence synthesized by identifying by peptide sequence Row.The purity thin layer chromatography of peptide fragment and high performance liquid chromatography are evaluated, and measure the concentration of epitope peptide.

Two, GFAP antigen

Present invention also offers a kind of GFAP antigen, it is by making in people's GFAP epitope peptide (1) and (2) of the present invention One be prepared from carrier protein couplet.Specifically, the invention provides GFAP antigen (1) and (2), described GFAP resists Former (1) is prepared from carrier protein couplet by making people's GFAP epitope peptide (1) of the present invention；Described GFAP antigen (2) By making people's GFAP epitope peptide (2) of the present invention be prepared from carrier protein couplet.The GFAP antigen of the present invention has Immunogenicity and specificity, be a kind of immunogen, can be used to immune animal thus prepare specific GFAP antibody.In the present invention In, the example of available carrier protein includes KLH(keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin OVA etc.. Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is preferable, and closes with immune animal relationship System farther out, is difficult to cause cross reaction as carrier protein with it, is therefore preferred.

Three, GFAP monoclonal antibody, GFAP polyclonal antibody and people's GFAP external diagnosis reagent case

Present invention also offers people's GFAP monoclonal antibody and people's GFAP polyclonal antibody, described antibody can be utilized respectively GFAP antigen (1) and (2) (immunogen) immune animal of the present invention are prepared.Preparation method can use the conventional skill of this area Art, specifically can be found in embodiment 2.

The GFAP monoclonal antibody of the present invention and polyclonal antibody may be used for preparing people's GFAP external diagnosis reagent case, should GFAP in tissue, cell or body fluid can be detected by test kit based on immunization method, preferably in blood preparation GFAP detect.

Therefore, the invention provides a kind of people's GFAP external diagnosis reagent case, it comprises people's GFAP monoclonal of the present invention Antibody or polyclonal antibody.

It is currently known and can be used for the immunization experiment method of Clinical Laboratory and mainly include following several: ELISA method, chemiluminescence Method, fluorescent chromatographic method, colloid gold immune algoscopy etc..

And ELISA method includes following several types: double antibody sandwich method detection antigen, dual-antigen sandwich method detection antibody, Indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, capture is coated method and surveys antibody etc..

People's GFAP external diagnosis reagent case of the present invention preferably employs ELISA double antibody sandwich method to detect GFAP albumen. This test kit can comprise coated antibody, binding antibody, the second antibody of enzyme labelling and/or the instrument of necessity and reagent etc..

Preferably, described people's GFAP external diagnosis reagent case uses people's GFAP monoclonal antibody of the present invention as being coated Antibody.Here, term " coated antibody " refers to the antibody being coated in the ELISA Plate of solid phase.Examine additionally, described people GFAP is external Disconnected test kit further preferably comprises people's GFAP polyclonal antibody using as binding antibody, wherein, when described binding antibody derives from this Invention people's GFAP epitope peptide (1) and (2) in one time, described coated antibody derives from described epitope peptide (1) (2) another one in.Here, term " binding antibody " refers to tie with determined antigen and enzyme-labeled secondary antibody in test kit The specific antibody closed.Described test kit can also comprise the second antibody of enzyme labelling, and this second antibody can be goat anti-rabbit igg Antibody, described enzyme labelling can be horseradish peroxidase, alkali phosphatase etc..

In the test kit of the present invention, it is also possible to comprise any reagent needed for detection or instrument, the most pre-coated plate, wash Wash liquid, developer, stop buffer etc..

Four, for the fluorescence immune chromatography test paper of detection by quantitative people's GFAP albumen

Present invention also offers a kind of fluorescence immune chromatography test paper of people GFAP albumen in detection by quantitative determinand, should Reagent paper detects described people's GFAP albumen by double antibody sandwich method, wherein:

Described double antibody sandwich method uses the GFAP monoclonal antibody being marked with fluorescent microsphere as capture antibody, institute State a GFAP monoclonal antibody and derive from the one in people's GFAP epitope peptide (1) and (2)；And

Described double antibody sandwich method also uses the 2nd GFAP monoclonal antibody as detection antibody, described 2nd GFAP Dan Ke Grand antibody sources another one in people's GFAP epitope peptide (1) and (2)；

Described people's GFAP epitope peptide (1) and (2) is respectively as follows:

(2)Tyr-Gln-Glu-Ala-Leu-Ala-Arg-Leu-Glu-Glu-Glu-Gly-Gln-Ser-Leu-Lys- Asp。

In the present invention, in double antibody sandwich method fluorescence immune chromatography test paper, " capture antibody " refers to first special The opposite sex identifies the antibody of determined antigen, and it is generally arranged on pad；" detection antibody " refers to that another kind can be known by specificity The antibody of other determined antigen, it identifies the different epitope on determined antigen molecule respectively from capture antibody, and it is the most solid It is scheduled on the detection zone of reaction film.

In the present invention, a described GFAP monoclonal antibody can be prepared from by a GFAP antigen, and this is first years old GFAP antigen can be prepared from carrier protein couplet by making the one in described people's GFAP epitope peptide (1) and (2)； And described 2nd GFAP monoclonal antibody can be prepared from by the 2nd GFAP antigen, the 2nd GFAP antigen can be by making Another one in described people's GFAP epitope peptide (1) and (2) is prepared from carrier protein couplet.

In the present invention, the example of available carrier protein includes KLH(keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin OVA etc..Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is preferable, And with immune animal sibship farther out, it is difficult to cause cross reaction as carrier protein with it, is therefore preferred.

Preferably, the particle diameter of fluorescent microsphere used in the fluorescence immune chromatography test paper of the present invention is 320nm to 400nm, Being preferably 360nm, the fluorescent material on fluorescent microsphere can be Fluorescein isothiocyanate, RB 200, tetramethyl different sulfur cyanogen Acid rhodamine or X-rhodamine etc., the most preferably X-rhodamine (is purchased from Shanghai Jing Chun company).The microsphere material of fluorescent microsphere Material can be the copolymerization formed with other monomer copolymerization by polystyrene, polymethyl methacrylate or methyl methacrylate Thing, the example of other monomer is styrene etc..The excitation wavelength of fluorescent microsphere can be 350～600nm, preferably 390nm；Send out Ejected wave length can be 500～700nm, preferably 615nm.

In the present invention, the maximum excitation wavelength of fluorescent microsphere is relatively big with transmitting wavelength difference, illustrates that fluorescent microsphere has relatively Big Stokes (Stokes) displacement, so, the ambient interferences of fluorescent test paper is relatively low, does immunochromatography label with this microsphere There is stronger advantage.

In a specific embodiment, the fluorescence immune chromatography test paper of the present invention has base plate, and at this base plate Chromatography direction when upper edge uses is provided with sample pad, pad, reaction film, absorbent filter, described sample with the way of contact successively Product pad is for loading testing sample in use, and it is mono-that described pad is provided with the described GFAP being marked with fluorescent microsphere Clonal antibody, described reaction film includes that detection zone and quality control region, described detection zone are coated with described 2nd GFAP monoclonal antibody, Described quality control region is coated with can be combined anti-anti-with the described GFAP monoclonal antibody specificity being marked with fluorescent microsphere Body.

Preferably, the sample pad of the fluorescence immune chromatography test paper of the present invention, pad, reaction film, absorbent filter can edge Chromatography direction during use overlaps successively and is arranged on base plate.On reaction film, spaced detection zone and quality control region can be, But being not limited to, form, detection zone and the quality control region such as line, band, block are preferably by 3mm to 8mm.

In the present invention, reaction film is preferably the most not fluorescent celluloid under the wavelength more than 550nm Film.Additionally, base plate does not the most have photoluminescent property.

Generally, conventional chromatographic test paper assembly (reaction film, base plate etc.) has the obvious fluorescence back of the body under 550nm wavelength Scape, this produces the biggest interference to the detection of fluorescence signal.The present invention does not sends out under the wavelength more than 550nm by using The nitrocellulose filter of fluorescence and the base plate of low Poison character, thus overcome the defect of conventional fluorescent reagent paper.Additionally, the present invention Fluorescent material X-rhodamine used can produce stronger fluorescence signal, thus substantially increases fluorescence signal-to-background ratio further, makes Obtain and can distinguish signal and background well, and then improve detection sensitivity.

In the present invention, the material of sample pad and pad can use material commonly used in the art, such as, sample pad Can be glass fibre with pad.

Of the present invention can be combined with the GFAP monoclonal antibody specificity being marked with fluorescent microsphere anti-anti- Body can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, the most preferably sheep anti-mouse igg monoclonal anti Body, compared with polyclonal antibody, monoclonal antibody specificity is higher.

In a specific embodiment, the fluorescence immune chromatography test paper of the present invention in use, drips in sample pad Adding sample liquid (blood sample as containing GFAP), under capillarity, sample liquid moves to absorbent filter one end, at pad Forming immune complex with the described GFAP monoclonal antibody being marked with fluorescent microsphere, this immune complex moves further Dynamic, detection line is combined with described 2nd GFAP monoclonal antibody the immune complex forming double-antibody sandwich, and is not formed The a GFAP monoclonal antibody then anti antibody on nature controlling line being marked with fluorescent microsphere of immune complex is combined.This process Need 10 minutes to 15 minutes, afterwards, detect with fluorescence detector, if band does not occurs at nature controlling line, then examination is described Paper lost efficacy；If band occurs at nature controlling line, and detect and band does not occurs at line, then without people's GFAP albumen in explanation sample； If band all occurring on nature controlling line and detection line, then containing people's GFAP albumen in explanation sample.

In yet another aspect, the invention provides and a kind of prepare the fluorescence of people GFAP albumen in detection by quantitative determinand The method of immune chromatography test paper, it comprises the following steps:

1) offer is marked with a GFAP monoclonal antibody of fluorescent microsphere；

2) provide pad, on described pad, be wherein coated the described GFAP Dan Ke being marked with fluorescent microsphere Grand antibody；

3) provide reaction film, wherein on described reaction film, fix the 2nd GFAP Dan Ke along the interval, chromatography direction when using Grand antibody and anti antibody, to form detection zone and quality control region respectively；With

4) on base plate, successively sample pad, described pad, described is set with the way of contact along chromatography direction when using Reaction film, absorbent filter, thus make described fluorescence immune chromatography test paper.

The method of the present invention can also include the step 5) that the fluorescence immune chromatography test paper made cuts into proper width.

The present inventor is groped by substantial amounts of test, optimize preparation the present invention for detecting people's GFAP albumen The condition of each step of method of fluorescence immune chromatography test paper so that the fluorescence immune chromatography test paper of the present invention can Obtain for people's GFAP albumen and meet the result that Clinical detection requires, i.e. detection range width, specificity are high, sensitivity is good.

It is preferred, therefore, that in the method for the invention, described step 1) includes:

A) carbodiimide activation fluorescent microsphere is used, it is preferable that by aqueous dispersions or the MES buffer dispersion liquid of fluorescent microsphere Mix through ultrasonic Treatment and with carbodiimide, thus activate described fluorescent microsphere；

B) fluorescent microsphere of the activation that washing step a) is obtained, it is preferable that the fluorescence of the activation that step a) is obtained Microsphere N-hydroxy thiosuccinimide-citrate buffer solution washing, dispersion, and through ultrasonic Treatment；

C) fluorescent microsphere labelling the oneth GFAP monoclonal antibody obtained by step b), it is preferable that step b) is obtained The fluorescent microsphere obtained and GFAP monoclonal antibody mixing, with BSA-ethanolamine buffer blind, centrifugal, with BSA-tween water Solution disperses, and through ultrasonic Treatment, thus obtains being marked with a GFAP monoclonal antibody of fluorescent microsphere.

In a specific embodiment of the present invention, described step 1) includes:

A) take the fluorescent microsphere aqueous dispersions of 1 (w/v) %, 10000rpm to 15000rpm low temperature (such as 10 DEG C) centrifugal 5 to 10 minutes, remove supernatant, precipitate is distributed in distilled water or the first wash buffer (the MES aqueous solution of 0.1M) of 500 μ l, ultrasonic Ripple (240W) processes 1 to 2 minute, repeats above procedure three times, adds carbodiimide 10mg to 50mg, stirs 10～15 minutes, Thus activate described fluorescent microsphere；

B) fluorescent microsphere of the activation that washing step a) is obtained, it is preferable that the fluorescence of the activation that step a) is obtained Microsphere is centrifuged 5 to 10 minutes under 1000rpm to 15000rpm, and precipitate is distributed to 1ml coupling buffer (20～100mM N-hydroxy thiosuccinimide-citrate buffer solution)) in, ultrasound wave (240W) processes 1 to 2 minute, repeat above procedure Three times；

C) fluorescent microsphere labelling the oneth GFAP monoclonal antibody obtained by step b), it is preferable that according to 1 μ l to 3 μ l The ratio of the fluorescent microsphere of activation, the fluorescent microsphere that step b) is obtained and a GFAP described in antibody (8mg/ml)/100 μ l Monoclonal antibody mixes, and stirs 1.5～3 hours (preferably 2 hours), add 1ml Block buffer (1 (w/ under room temperature (25 DEG C) V) %BSA-0.05M ethanolamine), continue stirring 1 hour, under 10000rpm to 15000rpm centrifugal 5 to 10 minutes, repeat from The heart 3 times, is distributed to precipitate in 500 μ l wash buffers at end (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution), ultrasonic Ripple (240W) processes 1 to 2 minute, is settled to 500 μ l with described whole wash buffer.

Preferably, in the method for the invention, described step 2) including: a GFAP Dan Ke of fluorescent microsphere will be marked with Grand antibody antibody diluent (1% (w/v) BSA-0.01MPBS(pH7.2) buffer) be diluted, be diluted to 0.5～ 2mg/ml, preferably 1mg/ml, then with micropipettor even application on pad, post-drying or vacuum freezing do Dry.The step 2 of method in the present invention) in, being coated of the described GFAP monoclonal antibody being marked with fluorescent microsphere is dense Degree is 0.5～2mg/ml, preferably 1mg/ml.

Preferably, described step 3) includes: with metal spraying machine, described 2nd GFAP monoclonal antibody and anti antibody are drawn nitre Using as detection zone and quality control region on acid cellulose film (solid phase carrier), make detection zone and quality control region is spaced apart 3mm to 8mm, The concentration of described 2nd GFAP monoclonal antibody and described anti antibody is respectively 0.5～2mg/ml, preferably 1mg/ml.

Preferably, result detection utilize special fluorescence detector (be purchased from Anqun Bioengineering Co., Ltd., Shenzhen, Model AQ-3000) quality control region and detection zone are detected, in the ratio of detection zone and quality control region fluorescence intensity and testing sample The content of GFAP be directly proportional.Use the absolute of detection zone and the ratio of quality control region fluorescence intensity rather than direct employing detection zone Fluorescent value can be reduced as far as the impact of reaction condition, substrate etc., and avoid ambient interferences as far as possible.

Mode below by way of example further explains and describes present disclosure, but these examples are understood not to Restriction to protection scope of the present invention.

Embodiment

Except as otherwise noted, the following stated solution is aqueous solution, and the percent in solution is percentage by volume.

Embodiment 1:GFAP epitope peptide (1) and the preparation of (2).

Preparation method chemical synthesis: utilize the many automatic peptide synthesizers of American AB I431A type, closed respectively by solid phase method Become GFAP epitope peptide (1) and (2).The purity high performance liquid chromatography of epitope peptide is evaluated, and measures peptide fragment Concentration.The epitope peptide (1) of the present invention and the molecular weight of (2) are respectively 2690.27,1979.35, utilize mass spectrum to carry out really Fixed, measure the peptide sequence synthesized by identifying by peptide sequence.

One, GFAP epitope peptide (1) and the synthesis of (2)

Above-mentioned peptide fragment uses Solid phase synthesis.The main thought of Solid phase peptide synthesis is: first by the carboxyl of peptide chain to be synthesized The carboxyl of end amino acid is connected with the same insoluble macromolecular compound of covalent bond form (resin), then ties with this The aminoacid being combined on solid phase carrier is as moiety, anti-through sloughing amino protecting group the activated carboxyl component with excess Should, spreading peptide chain.Such step can the most repeatedly go on, and finally reaches the length of the peptide chain of required synthesis. This building-up process is as follows.

The GFAP epitope peptide (1) of the present invention and the respective concrete preparation process of (2) are as follows:

The most raw materials used:

HMP resin (the many polyvinyl resins of P-hydroxymethyl phenoxy methyl, be purchased from sigma company)

Fmoc-AA (aminoacid of 9-fluorenylmethoxycarbonyl carbonyl acyl group protection, be purchased from Merck company)

NMP(N-methyl ketopyrrolidine, is purchased from sigma company)

DCM(dichloromethane, is purchased from Central Plains chemical company)

MeoH(methanol, is purchased from Central Plains chemical company)

Piperidine(piperidines, is purchased from sigma company)

DMAP(dimethyl aminopyridine, is purchased from sigma company)

HOBT(hydroxybenzotriazole, is purchased from sigma company)

DCC(dicyclohexylcarbodiimide, is purchased from sigma company)

TFA(trifluoroacetic acid, is purchased from sigma company)

EDT(1,2-dithioglycol, is purchased from sigma company)

Thioanisole, is purchased from Guangzhou Wei Bai Chemical Co., Ltd.

Crystalline phenol, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

2. use instrument:

Many automatic peptide synthesizers, model 431A, it is purchased from ABI company

Rotary Evaporators, model R-201, it is purchased from Shanghai Shen Shun company

High performance liquid chromatograph, Waters600, it is purchased from Waters, US

Freezer dryer, model VFD-2000, it is purchased from Beijing rich doctor Kanggong department

3. synthetic method and process:

Weighing HMP resin 100mg, replacing equivalent is 1.0meq, will be placed in American AB I431A type polypeptide certainly by 0.1mmol In the reaction chamber of dynamic synthesizer, synthesizer automatically being coupled together in a different order by specific aminoacid, Conjugate ratio reaches 99%.React as follows:

(1) amino acid whose activation (HOBt/DCC method)

(2) aminoacid is connected to resin

(3) amino acid whose Fmoc protection group is sloughed

(4) another amino acid whose activation (HOBt/DCC method)

(5) coupling

(6) step (3) to (5) is repeated until end of synthesis.

Respectively obtain the peptide resin 214mg and the peptide resin 178mg of GFAP peptide fragment (2) of GFAP peptide fragment (1).

(7) peptide resin:

Use TFA(trifluoroacetic acid) cutting peptide chain, with EDT(2.5 volume %), thioanisole (2.5 volume %) make scavenger, At room temperature reaction 3.0 hours, remove cutting reagent, then extract with ether, respectively obtain GFAP peptide fragment (1) and the crude product of (2).

Two, GFAP epitope peptide (1) and the purification of (2) crude product:

Employing high performance liquid chromatography is isolated and purified:

Condition: chromatographic column: C810 × 100mm, is purchased from Waters, US

Chromatograph: Waters600, Waters, US

Flowing phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution

B:0.1%TFA(trifluoroacetic acid) in 60% acetonitrile

Detection wavelength: 214nm

Flow velocity: 4ml/ minute

Gradient: 20-60%B, 30 minutes

HPLC(high performance liquid chromatography) analyze

Chromatographic column: C184.6 × 150mm, is purchased from Waters, US

Flowing phase: A:0.1%TFA(trifluoroacetic acid) aqueous solution

B:0.1%TFA(trifluoroacetic acid) in acetonitrile

Detection wavelength: 214nm

Flow velocity: 1ml/ minute

Gradient: 0-60%B, 30 minutes

The GFAP epitope peptide (1) of the peptide fragment analysis result display present invention and the purity of (2) are more than 95%.

Three, GFAP epitope peptide (1) and the qualification of (2)

1. utilize mass spectrum to measure GFAP epitope peptide (1) and the molecular weight of (2) of purification gained respectively.

(1) reagent raw material

TFA(trifluoroacetic acid, is purchased from sigma company)

HCCA(alpha-cyano-4-hydroxycinnamic acid, is purchased from sigma company)

Acetonitrile (is purchased from Chemical Reagent Co., Ltd., Sinopharm Group)

(2) instrument

Matrix Assisted Laser Desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS(model: REFLEX III, Germany Bruker company)；

(3) matrix liquid: α-CCA is dissolved in the 50%ACN solution containing 0.1%TFA, makes saturated solution, centrifugal, take Clearly；

(4) instrument testing conditions: reflection detection mode；Flight pipe range 3m；Nitrogen laser: wavelength 337nm, accelerating potential 20KV；Reflected voltage 23KV.

(5) operating procedure: take the above-mentioned purified polypeptide of 1 μ L (1) and the sample of (2) respectively, in the respective saturated substrate with 1 μ L Clear liquid mixing equal-volume mixes, and takes 1 μ L point respectively on sample target, detects in feeding ion source.

As a result, the molecular weight recording gained GFAP epitope peptide (1) is 2690.4, dividing of GFAP epitope peptide (2) Son amount is 1982.5, consistent with theoretical molecular 2690.27,1979.35, it was demonstrated that synthesize product for the purpose of polypeptide is.

2. measured by peptide sequence and identify gained GFAP epitope peptide (1) and the sequence of (2) respectively.

(1) principle: the ultimate principle of polypeptid acid sequence analysis is Edman degraded, is that a circulating chemistry is anti- Answer process.Including three main chemical steps: (1) coupling: isothiocyanic acid benzene fat is anti-with the N-end residue of proteins and peptides Should, form phenylamino formyl sulfide (PTC) derivant, i.e. PTC-peptide.(2) cyclisation cracking: PTC-peptide cyclisation cracking.(3) convert: thiophene Azoles purine ketone phenylamino (ATZ) is converted into benzene different sulfur urine amino acid (PTH-aminoacid).Stay in the solution decrease an aminoacid The peptide of residue repeats and carries out above-mentioned course of reaction, and whole sequencing procedure is the most all automatically to be carried out by sequenator.

(2) instrument: American AB I company 491 type protein/polypeptide-terminal amino acid sequenator

(3) reagent raw material

Phenyl isothiocyanate PITC, is purchased from sigma company

Normal heptane, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

Trimethylamine TMA aqueous solution, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

TFA(trifluoroacetic acid, is purchased from sigma company)

Ethyl acetate, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

Chlorobutane, is purchased from sigma company

Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group

(4) measure

Carry out by instrument description.

Result: identified, the sequence of gained GFAP epitope peptide (1) and (2) is respectively as follows:

(1)T-V-R-Q-K-L-Q-D-E-T-N-L-R-L-E-A-E-N-N-L-A-A-Y；With

(2)Y-Q-E-A-L-A-R-L-E-E-E-G-Q-S-L-K-D。

This result is consistent with target section of synthesized peptide.

Embodiment 2: respectively GFAP epitope peptide (1) and (2) of embodiment 1 gained are connected with carrier protein with preparation GFAP antigen (1) and (2), utilize gained antigen (1) and (2) immune animal respectively, thus utilize antigen (1) to prepare specific Monoclonal antibody and polyclonal antibody, and utilize antigen (2) to prepare specific monoclonal antibody and polyclonal antibody.

1. the preparation of antigen: use BDB(Bis-diazotizedbenzidine dichloride) method is by GFAP peptide fragment (1) (2) respectively with carrier protein KLH(keyhole limpet hemocyanin) be connected and be prepared as GFAP antigen (1) and (2).

Take GFAP peptide fragment (1) or (2) 10.0mg, dissolve with 1ml0.1M PBS (pH7.4)；KLH10mg, uses 0.2M borate buffer solution (pH9.0) 20ml dissolves；Then both are mixed, be cooled to 0 DEG C, take BDBCl₂110 μ L, under room temperature Reaction 1.5h, subpackage after dialysed overnight ,-20 DEG C of preservations.

In the present embodiment, the formula of PBS is: the Na of 0.2mol/L₂HPO₄81ml adds 0.2mol/L's NaH₂PO₄19ml mixes.

The formula of borate buffer solution is: 0.05mol/L Borax 80ml, adds 0.2mol/L boric acid 20ml and mixes.

2. immune animal prepares monoclonal antibody:

2.1. the GFAP antigen (1) and (2) (immunogen) that take above-mentioned preparation (are purchased with isopyknic Freund's complete adjuvant respectively From Shanghai Yuan Ju biotech firm) be sufficiently mixed after, immunity Balb/c mice, 50 μ g antigens/only, subcutaneous multi-point injection.Survey after 4 weeks Serum titer, selects the mice booster immunization again that immunoreactivity is good: take antigen abundant with isopyknic incomplete Freund's adjuvant After mixing, only, subcutaneous multi-point injection, the number of times of booster immunization is 6 times to antigen dose 25 μ g/, continuous booster immunization two before merging Secondary, extracting spleen cell and Sp2/0 myeloma cell are according to a conventional method with 50%PEG(MW4000 afterwards) (purchased from Central Plains chemical company) Mediation is merged, and selects to cultivate with HAT conditioned medium (purchased from sigma company).CO is put into after fusion₂In incubator 37 DEG C cultivate after 9～11 days, the cell clone that appearance is bigger in the hole in.Within 11 days, start to screen with indirect ELISA.Positive to primary dcreening operation Hole utilize limiting dilution assay to carry out 4 time cloningizations to cultivate (even if a large amount of schizogamy of the cell after Shai Xuan), amplification afterwards is carefully Born of the same parents, frozen, preparation ascites.

2.2. only being processed by Balb/c mice norphytane (purchased from sigma company) 0.5ml/, pneumoretroperitoneum inoculation in a week is miscellaneous Hand over oncocyte 2 × 10⁶Individual/only, collect ascites after 10 days.

2.3. antibody titer is measured: measure the monoclonal antibody utilizing GFAP antigen (1) to prepare with indirect ELISA method (1) titer, the titer of result display monoclonal antibody reaches more than 1:32000.

The titer utilizing monoclonal antibody (2) prepared by GFAP antigen (2) also utilizes identical method to be measured, its effect Valency also reaches more than 1:32000.

3. immune animal prepares polyclonal antibody:

3.1. three monthly ages, body weight are selected to be about the New Zealand white rabbit of about 2kg as immune animal.In fundamental immunity, GFAP antigen (1) and (2) (immunogen) of above-mentioned for 1-2mg preparation are mixed with isopyknic Freund's complete adjuvant respectively-fully Multiple spot subcutaneous injection is carried out at rabbit back after emulsifying.Every 4 weeks booster immunizations once, antigen is abundant with incomplete Freund's adjuvant After emulsifying, with 100 μ g/ only in back multiple spot subcutaneous injection.Carotid artery blood-letting in 10th day after final boost, separates serum.

3.2. antibody titer is measured: measure the polyclonal antibody (1) utilizing GFAP antigen (1) to prepare with indirect elisa method Titer, result display antibody titer reach more than 1:32000.

The titer utilizing polyclonal antibody (2) prepared by GFAP antigen (2) also utilizes identical method to be measured, its effect Valency also reaches more than 1:32000.

3.3. take blood and separate serum: carotid artery intubates and takes blood, separates serum.

The most isolated and purified antibody: after ammonium sulfate precipitation, then through Protein G(purchased from sigma company) affinity purification.

5. lyophilizing after antibody subpackage, cryopreservation.

Embodiment 3: people's GFAP monoclonal antibody (1) and the specificity identification of (2)

Detect with ELISA.Respectively with people's GFAP albumen, S-100B albumen, neuronspecific enolase NSE (being purchased from Shanghai Lian Shuo company), for detecting antigen coated elisa plate, detects prepared GFAP monoclonal respectively by ELISA Antibody (1) and (2) and the specific reaction of this people's GFAP albumen, make negative control with normal BALB/c mouse serum, and PBS liquid is made Blank.

Result: it is positive (P/N > 2.1) that GFAP monoclonal antibody (1) and (2) are the most only reacted with GFAP, and and S-100B Albumen, neuronspecific enolase NSE reaction is feminine gender, and GFAP monoclonal antibody (1) and (2) difference of the present invention are described There is specificity.

Embodiment 4: people's GFAP polyclonal antibody (1) and the specificity identification of (2)

The method identical with above-mentioned qualification monoclonal antibody specificity is utilized to identify.

Result shows: it is positive (P/N > 2.1) that GFAP polyclonal antibody (1) and (2) are reacted with GFAP respectively, and and S- 100B albumen, neuronspecific enolase NSE reaction is feminine gender, and GFAP polyclonal antibody (1) and (2) of the present invention are described It is respectively provided with specificity.

Embodiment 5: utilize GFAP monoclonal antibody and GFAP polyclonal antibody to prepare GFAP external diagnosis reagent case.

In the present embodiment, using the monoclonal antibody (1) that utilizes GFAP epitope peptide (1) to prepare in embodiment 2 as Coated antibody in this test kit；Using the polyclonal antibody (2) that utilizes GFAP epitope peptide (2) to prepare in embodiment 2 as Binding antibody.

Preparation and the operation of GFAP external diagnosis reagent case are as follows:

The most various buffer and the preparation of reagent:

A, it is coated buffer: the CB(carbonate buffer solution of 0.050M, pH9.6)

Na2CO₃: 16.0 grams

NaHCO₃: 29.0 grams

Distill water-soluble to 1000ml

B, the 10 × PBS-Tween20 of sample/lavation buffer solution: pH7.2

Na₂HPO₄·12H₂O:58 gram

KH₂PO₄: 4 grams

NaCl:100 gram

KCl:4 gram

Distill water-soluble to 1000ml

Add Tween20:20ml

C, enzyme marker diluent:

10 × PBS-Tween20:10ml

FCS(calf serum): 20ml

Distill water-soluble to 1000ml

Enzyme stabilizers (is purchased from Shanghai Xi Bao company): 1 gram

Biological preservative (is purchased from Shanghai Xi Bao company): 1ml

D, developer A:

Citric acid: 35.5 grams

Urea peroxide: 10 grams

Distill water-soluble to 1000ml

Tween20:10ml

E, developer B:

Citric acid: 120 grams

EDTA-2Na:1 gram

TMB 2HCl:2 gram

Distill water-soluble to 1000ml

F, stop buffer: 2M H₂SO₄

Concentrated sulphuric acid (95-98%): 22.2ml

Distilled water: 177.3ml

Concentrated sulphuric acid is slowly dropped in distilled water by timing, and limit edged shakes up.

The preparation of the most pre-coated plate:

In the carbonate buffer solution of the 0.05M that GFAP monoclonal antibody (1) is dissolved in pH=9.6, make pre-coated liquid, The upper every hole of ELISA Plate (being purchased from Shenzhen Jin Canhua company) adds 100 μ l by 0.1 μ g/ hole, puts 4 DEG C and places 18-24 hour, takes Go out, get rid of and be coated liquid, washing, after BSA closing 16 hours, dried overnight, load evacuation in aluminide-coating bag seal, be placed in 4 DEG C Preserve.

3. binding antibody (GFAP polyclonal antibody (2)) and the goat anti-rabbit igg of enzyme connection thing (horseradish peroxidase) labelling The dilution ratio of antibody (purchased from Beijing company of Zhong Shan Golden Bridge) is determined by square formation titration experiments.

4. the composition of test kit:

Pre-coated plate: 48/96 hole

GFAP calibration object (raw material is purchased from Shanghai Lian Shuo company): 7: 7 × 1.0ml(concentration is respectively 25ng/ml, 10ng/ Ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0.5ng/ml, 0.25ng/ml)

GFAP binding antibody: 1 × 10ml(dilutes through 1:5000)

Enzyme connection thing: 1 × 10ml(dilutes through 1:5000)

Concentrated cleaning solution (25 × PBS-Tween20): 1 × 20ml

Developer A:1 × 6.0ml

Developer B:1 × 6.0ml

Stop buffer: 1 × 6.0ml

5. the operating procedure of test kit:

In each hole of pre-coated plate, it is separately added into blood sample to be checked and standard substance 100 μ l/ hole, is diplopore, incubates for 37 DEG C Educate 60 minutes, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add GFAP binding antibody 100 μ l/ hole, incubate for 37 DEG C Educate 30 minutes, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add enzyme connection thing 100 μ l/ hole again, hatch 30 for 37 DEG C Minute, wash 5 times with 1 × lavation buffer solution, pat dry.Add developer A, B liquid, each 50 μ l in every hole, mixing, hatch 15 points for 37 DEG C Clock.Add stop buffer 50 μ l/ hole and terminate reaction, join detector (model RT-6000 is purchased from Lei Du company) with enzyme and use dual wavelength (450nm, 620nm) detects absorbance.

6. result judges:

Table 1: standard concentration and corresponding mean light absorbency (OD) value

Concentration ng/ml	0.25	0.5	1	2	5	10	25
								Mean OD value	0.068	0.123	0.213	0.385	0.714	1.237	2.156

Standard curve, the R of standard curve is drawn with the logarithm value of standard concentration and corresponding absorbance²=0.977。

The GFAP concentration results in the specimen detected is calculated according to standard curve.

30 example acute cerebral insult patients and 81 example healthy persons are carried out serum GFAP detection, patients with brain injury in a manner described GFAP content in serum apparently higher than normal healthy controls group, difference statistically significant (P < 0.01), be shown in Table 2.

2: two groups of sample GFAP concentration of table compare

Embodiment six: for detecting the preparation of the fluorescence immune chromatography test paper of people GFAP albumen in determinand.

One, it is marked with the preparation of the monoclonal antibody of fluorescent microsphere and is coated pad

1, the preparation of the monoclonal antibody of fluorescent microsphere it is marked with

1.1, the activation of fluorescent microsphere:

Take fluorescent microsphere (purchased from the Guangzhou Growth hormone secretagogue company) aqueous dispersions of 500 μ l, content 1 (w/v) %, at 10 DEG C, with 12000rpm is centrifuged 10 minutes, removes supernatant, and precipitate is distributed to the distilled water of 500 μ l or first wash buffer (the MES water of 0.1M Solution) in, ultrasound wave (240W) processes 2 minutes, repeats above procedure three times, adds carbodiimide (purchased from Shanghai Jing Chun company) 50mg, stirs 15 minutes, thus activates described fluorescent microsphere.

1.2, with the fluorescent microsphere traget antibody that activated:

The fluorescent microsphere activated is centrifuged 10 minutes under 12000rpm, removes supernatant, precipitate is distributed to 1ml coupling In buffer (citrate buffer solution of the N-hydroxy thiosuccinimide of 50mM), ultrasound wave (240W) processes 2 minutes, repeats Above procedure three times, it is thus achieved that be dispersed with the buffer 1ml of fluorescent microsphere.Activate according to 3 μ l antibody (8mg/ml)/100 μ l The ratio of fluorescent microsphere, is added thereto to the GFAP monoclonal antibody (1) by embodiment 2 preparation, at normal temperatures stirring 2 hours, Add 1ml Block buffer (1 (w/v) %BSA-0.05M ethanolamine), continue stirring 1 hour, afterwards, centrifugal under 12000rpm 10 minutes, repeated centrifugation 3 times, precipitate is distributed to 500 μ l wash buffer at end (0.5 (w/v) %BSA-0.11 (v/v) % tweens Aqueous solution) in, ultrasound wave (240W) processes 2 minutes, is settled to 500 μ l with above-mentioned whole wash buffer.

2, it is coated pad

By GFAP monoclonal antibody (1) antibody diluent (1% (w/v) BSA-being marked with fluorescent microsphere of above-mentioned preparation 0.01M PBS(pH7.2) buffer) it is diluted to 1mg/ml, it is thus achieved that working solution, then (it is purchased from micropipettor Labsystems company) by the amount even application of 4 μ l/cm on pad, afterwards by 37 DEG C of oven for drying, under 45% humidity Save backup.

Two, the preparation of reaction film

GFAP monoclonal antibody (2) and sheep anti-mouse igg monoclonal antibody according to embodiment 2 preparation are (purchased from China fir in Beijing Company of Golden Bridge) it is diluted to 1mg/ml respectively with the PBS of 50mM pH7.2, by metal spraying machine (purchased from Hangzhou Feng Hang company) Detection line and nature controlling line spacing parameter are set to 6mm, package amount are respectively set to 1.0 μ l/cm, with metal spraying machine at cellulose nitrate Drawing upper GFAP monoclonal antibody (2) and sheep anti-mouse igg monoclonal antibody on element film, room temperature dries standby.

Three, the assembling of reagent paper and cutting

On base plate, mutually overlap joint pastes sample pad, pad, reaction film and absorbent filter successively, obtains test paper plate, will It cuts into the test strips that width is 5mm.

Four, the preparation of GFAP fluorescence immunoassay detection card:

Being fixed on plastic bottom card by the reagent paper of above-mentioned well cutting, reagent paper surface face card compresses, and face is stuck in test strips Well and observation window is had on the position of sample pad and reaction film.Detection card loads in aluminium foil bag after assembling, and adds and is dried Agent sealing preserves, and can preserve more than 1 year under the conditions of drying at room temperature.

Five, the detection of sample

GFAP standard substance (purchased from Shanghai Lian Shuo company) sample diluting liquid (1% (w/v) BSA-0.01M PBS(pH7.2) Buffer) be configured to the calibration object of following series concentration: 100ng/ml, 50ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, The 50 above calibration objects of μ l are added drop-wise to add by 2ng/ml, 1ng/ml, 0.5ng/ml, 0.2ng/ml, 0.1ng/ml, 0ng/ml respectively On sample hole, with fluorescence detector (purchased from Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) detection after 10 minutes, Fluorescence can be collected on detection line and nature controlling line position.Fluorescence with sample concentration as abscissa, at detection line and nature controlling line The ratio of intensity is that vertical coordinate draws calibration curve, R²It is 0.995.Nature controlling line is for reagent paper Effective judgement and to detection line Signal makees corresponding correction, and as band does not occurs in nature controlling line, then explanation reagent paper lost efficacy.

Take the blood sample to be checked of 50 μ l, be added drop-wise on well, detect with fluorescence detector after 10 minutes, if detection line goes out Existing band, illustrates that its concentration can obtain according to calibration curve containing GFAP in sample.

Six, GFAP fluorescence immunoassay reagent paper performance evaluation

1. evaluate the index of reagent paper performance

1) range of linearity: each concentration calibration product duplicate detection 3 times, draws calibration curve, through data matching and statistical analysis, Reagent paper linear detection range of the present invention is 0.2ng/ml-100ng/ml.

2) minimum detectability: GFAP null value blood sample (without GFAP composition) (purchased from Shanghai Lian Shuo company) is divided into 20 parts and carries out Detection, calculates mean concentration and 2 times of standard deviation sums, obtains reagent paper lowest detection of the present invention and be limited to 0.04ng/ml.

3) precision: with the GFAP fluorescence immunoassay reagent paper of the present invention detect respectively GFAP concentration be respectively 50ng/ml, The blood sample of 12.5ng/ml, 2.50ng/ml, duplicate detection 10 times, carry out withinrun precision mensuration.Every day is to above-mentioned 3 concentration Sample is measured, 1 day 1 time, surveys 20 days continuously, carries out betweenrun precision mensuration, and result is as shown in table 3 below:

Table 3

Batch in the CV(coefficient of variation) and batch between CV be respectively less than 8%, illustrate that this reagent accurate is good.

Additionally, as seen from the above table, the range of linearity width of this detection paper GFAP, sensitivity are good.

Claims

1. a people GFAP epitope peptide, the aminoacid sequence of wherein said GFAP epitope peptide is one of both:

(1)Thr-Val-Arg-Gln-Lys-Leu-Gln-Asp-Glu-Thr-Asn-Leu-Arg-Leu-Glu-Ala-Glu- Asn-Asn-Leu-Ala-Ala-Tyr；

(2)Tyr-Gln-Glu-Ala-Leu-Ala-Arg-Leu-Glu-Glu-Glu-Gly-Gln-Ser-Leu-Lys-Asp。

2. a GFAP antigen, it is by making people's GFAP epitope peptide (1) described in claim 1 and carrier protein couplet It is prepared from.

3. a GFAP antigen, it is by making people's GFAP epitope peptide (2) described in claim 1 and carrier protein couplet It is prepared from.

4. a people GFAP antibody, it is the monoclonal antibody or many grams being prepared from by the GFAP antigen described in claim 2 Grand antibody.

5. a people GFAP antibody, it is the monoclonal antibody or many grams being prepared from by the GFAP antigen described in claim 3 Grand antibody.

6. according to the people's GFAP antibody described in claim 4 or 5 in the purposes prepared on people's GFAP external diagnosis reagent case.

7. a people GFAP external diagnosis reagent case, its people's GFAP antibody comprised described in claim 4 or 5 is anti-as being coated Body.

People's GFAP external diagnosis reagent case the most according to claim 7, wherein said test kit also comprises binding antibody, institute Stating binding antibody is the people's GFAP antibody described in claim 4 or 5, and when described binding antibody derives from described people GFAP During one in epitope peptide (1) and (2), described coated antibody derives from described people's GFAP epitope peptide (1) and (2) In another one.

People's GFAP external diagnosis reagent case the most according to claim 8, wherein said test kit also comprises the of enzyme labelling Two antibody.

People's GFAP external diagnosis reagent case the most according to claim 7, wherein said coated antibody is monoclonal antibody.

11. people's GFAP external diagnosis reagent cases according to claim 8, wherein said binding antibody is polyclonal antibody.