CN105652011B

CN105652011B - Detect fluorescence immune chromatography test paper of the albumen of people HSP90 α 2 and preparation method thereof

Info

Publication number: CN105652011B
Application number: CN201410668302.6A
Authority: CN
Inventors: 朱建安
Original assignee: ANQUN BIOENGINEERING Co Ltd SHENZHEN
Current assignee: ANQUN BIOENGINEERING Co Ltd SHENZHEN
Priority date: 2014-11-20
Filing date: 2014-11-20
Publication date: 2017-09-22
Anticipated expiration: 2034-11-20
Also published as: CN105652011A

Abstract

The present invention relates to fluorescence immune chromatography test paper of the detection albumen of people HSP90 α 2 and preparation method thereof.The test paper detects the albumen of people HSP90 α 2 by double antibody sandwich method, the double antibody sandwich method uses the first HSP90 alpha 2 monoclonal antibodies for being marked with fluorescent microsphere as capture antibody, and the first HSP90 alpha 2 monoclonal antibodies are from one of sequence described in sequence table SEQ ID NO.1 and SEQ ID NO.2；And the double antibody sandwich method is using the 2nd HSP90 alpha 2 monoclonal antibodies as detection antibody, and the 2nd HSP90 alpha 2 monoclonal antibodies are from the other of sequence described in sequence table SEQ ID NO.1 and SEQ ID NO.2.Fluorescence immune chromatography test paper of the invention is easy to operate, quick, detection range is wide, specific high, sensitivity is good.

Description

Detect fluorescence immune chromatography test paper of people's HSP90 α -2 albumen and preparation method thereof

Technical field

The invention belongs to chemiluminescent polypeptide and field of immunology, and in particular to the α -2 of human heat shock protein 90 (HSP90 α -2) resists Former epitope peptide, HSP90 α -2 specific antigens and corresponding monoclonal antibody or Anti-TNF-α prepared with the epitope peptide The application of body, the antibody on people's HSP90 α -2 external diagnosis reagent cases are prepared, people's HSP90 α -2 external diagnosis reagent cases, with And it is a kind of for fluorescence immune chromatography test paper of people's HSP90 α -2 albumen in quantitatively detection determinand and preparation method thereof.

Background technology

In recent years, the research to tumor markers has caused the attention of many researchers, and searching one kind is reliable, non-to invade Generation entering formula, can reflecting tumour, the tumor markers of Proliferation, Differentiation are the common aspirations of numerous researchers.By to blood The research of liquid, tissue fluid, has now been found that some tumor markerses can be predicted to the generation of tumour, Proliferation, Differentiation.Wherein, The α -2 of human heat shock protein 90 (HSP90 α -2) is exactly a kind of such tumor markers.

Heat shock protein72-2 is one of important albumen in heat-shock protein family, and it joins as molecular chaperones Conformation and function with regulating and controlling, maintaining intracellular multiple protein, play important in terms of regulation cell growth, differentiation and apoptosis Effect.In recent years find, Heat shock protein72-2 has as chaperone to the malignant transformation of tumour, growth, propagation and invasion and attack Important effect.There are many reports to confirm that in body tumor tissue, the expression of heat shock protein is changed.In difference In the tumor tissues at position, the change of heat shock protein expression is not consistent.Have now been found that HSP90 α in affinity antibody to SpA- The grade malignancy of 2 contents and tumour, especially shifts positive correlation.Therefore, HSP90 α -2 hold promise as diagnosing tumor and prognosis Mark.

The optimal method for detecting HSP90 α -2 levels in serum is immune detection.Therefore, finding suitably has HSP90 α -2 epitope peptides of immunogenicity, prepare specific HSP90 α -2 antigens and antibody emphasis.

The determinand that fluorescence immune chromatography method is surveyed in blood is easy to operate, quick, only needs 10 minutes with regard to that can complete sample Detection, and detection range is wide, and sensitivity is good, can the assisted diagnosis state of an illness in time rapidly, monitor prognosis.Chinese patent application No.200910117820.8 discloses the preparation method and quantitative detecting method of a kind of fluorescent micro-ball immune chromatography test paper strip；In State patent application No.200910047352.1 discloses a kind of fluorescence immune chromatography test paper and its preparation method and application.However, There is presently no the fluorescence immune chromatography test paper for people's HSP90 α -2 albumen.

The content of the invention

For solve it is above-mentioned the problems of in the prior art, be used for the invention provides one kind in quantitatively detection determinand Fluorescence immune chromatography test paper of people's HSP90 α -2 albumen and preparation method thereof.

Specifically, the invention provides：

A kind of to be used for the fluorescence immune chromatography test paper of people's HSP90 α -2 albumen in quantitatively detection determinand, the test paper passes through double Antibody sandwich detects people HSP90 α -2 albumen, wherein：

The double antibody sandwich method is anti-as capture using the first HSP90 α -2 monoclonal antibodies for being marked with fluorescent microsphere Body, the first HSP90 α -2 monoclonal antibodies are from one of people's HSP90 α -2 epitope peptides (1) and (2)；And

The double antibody sandwich method is used as detection antibody, the 2nd HSP90 using the 2nd HSP90 α -2 monoclonal antibodies α -2 monoclonal antibodies are from the other of people's HSP90 α -2 epitope peptides (1) and (2)；

People HSP90 α -2 epitope peptides (1) and (2) are respectively：

(1)Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-G ly-Ser- Asp-Glu；

(2)Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Se r-Ala- Ala-Val-Thr-Glu。

Preferably, the first HSP90 α -2 monoclonal antibodies are prepared from by the first HSP90 α -2 antigens, and this One HSP90 α -2 antigens are by making one of people HSP90 α -2 epitope peptides (1) and (2) and carrier protein couplet It is prepared from；And

2nd HSP90 α -2 monoclonal antibodies are prepared from by the 2nd HSP90 α -2 antigens, the 2nd HSP90 α -2 antigens are by preparing the other of people HSP90 α -2 epitope peptides (1) and (2) and carrier protein couplet Into.

Preferably, the particle diameter of the fluorescent microsphere is 320nm to 400nm.

Preferably, the fluorescent material on the fluorescent microsphere is that fluorescein isothiocynate, RB 200, tetramethyl are different Thiocyanic acid rhodamine or X- rhodamines etc., wherein preferably X- rhodamines；The micro-sphere material of the fluorescent microsphere be polystyrene, The copolymer of polymethyl methacrylate or methyl methacrylate.

Preferably, the excitation wavelength of the fluorescent microsphere is 350~600nm, preferably 390nm；Launch wavelength be 500~ 700nm, preferably 615nm.

Preferably, the fluorescence immune chromatography test paper has bottom plate, and on which floor plate along chromatography direction when using It is provided with successively with the way of contact：Sample pad, pad, reaction film, absorbent filter, the pad are provided with described mark There are the first HSP90 α -2 monoclonal antibodies of fluorescent microsphere, the reaction film includes detection zone and quality control region, the detection zone bag There are the 2nd HSP90 α -2 monoclonal antibodies, the quality control region is coated with can have with described mark first The antiantibody that HSP90 α -2 monoclonal antibody specificities are combined.

Preferably, the reaction film is the substantially not fluorescent nitrocellulose filter under the wavelength more than 550nm.

Preferably, the bottom plate does not have photoluminescent property substantially.

Preferably, the antiantibody be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, wherein it is preferred that For sheep anti-mouse igg monoclonal antibody.

The described fluorescence for being used to quantitatively detect people's HSP90 α -2 albumen in determinand is prepared present invention also offers a kind of The method of immune chromatography test paper, it comprises the following steps：

1) the first HSP90 α -2 monoclonal antibodies for being marked with fluorescent microsphere are provided；

2) pad is provided, wherein being coated with the first HSP90 α -2 that described mark has on the pad Monoclonal antibody；

3) reaction film is provided, wherein fixing the 2nd HSP90 α -2 along chromatography direction interval when using on the reaction film Monoclonal antibody and antiantibody, to form detection zone and quality control region respectively；

4) sample pad is set with the way of contact successively along chromatography direction when using on bottom plate, it is the pad, described Reaction film, absorbent filter, so that the fluorescence immune chromatography test paper is made.

Preferably, the step 1) include：

A) carbodiimide activation fluorescent microsphere is used, it is preferable that by the aqueous dispersions of fluorescent microsphere or MES buffer solution dispersion liquids Mixed through ultrasonication and with carbodiimide, so as to activate the fluorescent microsphere；

B) fluorescent microsphere for the activation that washing step a) is obtained, it is preferable that by the fluorescence of the step a) activation obtained Microballoon is washed with N- hydroxy thiosuccinimides-citrate buffer solution, disperseed, and through ultrasonication；

C) HSP90 α -2 monoclonal antibodies of fluorescent microsphere mark the first that step b) is obtained are used, it is preferable that by step b) The fluorescent microsphere obtained is mixed with the first HSP90 α -2 monoclonal antibodies, with BSA- monoethanolamine buffer blinds, is centrifuged, is used BSA- Tween solutions disperse, through ultrasonication, so that the first HSP90 α -2 monoclonals for obtaining being marked with fluorescent microsphere resist Body.

Preferably, in the step 2) in, the first HSP90 α -2 monoclonal antibodies that described mark has Coating concentration is 0.5~2mg/ml.

Preferably, in the step 3) in, the detection zone and the quality control region interval 3mm to 8mm.

Preferably, in the step 3) in, the coating of the 2nd HSP90 α -2 monoclonal antibodies and the antiantibody is dense Degree is respectively 0.5~2mg/ml.

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

1. people's HSP90 α -2 epitope peptides of the present invention have good antigenicity, the antigen (immunogene) prepared with it Immune animal can produce the monoclonal antibody and polyclonal antibody of high degree of specificity.

2. with HSP90 α -2 monoclonal antibodies that prepare of the present invention and polyclonal antibody can high special with blood sample HSP90 α -2 in this are combined.

3. people HSP90 α -2 external diagnosis reagent cases of the present invention can effectively detect the water of the HSP90 α -2 in serum It is flat, it can be used to judge the grade malignancy of tumour, and transfer and prognosis are predicted.

4. fluorescence analysis are combined by the present invention with flash chromatography immunological technique to be used to quantitatively detect there is provided one kind The fluorescence immune chromatography test paper of people HSP90 α -2 albumen in determinand, people's HSP90 α -2 eggs in determinand are detected with the test paper In vain, it is easy to operate, quick, 10 minutes are only needed with regard to sample detection can be completed, and detection range is wide, specific high, sensitivity is good, Can the assisted diagnosis state of an illness in time rapidly, monitor prognosis.

5. the present invention is during the fluorescence immune chromatography test paper of people HSP90 α -2 albumen is prepared, by substantial amounts of Experiment is groped, and optimizes the preparation condition of each side so that when being detected with the fluorescence immune chromatography test paper of the present invention, fluorescence Signal-to-background ratio is greatly improved, so as to improve detection sensitivity and credible result degree；In addition, the present invention also passes through the detection zone of test paper Change with the fluorescence intensity ratio of quality control region carrys out the content of HSP90 α -2 in response sample, and this is only examined with traditional chromatographic technique The absolute fluorescence intensity for looking into detection zone is compared, and the influence of external condition and background etc. is reduced to the full extent, is further improved Testing result confidence level.

Brief description of the drawings

Fig. 1 schematically shows the structural representation of the fluorescence immune chromatography test paper of one embodiment of the invention.

Embodiment

Description below by way of embodiment and the invention will be further described referring to the drawings, but this is not pair The limitation of the present invention, those skilled in the art are according to the basic thought of the present invention, and various modifications may be made or improves, but only The basic thought of the present invention is not departed from, within the scope of the present invention.

First, people HSP90 α -2 epitope peptides

People's HSP90 α -2 albumen specifically described herein be it is known in the art, its amino acid sequence be it is known in the art, It can be found in the specialized databases such as NCBI.

The invention provides people's HSP90 α -2 epitope peptides (1) and (2), its amino acid sequence is respectively such as sequence table SEQ Shown in ID No.1 and SEQ ID No.2, it is：

(1)Y-E-K-E-S-E-D-K-P-E-I-E-D-V-G-S-D-E；With

(2)Y-R-G-I-D-E-D-D-P-T-A-D-D-T-S-A-A-V-T-E。

The present inventor gropes by substantial amounts of theoretical research and experiment, and final screening, which obtains two kinds, to be had well Antigenic epitope peptide.

HSP90 α -2 epitope peptides (1) include people HSP90 α -2 albumen (NCBI accession number NP_005339.3) N-terminal the The peptide fragment of 249 to the 265th, and Y is added in the N sections of the peptide fragment, so as to constitute the HSP90 α -2 for including 18 amino acid Epitope peptide (1).

HSP90 α -2 epitope peptides (2) include people HSP90 α -2 albumen (NCBI accession number NP_005339.3) C-terminal the 697 to the 714th peptide fragments, and Y and R is added in the N sections of the peptide fragment, so as to constitute the HSP90 α -2 for including 20 amino acid Epitope peptide (2).

It is strong and the characteristics of be readily synthesized that the two peptide fragments are respectively provided with hydrophily, antigenicity.

At present, present invention research finds that HSP90 α -2 epitope peptides of the invention have following function：

1. with antigenicity；2. produce specific antibody as immunogene stimulating animal after being connected with carrier protein；3. The antibody prepared with epitope peptide can be combined specifically with people HSP90 α -2.

The preparation method of HSP90 α -2 epitope peptides of the present invention can use chemical synthesis：Utilize American AB I431A types Many automatic peptide synthesizers, pass through Solid phase synthesis epitope peptide.The epitope peptide (1) of the present invention and the molecular weight of (2) point Not Wei 2404.35,2483.39, can be determined with mass spectrum, and pass through peptide sequence determine identification synthesized by epitope peptide Sequence.The purity of peptide fragment can be evaluated with thin-layer chromatography and high performance liquid chromatography, and determine the concentration of epitope peptide.

2nd, HSP90 α -2 antigens

Present invention also offers HSP90 α -2 antigens, its by make the present invention people's HSP90 α -2 epitope peptides (1) and One of (2) it is prepared from carrier protein couplet.Specifically, the invention provides HSP90 α -2 antigens (1) and (2), HSP90 α -2 antigens (1) are by preparing people's HSP90 α -2 epitope peptides (1) and the carrier protein couplet of the present invention Into；HSP90 α -2 antigens (2) are by making people's HSP90 α -2 epitope peptides (2) of the present invention be prepared with carrier protein couplet Form.HSP90 α -2 antigens of the present invention have immunogenicity and specificity, are a kind of immunogenes, can be used to immune animal so as to Prepare specific HSP90 α -2 antibody.In the present invention, the example of available carrier protein includes KLH (keyhole blood indigo plant eggs In vain), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc..Because KLH (keyhole limpet hemocyanin) immunogenicity is strong, binding site Many, immune effect preferably, and with immune animal affiliation farther out, is difficult to cause cross reaction with it as carrier protein, Therefore it is preferred.

3rd, HSP90 α -2 monoclonal antibodies, HSP90 α -2 polyclonal antibodies and people's HSP90 α -2 external diagnosis reagent cases

Present invention also offers people's HSP90 α -2 monoclonal antibodies and people's HSP90 α -2 polyclonal antibodies, the antibody can be with Animal is immunized each with HSP90 α -2 antigens (1) or (2) (immunogene) of the present invention to be prepared.Preparation method can be using this The routine techniques in field, for details, reference can be made to embodiment 2.

HSP90 α -2 monoclonal antibodies and polyclonal antibody of the present invention can be used for preparing people's HSP90 α -2 in-vitro diagnosis Kit, the kit can be detected based on immunization method to the HSP90 α -2 in tissue, cell or body fluid, preferably HSP90 α -2 in blood preparation are detected.

Therefore, the invention provides a kind of people HSP90 α -2 external diagnosis reagent cases, its people HSP90 α comprising the present invention - 2 monoclonal antibodies or polyclonal antibody.

The immunization experiment method available for clinical examination is currently known mainly including following several：ELISA method, chemiluminescence Method, fluorescent chromatographic method, colloid gold immune determination method 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 and surveys antibody, competition law survey antigen, capture coating method survey antibody etc..

People's HSP90 α -2 external diagnosis reagent cases of the present invention are preferred to use ELISA double antibody sandwich methods to detect HSP90 α -2 albumen.The kit can include coated antibody, binding antibody, the secondary antibody of enzyme mark and/or necessary instrument and examination Agent etc..

Preferably, people HSP90 α -2 external diagnosis reagent cases are using people's HSP90 α -2 monoclonal antibodies of the invention It is used as coated antibody.Here, term " coated antibody " refers to be coated in the antibody on the ELISA Plate of solid phase.In addition, the people HSP90 α -2 external diagnosis reagent cases further preferably comprising people's HSP90 α -2 polyclonal antibodies using as binding antibody, wherein, work as institute When stating binding antibody from one of people's HSP90 α -2 epitope peptides (1) and (2) of the present invention, the coated antibody comes Come from the other of the epitope peptide (1) and (2).Here, term " binding antibody " refer in kit can with it is to be measured The specific antibody that antigen and enzyme-labeled secondary antibody are combined.The secondary antibody that the kit can also be marked comprising enzyme, should Secondary antibody can be goat anti-rabbit igg antibody, and the enzyme mark can be horseradish peroxidase, alkaline phosphatase etc..

In the kit of the present invention, for example pre-coated plate, it can also be washed comprising any reagent needed for detection or instrument Wash liquid, developer, terminate liquid etc..

4th, for quantitatively detecting the fluorescence immune chromatography test paper of people's HSP90 α -2 albumen

It is used for the fluorescence immune chromatography examination of people's HSP90 α -2 albumen in quantitatively detection determinand present invention also offers a kind of Paper, the test paper detects people HSP90 α -2 albumen by double antibody sandwich method, wherein：

The double antibody sandwich method is also used as detection antibody, described second using the 2nd HSP90 α -2 monoclonal antibodies HSP90 α -2 monoclonal antibodies are from the other of people's HSP90 α -2 epitope peptides (1) and (2)；

People HSP90 α -2 epitope peptides (1) and (2) are respectively：

(1)Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser- Asp-Glu；

In the present invention, in double antibody sandwich method fluorescence immune chromatography test paper, " capture antibody " is to refer to spy first The antibody of opposite sex identification determined antigen, it is generally arranged on pad；" detection antibody " refer to it is another can specificity know The antibody of other determined antigen, it recognizes the different epitopes on determined antigen molecule from capture antibody respectively, and it is generally solid It is scheduled on the detection zone of reaction film.

In the present invention, the first HSP90 α -2 monoclonal antibodies can be prepared from by the first HSP90 α -2 antigens, First HSP90 α -2 antigens can be by making one of people HSP90 α -2 epitope peptides (1) and (2) and carrier egg White coupling is prepared from；And the 2nd HSP90 α -2 monoclonal antibodies can be prepared from by the 2nd HSP90 α -2 antigens, 2nd HSP90 α -2 antigens can be by making the other of people HSP90 α -2 epitope peptides (1) and (2) and carrier Albumen coupling is prepared from.

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

Preferably, the particle diameter of fluorescent microsphere used in fluorescence immune chromatography test paper of the invention is 320nm to 400nm, Fluorescent material on preferably 360nm, fluorescent microsphere can be fluorescein isothiocynate, RB 200, the different sulphur cyanogen of tetramethyl Sour rhodamine or X- rhodamine etc., wherein preferably X- rhodamines (being purchased from Shanghai Jing Chun companies).The microballoon material of fluorescent microsphere The copolymerization that material can be formed for polystyrene, polymethyl methacrylate or by methyl methacrylate and other monomer copolymerizations Thing, the example of other monomers is styrene etc..The excitation wavelength of fluorescent microsphere can be 350~600nm, preferably 390nm；Hair Ejected wave length can be 500~700nm, preferably 615nm.

In the present invention, the maximum excitation wavelength and launch wavelength difference of fluorescent microsphere are larger, illustrate fluorescent microsphere have compared with Big Stokes (Stokes) displacement, so, the ambient interferences of fluorescent test paper are relatively low, and immunochromatography label is done with the microballoon There is stronger advantage.

In a specific embodiment, fluorescence immune chromatography test paper of the invention has bottom plate, and in the bottom plate Chromatography direction when upper edge is used is provided with the way of contact successively：Sample pad, pad, reaction film, absorbent filter, the sample Product pad is used to load testing sample when in use, and the pad is provided with the first HSP90 that described mark has α -2 monoclonal antibodies, the reaction film includes detection zone and quality control region, and it is single that the detection zone is coated with the 2nd HSP90 α -2 Clonal antibody, it is special that the quality control region is coated with the first HSP90 α -2 monoclonal antibodies that can have with described mark Property combine antiantibody.

Preferably, the sample pad of fluorescence immune chromatography test paper of the invention, pad, reaction film, absorbent filter can edges Chromatography direction when using is overlapped successively to be arranged on bottom plate (referring to Fig. 1).Spaced detection zone and Quality Control on reaction film Area can be, but be not limited to, the form such as line, band, block, and detection zone and quality control region are preferably by 3mm to 8mm.

In the present invention, reaction film is preferably the substantially not fluorescent nitrocellulose under the wavelength more than 550nm Film (is for example purchased from Shanghai outstanding person one, model GFCP203000).Herein, the implication " not fluoresced substantially " refers in phase Do not launch fluorescence under the illumination for answering wavelength (being greater than 550nm wavelength), or only launch micro, not substantially influence inspection Survey the fluorescence of result.In addition, bottom plate does not have photoluminescent property preferably substantially, it is, for example, substantially without photoluminescent property PVC backboards (are for example purchased from Shanghai outstanding person one, model HF000MC100).Herein, it is " substantially without photoluminescent property " Refer to do not have photoluminescent property, or only have it is low, not substantially influence testing result photoluminescent property.

Generally, conventional chromatographic test paper component (reaction film, bottom plate etc.) under 550nm wavelength there is obvious fluorescence to carry on the back Scape, this detection to fluorescence signal produces very big interference.The present invention is not sent out substantially by using under the wavelength more than 550nm The nitrocellulose filter of fluorescence and the bottom plate of low Poison property, so as to overcome the defect of conventional fluorescent test paper.In addition, of the invention Fluorescent material X- rhodamines used can produce stronger fluorescence signal, so as to further substantially increase fluorescence signal-to-background ratio, make Signal and background can be distinguished well by obtaining, and then improve detection sensitivity.

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

It is of the present invention to be combined with being marked with the first HSP90 α -2 monoclonal antibody specificities of fluorescent microsphere Antiantibody can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, wherein preferably sheep anti-mouse igg Dan Ke Grand antibody, compared with polyclonal antibody, monoclonal antibody specificity is higher.

In a specific embodiment, fluorescence immune chromatography test paper of the invention when in use, drips in sample pad Plus sample liquid (blood sample such as containing HSP90 α -2), under capillarity, sample liquid is moved to absorbent filter one end, is being combined The first HSP90 α -2 monoclonal antibodies formation immune complex having at pad with described mark, the immune complex It is further mobile, the immune compound of double-antibody sandwich is combined to form with the 2nd HSP90 α -2 monoclonal antibodies in detection line Thing, and the first HSP90 α -2 monoclonal antibodies that the mark for not forming immune complex has then with it is anti-on nature controlling line Antibody binding.This process needs 10 minutes to 15 minutes, afterwards, is detected with fluorescence detector, if do not gone out at nature controlling line Existing band, then illustrate that test paper fails；If occurring band at nature controlling line, and do not occur band at detection line, then illustrate in sample Without people's HSP90 α -2 albumen；If there is band on nature controlling line and detection line, illustrate the α of HSP90 containing someone in sample- 2 albumen.

In another aspect, prepared the invention provides a kind of for quantitatively detecting people HSP90 α -2 albumen in determinand The method of fluorescence immune chromatography test paper, it comprises the following steps：

3) reaction film is provided, wherein fixing the 2nd HSP90 α -2 along chromatography direction interval when using on the reaction film Monoclonal antibody and antiantibody, to form detection zone and quality control region respectively；With

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

The present inventor by it is substantial amounts of experiment grope, optimize prepare the present invention be used for detect people HSP90 α -2 The condition of each step of the method for the fluorescence immune chromatography test paper of albumen, so that the fluorescence immune chromatography test paper of the present invention The result for meeting clinical detection requirement can be obtained for people HSP90 α -2 albumen, i.e. detection range is wide, specific high, sensitive Spend.

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

In the specific embodiment of the present invention, the step 1) include：

A) 1 (w/v) % fluorescent microsphere aqueous dispersions, 10000rpm to 15000rpm low temperature (such as 10 DEG C) centrifugations 5 are taken To 10 minutes, supernatant is removed, sediment is distributed in 500 μ l distilled water or first wash buffer (the 0.1M MES aqueous solution), is surpassed Sound wave (240W) is handled 1 to 2 minute, repeats above procedure three times, is added carbodiimide 10mg to 50mg, is stirred 10~15 points Clock, so as to activate the fluorescent microsphere；

B) fluorescent microsphere for the activation that washing step a) is obtained, it is preferable that by the fluorescence of the step a) activation obtained Microballoon is centrifuged 5 to 10 minutes under 1000rpm to 15000rpm, and sediment is distributed into 1ml coupling buffers (20~100mM N- hydroxy thiosuccinimides-citrate buffer solution)) in, ultrasonic wave (240W) handle 1 to 2 minute, repeat above procedure Three times；

C) HSP90 α -2 monoclonal antibodies of fluorescent microsphere mark the first that step b) is obtained are used, it is preferable that according to 1 μ l extremely The ratio of the fluorescent microsphere activated described in 3 μ l antibody (8mg/ml)/100 μ l, by the step b) fluorescent microspheres obtained and first HSP90 α -2 monoclonal antibodies are mixed, and 1.5~3 hours (preferably 2 hours) are stirred under room temperature (25 DEG C), add 1ml closing bufferings Liquid (1 (w/v) %BSA-0.05M monoethanolamines), continues to stir 1 hour, 5 to 10 points is centrifuged under 10000rpm to 15000rpm Sediment is distributed to whole wash buffer (0.5 (w/v) %BSA-0.11 (v/v) the % tween water of 500 μ l by clock, repeated centrifugation 3 times Solution) in, ultrasonic wave (240W) is handled 1 to 2 minute, and 500 μ l are settled to the whole wash buffer.

Preferably, in the method for the invention, the step 2) include：The first HSP90 α -2 of fluorescent microsphere will be marked with Monoclonal antibody is diluted with antibody diluent (1% (w/v) BSA-0.01MPBS (pH7.2) buffer solution), to be diluted to 0.5 ~2mg/ml, preferably 1mg/ml, then with micropipettor even application on pad, drying afterwards or vacuum refrigeration are dry It is dry.In the step 2 of the method for the present invention) in, the bag for the first HSP90 α -2 monoclonal antibodies that described mark has It is 0.5~2mg/ml, preferably 1mg/ml by concentration.

Preferably, the step 3) include：The 2nd HSP90 α -2 monoclonal antibodies and antiantibody are drawn with metal spraying machine On to nitrocellulose filter (solid phase carrier) using as detection zone and quality control region, make detection zone and quality control region at intervals of 3mm extremely The concentration of 8mm, the 2nd HSP90 α -2 monoclonal antibodies and the antiantibody is respectively 0.5~2mg/ml, preferably 1mg/ ml。

Preferably, as a result detection using 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 and testing sample of detection zone and quality control region fluorescence intensity HSP90 α -2 content be directly proportional.Using detection zone with the ratio of quality control region fluorescence intensity rather than directly using detection zone Absolute fluorescence value can be reduced as far as the influence of reaction condition, matrix etc., and avoid ambient interferences as far as possible.

Present disclosure is further explained and described in mode below by way of example, but these examples are understood not to Limitation to protection scope of the present invention.

Embodiment

Unless otherwise indicated, the percentage that solution as described below is in the aqueous solution, solution is percentage by volume.

Embodiment 1：HSP90 α -2 epitope peptides (1) and the preparation of (2).

Preparation method chemical synthesis：Using many automatic peptide synthesizers of American AB I431A types, closed respectively by solid phase method Into HSP90 α -2 epitope peptides (1) and (2).The purity of epitope peptide is evaluated with high performance liquid chromatography, and determines peptide The concentration of section.The epitope peptide (1) and the molecular weight of (2) of the present invention is respectively 2404.35,2483.39, is carried out using mass spectrum It is determined that, the peptide sequence synthesized by identification is determined by peptide sequence.

First, the synthesis of HSP90 α -2 epitope peptides (1) and (2)

Above-mentioned peptide fragment uses Solid phase synthesis.The main thought of Solid phase peptide synthesis is：The carboxyl that peptide chain will first be synthesized The carboxyl of end amino acid is connected with the same insoluble high-molecular compound (resin) of covalent bond form, is then tied with this The amino acid on solid phase carrier is closed as moiety, it is anti-by the activated carboxyl component for sloughing amino protecting group and same excess Should, spreading peptide chain.Such step repeatedly can repeatedly go on, the length of the peptide chain of synthesis required for finally reaching. This building-up process is as follows.

HSP90 α -2 epitope peptides (1) of the present invention and the respective specific preparation process of (2) are as follows：

1. it is raw materials used：

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

Fmoc-AA (amino acid of 9- fluorenylmethoxycarbonyl carbonyls acyl group protection, be purchased from Merck companies)

NMP (N-methyl pyrrolidones is purchased from sigma companies)

DCM (dichloromethane is purchased from Central Plains chemical company)

MeoH (methanol is purchased from Central Plains chemical company)

Piperidines (Piperidine is purchased from sigma companies)

DMAP (dimethyl aminopyridine is purchased from sigma companies)

HOBT (hydroxybenzotriazole is purchased from sigma companies)

DCC (dicyclohexylcarbodiimide is purchased from sigma companies)

TFA (trifluoroacetic acid is purchased from sigma companies)

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

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

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 is purchased from ABI companies

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

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

Freeze drier, model VFD-2000 is purchased from the rich doctor Kanggong department in Beijing

3. synthetic method and process：

HMP resin 100mg are weighed, substitution equivalent is 1.0meq, i.e., 0.1mmol is placed in American AB I431A types polypeptide certainly In the reaction chamber of dynamic synthesizer, specific amino acid is connected in a different order automatically by synthesizer, Conjugate ratio reaches 99%.Reaction is as follows：

(1) activation (HOBt/DCC methods) of amino acid

(2) amino acid is connected to resin

(3) the Fmoc protection groups of amino acid are sloughed

(4) activation (HOBt/DCC methods) of another amino acid

(5) it is coupled

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

Respectively obtain the peptide resin 178mg of peptide resin 214mg and HSP90 α -2 peptide fragments (2) of HSP90 α -2 peptide fragments (1).

(7) peptide resin：

Peptide chain is cut with TFA (trifluoroacetic acid), is removed with EDT (2.5 volume %), thioanisole (2.5 volume %) Agent, reacts 3.0 hours at room temperature, removes cutting reagent, then is extracted with ether, respectively obtains HSP90 α -2 peptide fragments (1) and (2) Crude product.

2nd, the purifying of HSP90 α -2 epitope peptides (1) and (2) crude product：

Purified using high performance liquid chromatography separation：

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

Chromatograph：Waters600, Waters, US

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

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

Detection wavelength：214nm

Flow velocity：4ml/ minutes

Gradient：20-60%B, 30 minutes

HPLC (high performance liquid chromatography) is analyzed

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

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

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

Detection wavelength：214nm

Flow velocity：1ml/ minutes

Gradient：0-60%B, 30 minutes

The purity of peptide fragment analysis result display HSP90 α -2 epitope peptides (1) of the invention and (2) is more than 95%.

3rd, the identification of HSP90 α -2 epitope peptides (1) and (2)

1. determine HSP90 α -2 epitope peptides (1) obtained by purifying and the molecular weight of (2) respectively using mass spectrum.

(1) reagent raw material

TFA (trifluoroacetic acid is purchased from sigma companies)

HCCA (alpha-cyano -4- hydroxycinnamic acids, be purchased from sigma companies)

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

(2) instrument

MALDI-TOF-MS instrument MALDI-TOF-MS (models：REFLEX III, Germany Bruker companies)；

(3) matrix liquid：α-CCA are dissolved in the 50%ACN solution containing 0.1%TFA, saturated solution is made, centrifuges, takes Clearly；

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

(5) operating procedure：Take the 1 above-mentioned purified polypeptides of μ L (1) and the sample of (2) respectively, each with 1 μ L saturation matrix The isometric mixing of clear liquid mixing, takes 1 μ L points on sample target respectively, is detected in feeding ion gun.

As a result, the molecular weight for measuring gained HSP90 α -2 epitope peptides (1) is 2404.5, HSP90 α -2 epitope peptides (2) molecular weight is 2483.5, consistent with theoretical molecular 2404.35,2483.39, it was demonstrated that synthesis polypeptide is purpose product.

2. the sequence of HSP90 α -2 epitope peptides (1) and (2) as obtained by peptide sequence determines identification respectively.

(1) principle：The general principle of polypeptid acid sequence analysis is Edman degradeds, is that a circulating chemistry is anti- Answer process.Including three main chemical steps：(1) it is coupled：The N- ends residue of phenyl isothiocyanate and proteins and peptides is anti- Should, form phenylamino formyl sulfide (PTC) derivative, i.e. PTC- peptides.(2) cyclisation cracking：The cyclisation cracking of PTC- peptides.(3) convert：Thiophene Azoles purine ketone phenylamino (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH- amino acid).Stay in the solution reduce an amino acid The peptide of residue repeats the above-mentioned course of reaction of progress, and whole sequencing procedure is carried out automatically by sequenator now.

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

(3) reagent raw material

Phenyl isothiocyanate PITC, is purchased from sigma companies

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

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

TFA (trifluoroacetic acid is purchased from sigma companies)

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

Chlorobutane, is purchased from sigma companies

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

(4) determine

Carried out by instrument specification.

As a result：Identified, the sequence of gained HSP90 α -2 epitope peptides (1) and (2) is respectively：

(1)Y-E-K-E-S-E-D-K-P-E-I-E-D-V-G-S-D-E；With

(2)Y-R-G-I-D-E-D-D-P-T-A-D-D-T-S-A-A-V-T-E。

The result is consistent with target section of synthesized peptide.

Embodiment 2：Respectively by HSP90 α -2 epitope peptides (1) of the gained of embodiment 1 and (2) be connected with carrier protein with HSP90 α -2 antigens (1) and (2) are prepared, animal is immunized respectively using gained antigen (1) and (2), so as to be prepared using antigen (1) Specific monoclonal antibody and polyclonal antibody, and prepare specific monoclonal antibody and polyclonal using antigen (2) Antibody.

1. the preparation of antigen：With BDB (Bis-diazotizedbenzidine dichloride) methods by HSP90 α -2 peptides Section (1) and (2) is connected that to be prepared into HSP90 α -2 anti-with carrier protein KLH (keyhole limpet hemocyanin) (derive from sigma companies) respectively Former (1) and (2).

HSP90 α -2 peptide fragments (1) or (2) 10.0mg are taken, is dissolved with 1ml0.1M PBSs (pH7.4)；KLH10mg, Dissolved with 0.2M borate buffer solutions (pH9.0) 20ml；Then both are mixed, is cooled to 0 DEG C, takes BDBCl₂110 μ L, room temperature Dispensed after lower reaction 1.5h, dialysed overnight, -20 DEG C of preservations.

In the present embodiment, the formula of PBS is：0.2mol/L Na₂HPO₄81ml adds 0.2mol/L's NaH₂PO₄19ml is mixed.

The formula of borate buffer solution is：0.05mol/L borax 80ml, plus 0.2mol/L boric acid 20ml are mixed.

2. immune animal prepares monoclonal antibody：

2.1. HSP90 α -2 antigens (1) and (2) (immunogene) of above-mentioned preparation is taken to be helped completely with isometric Freund respectively After agent (being purchased from Shanghai Yuan Ju biotech firms) is sufficiently mixed, immune Balb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection.4 Serum titer is surveyed after week, the good mouse of immunoreactivity booster immunization again is selected：Take antigen and isometric incomplete Freund After agent (being purchased from Shanghai Yuan Ju biotech firms) is sufficiently mixed, 25 μ of antigen dose g/, subcutaneous multi-point injection, time of booster immunization Number is 6 times, each At intervals of two to three weeks, before fusion in addition continuous booster immunization twice, per minor tick 1-2 week, afterwards extracting spleen cell and Sp2/0 myeloma cell is merged, is used in combination with 50%PEG (MW4000) (being purchased from Central Plains chemical company) mediation according to a conventional method HAT conditioned mediums (being purchased from sigma companies) selection culture.CO is put into after fusion₂In incubator 37 DEG C culture 9~11 days after, Occurs larger cell clone in hole.Start to be screened with indirect ELISA within 11 days.To primary dcreening operation, positive hole utilizes limiting dilution Method carries out 4 time cloning cultures (even if screening after a large amount of schizogamies of cell), afterwards amplifying cells, freeze, prepare ascites.

2.2. Balb/c mouse are only handled with norphytane (being purchased from sigma companies) 0.5ml/, pneumoretroperitoneum inoculation in one week is miscellaneous Hand over oncocyte 2 × 10⁶Individual/only, collect ascites after 10 days.

2.3. antibody titer is determined：The monoclonal prepared using HSP90 α -2 antigens (1) is determined with indirect ELISA method to resist The potency of body (1), as a result shows that the potency of monoclonal antibody reaches 1:More than 32000.

The potency of the monoclonal antibody (2) prepared using HSP90 α -2 antigens (2) is also measured using identical method, Its potency also reaches 1:More than 32000.

3. immune animal prepares polyclonal antibody：

3.1. the NZw for being about 2kg or so from three monthly ages, body weight is used as immune animal.In fundamental immunity, HSP90 α -2 antigens (1) and (2) (immunogene) of the above-mentioned preparations of 1-2mg (are purchased from isometric complete Freund's adjuvant respectively Shanghai Yuan Ju biotech firms) mixing-it is fully emulsified after rabbit back carry out multiple spot hypodermic injection.Every 4 weeks booster immunizations one It is secondary, booster immunization 6 times, after antigen and incomplete Freund's adjuvant (being purchased from Shanghai Yuan Ju biotech firms) are fully emulsified, with 100 μ g/ Only it is subcutaneously injected in back multiple spot.Arteria carotis bloodletting in 10th day after final boost, separates serum.

3.2. antibody titer is determined：The polyclonal antibody prepared using HSP90 α -2 antigens (1) is determined with indirect elisa method (1) potency, as a result shows that antibody titer reaches 1:More than 32000.

The potency of the polyclonal antibody (2) prepared using HSP90 α -2 antigens (2) is also measured using identical method, Its potency also reaches 1:More than 32000.

3.3. blood and separation serum are taken：Arteria carotis intubation takes blood, separates serum.

4. isolate and purify antibody：After ammonium sulfate precipitation, then through Protein G (being purchased from sigma companies) affinity purification.

5. freezed after antibody packing, Cord blood.

Embodiment 3：The specificity identification of people's HSP90 α -2 monoclonal antibodies (1) and (2)

Detected with ELISA.Respectively with people HSP90 α -2 albumen, S-100B albumen, neuronspecific enolase NSE (being purchased from Shanghai Lian Shuo companies) is detection antigen coat elisa plate, detected respectively by ELISA prepared HSP90 α- 2 monoclonal antibodies (1) and (2) and the specific reaction of people's HSP90 α -2 albumen, make negative with normal BALB/c mouse serum Control, PBS liquid makees blank control.

As a result：It is the positive (P/N that HSP90 α -2 monoclonal antibodies (1) and (2) are only reacted with HSP90 α -2 respectively>2.1), and It is feminine gender with S-100B albumen, neuronspecific enolase NSE reactions, illustrates HSP90 α -2 monoclonal antibodies of the present invention (1) and (2) respectively have specificity.

Embodiment 4：The specificity identification of people's HSP90 α -2 polyclonal antibodies (1) and (2)

Identified using with above-mentioned identification monoclonal antibody specificity identical method.

As a result show：HSP90 α -2 polyclonal antibodies (1) and (2) are respectively the positive (P/N with HSP90 α -2 reactions>2.1), And be feminine gender with S-100B albumen, neuronspecific enolase NSE reactions, illustrate HSP90 α -2 Anti-TNF-αs of the present invention Body (1) and (2) have specificity respectively.

Embodiment 5：HSP90 α -2 are prepared using HSP90 α -2 monoclonal antibodies and HSP90 α -2 polyclonal antibodies to examine in vitro Disconnected kit.

In the present embodiment, the monoclonal antibody (1) that will be prepared in embodiment 2 using HSP90 α -2 epitope peptides (1) It is used as the coated antibody in this kit；The polyclonal antibody that will be prepared in embodiment 2 using HSP90 α -2 epitope peptides (2) (2) as binding antibody.

The preparation and operation of HSP90 α -2 external diagnosis reagent cases are as follows：

1. the preparation of various buffer solutions and reagent：

A, coating buffer solution：0.050M, pH9.6 CB (carbonate buffer solution)

Na₂CO₃：16.0 grams

NaHCO₃：29.0 grams

Distillation is water-soluble to 1000ml

B, sample/lavation buffer solution：PH7.2 10 × PBS-Tween20

Na₂HPO₄·12H₂O：58 grams

KH₂PO₄：4 grams

NaCl：100 grams

KCl：4 grams

Distillation is water-soluble to 1000ml

Plus Tween20：20ml

C, enzyme marker dilution：

10×PBS-Tween20：10ml

FCS (calf serum)：20ml

Distillation is water-soluble to 1000ml

Enzyme stabilizers (are purchased from Shanghai Xi Bao companies)：1 gram

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

D, developer A：

Citric acid：35.5 grams

Urea peroxide：10 grams

Distillation is water-soluble to 1000ml

Tween20：10ml

E, developer B：

Citric acid：120 grams

EDTA-2Na：1 gram

TMB·2HCl：2 grams

Distillation is water-soluble to 1000ml

F, terminate liquid：2M H₂SO₄

The concentrated sulfuric acid (95-98%)：22.2ml

Distilled water：177.3ml

The concentrated sulfuric acid is slowly dropped into distilled water by timing, and side edged shakes up.

2. the preparation of pre-coated plate：

HSP90 α -2 monoclonal antibodies (1) are dissolved in pH=9.6 0.05M carbonate buffer solution, are made pre-coated Liquid, 100 μ l are added on ELISA Plate (being purchased from Shenzhen Jin Canhua companies) per hole by 0.1 μ g/ holes, put 4 DEG C of placement 18-24 small When, take out, get rid of coating buffer, wash with sample/lavation buffer solution, through 1 (w/v) %BSA-0.05M monoethanolamines closing 16 hours, It is fitted into after dried overnight in aluminide-coating bag and vacuumizes sealing, is placed in 4 DEG C of preservations.

3. binding antibody (HSP90 α -2 polyclonal antibodies (2)) and enzyme-linked thing (the goat-anti rabbit of horseradish peroxidase-labeled IgG antibody) dilution ratio of (be purchased from Beijing company of Zhong Shan Golden Bridge) determines by square formation titration experiments, horseradish peroxidase mark The goat anti-rabbit igg antibody of note uses enzyme marker diluted.

4. the composition of kit：

Pre-coated plate：48/96 hole

HSP90 α -2 calibration objects (raw material is purchased from Shanghai Lian Shuo companies)：7：7 × 1.0ml (concentration be respectively 25ng/ml, 10ng/ml、5ng/ml、2.5ng/ml、1ng/ml、0.5ng/ml、0.25ng/ml)

HSP90 α -2 binding antibodies：1 × 10ml is (through 1:5000 dilutions)

Enzyme-linked thing：1 × 10ml is (through 1:5000 dilutions)

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

Developer A：1×6.0ml

Developer B：1×6.0ml

Terminate liquid：1×6.0ml

5. the operating procedure of kit：

Blood sample to be checked and the μ l/ holes of standard items 100 are separately added into each hole of pre-coated plate, is diplopore, 37 DEG C incubate Educate 60 minutes, washed 5 times, patted dry with 1 × lavation buffer solution.The μ l/ holes of HSP90 α -2 binding antibodies 100,37 are added in each hole DEG C be incubated 30 minutes, washed 5 times, patted dry with 1 × lavation buffer solution.The enzyme-linked μ l/ holes of thing 100 are added in each hole again, 37 DEG C incubate Educate 30 minutes, washed 5 times, patted dry with 1 × lavation buffer solution.Developer A, B liquid is added, per each 50 μ l in hole, is mixed, 37 DEG C of incubations 15 minutes.Plus the μ l/ holes terminating reaction of terminate liquid 50, use double wave with enzyme detector (model RT-6000 is purchased from Lei Du companies) Long (450nm, 620nm) detection absorbance.

6. result judgement：

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	0.124	0.213	0.385	0.715	1.237	2.158

Standard curve, the R of standard curve are drawn with the logarithm value of standard concentration and correspondence absorbance²=0.978.

HSP90 α -2 concentration results in detected sample are calculated according to standard curve.

30 tumour patients and 81 healthy persons are carried out with serum HSP90 α -2 detections, affinity antibody to SpA in a manner described In HSP90 α -2 contents apparently higher than healthy control group, the statistically significant (P of difference<0.01) 2, are shown in Table.

Table 2：Two groups of sample HSP90 α -2 concentration compare

From data above, kit of the invention can effectively and specifically detect that the HSP90 α -2 in serum contain Amount, so as to detect HSP90 α -2 content differences between tumour patient and normal person, thus can determine whether the generation of tumour.

Embodiment six：Preparation for detecting the fluorescence immune chromatography test paper of people HSP90 α -2 albumen in determinand.

First, preparation and the coating pad of the monoclonal antibody of fluorescent microsphere are marked with

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

1.1st, the activation of fluorescent microsphere：

500 μ l, content 1 (w/v) % fluorescent microsphere (being purchased from Guangzhou Growth hormone secretagogue company) aqueous dispersions are taken, at 10 DEG C, Centrifuged 10 minutes with 12000rpm, remove supernatant, sediment is distributed to 500 μ l distilled water or first wash buffer (0.1M MES The aqueous solution) in, ultrasonic wave (240W) is handled 2 minutes, repeats above procedure three times, adds carbodiimide (purchased from the brilliant pure public affairs in Shanghai Department) 50mg, is stirred 15 minutes, so as to activate the fluorescent microsphere.

1.2nd, with the fluorescent microsphere labelled antibody activated：

The fluorescent microsphere activated is centrifuged 10 minutes under 12000rpm, supernatant is removed, sediment is distributed to 1ml couplings In buffer solution (citrate buffer solution of 50mM N- hydroxy thiosuccinimides), ultrasonic wave (240W) is handled 2 minutes, is repeated Above procedure three times, obtains the buffer solution 1ml for being dispersed with fluorescent microsphere.Activated according to 3 μ l antibody (8mg/ml)/100 μ l The ratio of fluorescent microsphere, adds HSP90 α -2 monoclonal antibodies (1) prepared by embodiment 2 thereto, 2 is stirred at normal temperatures small When, 1ml Block buffers (1 (w/v) %BSA-0.05M monoethanolamines) are added, continue to stir 1 hour, afterwards, under 12000rpm Sediment is distributed to whole wash buffer (0.5 (w/v) %BSA-0.11 (v/ of 500 μ l by centrifugation 10 minutes, repeated centrifugation 3 times V) % Tween solutions) in, ultrasonic wave (240W) is handled 2 minutes, and 500 μ l are settled to above-mentioned whole wash buffer.

2nd, it is coated with pad

HSP90 α -2 monoclonal antibodies (1) antibody diluent (1% (w/ that the mark of above-mentioned preparation is had V) BSA-0.01M PBS (pH7.2) buffer solution) 1mg/ml is diluted to, working solution is obtained, then (is purchased from micropipettor Labsystems companies) by 4 μ l/cm amount even application on pad (be purchased from Shanghai outstanding person one, article No. CFSP223000), it Afterwards with 37 DEG C of oven for drying, saved backup under 45% humidity.

2nd, the preparation of reaction film

HSP90 α -2 monoclonal antibodies (2) and sheep anti-mouse igg monoclonal antibody prepared according to embodiment 2 (is purchased from Beijing Company of Zhong Shan Golden Bridge) 1mg/ml is diluted to respectively with 50mM pH7.2 PBS, metal spraying machine is (public purchased from Hangzhou peak Hangzhoupro Department) detection line and nature controlling line spacing parameter be set to 6mm, package amount is respectively set to 1.0 μ l/cm, with metal spraying machine in nitre Upper HSP90 α -2 monoclonal antibodies (2) and sheep anti-mouse igg are drawn on acid cellulose film (being purchased from Shanghai outstanding person one, article No. GFCP203000) Monoclonal antibody, normal temperature dries standby.

3rd, the assembling and cutting of test paper

On bottom plate (be purchased from Shanghai outstanding person one, article No. HF000MC100) successively mutually overlap joint paste sample pad, it is pad, anti- Film and absorbent filter are answered, test paper plate is obtained, the test strips that width is 5mm are cut to.

4th, the preparation of HSP90 α -2 fluorescence immunoassays detection card：

The test paper of above-mentioned well cutting is fixed on plastic bottom card, test paper surface is compressed with face card, and face is stuck in test strips Well and observation window are provided with the position of sample pad and reaction film.Detection card is fitted into aluminium foil bag after assembling, and adds drying Agent sealing is preserved, and can be preserved more than 1 year under the conditions of drying at room temperature.

5th, the detection of sample

HSP90 α -2 standard items (being purchased from Shanghai Lian Shuo companies) sample diluting liquid (1% (w/v) BSA-0.01M PBS (pH7.2) buffer solution) it is configured to the calibration object of following series concentration：100ng/ml、50ng/ml、20ng/ml、10ng/ml、 5ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml, 0.2ng/ml, 0.1ng/ml, 0ng/ml, 50 μ l above calibration objects are dripped respectively It is added on well, with fluorescence detector (being purchased from Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) after 10 minutes Detection, can collect fluorescence in detection line and Quality Control line position.Using sample concentration as abscissa, at detection line and nature controlling line The ratio of fluorescence intensity is that ordinate draws calibration curve, R²For 0.995.Nature controlling line is used for test paper Effective judgement and to inspection Line signals make corresponding correction, and such as nature controlling line does not occur band, then illustrate that test paper fails.

50 μ l blood sample to be checked is taken, is added drop-wise on well, is detected after 10 minutes with fluorescence detector, if detection line goes out Existing band, illustrates α containing HSP90 in sample -2, its concentration can be obtained according to calibration curve.

6th, HSP90 α -2 fluorescence immunoassays test paper performance evaluation

1. evaluate the index of test paper performance

1) range of linearity：Each concentration calibration product repeat detection 3 times, draw calibration curve, through data fitting and statistical analysis, Test paper linear detection range of the present invention is 0.2ng/ml-100ng/ml.

2) minimum detectability：HSP90 α -2 null values blood sample (no HSP90 α -2 compositions) (being purchased from Shanghai Lian Shuo companies) is divided into 20 parts are detected, are calculated mean concentration and 2 times of standard deviation sums, are obtained test paper lowest detection of the present invention and be limited to 0.04ng/ml.

3) precision：Detect that HSP90 α -2 concentration is respectively respectively with the HSP90 α -2 fluorescence immunoassays test paper of the present invention 50ng/ml, 12.5ng/ml, 2.50ng/ml blood sample, repeat detection 10 times, carry out withinrun precision measure.Daily to above-mentioned 3 The sample of individual concentration is measured, 1 day 1 time, continuous to survey 20 days, carries out betweenrun precision measure, as a result as shown in table 3 below：

Table 3

CV is respectively less than 8% between criticizing interior CV (coefficient of variation) and criticizing, and illustrates that the reagent accurate is good.

In addition, as seen from the above table, this test paper detection HSP90 α -2 range of linearity is wide, sensitivity is good.

Claims

1. a kind of be used for the fluorescence immune chromatography test paper of people's HSP90 α -2 albumen in quantitatively detection determinand, the test paper passes through dual anti- Body sandwich method detects people HSP90 α -2 albumen, wherein：

The double antibody sandwich method uses the first HSP90 α -2 monoclonal antibodies for being marked with fluorescent microsphere as capture antibody, institute The first HSP90 α -2 monoclonal antibodies are stated from one of people's HSP90 α -2 epitope peptides (1) and (2)；And

The double antibody sandwich method is using the 2nd HSP90 α -2 monoclonal antibodies as detection antibody, and the 2nd HSP90 α -2 are single Clonal antibody is from the other of people's HSP90 α -2 epitope peptides (1) and (2)；

People HSP90 α -2 epitope peptides (1) and (2) are respectively：

(1)Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser-Asp- Glu；

(2)Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala- Val-Thr-Glu。

2. fluorescence immune chromatography test paper according to claim 1, wherein the first HSP90 α -2 monoclonal antibodies be by What the first HSP90 α -2 antigens were prepared from, the first HSP90 α -2 antigens are by making people HSP90 α -2 epitope peptides (1) it is prepared from one of (2) with carrier protein couplet；And

2nd HSP90 α -2 monoclonal antibodies are prepared from by the 2nd HSP90 α -2 antigens, and the 2nd HSP90 α -2 resist Original is by making the other of people HSP90 α -2 epitope peptides (1) and (2) are prepared from carrier protein couplet.

3. fluorescence immune chromatography test paper according to claim 1, wherein the particle diameter of the fluorescent microsphere be 320nm extremely 400nm。

4. fluorescence immune chromatography test paper according to claim 1, wherein the fluorescent material on the fluorescent microsphere is different sulphur Cyanic acid fluorescein, RB 200, TRITC or X- rhodamines；The micro-sphere material of the fluorescent microsphere The copolymer formed for polystyrene, polymethyl methacrylate or by methyl methacrylate and other monomer copolymerizations.

5. fluorescence immune chromatography test paper according to claim 1, wherein the excitation wavelength of the fluorescent microsphere be 350~ 600nm；Launch wavelength is 500~700nm.

6. fluorescence immune chromatography test paper according to claim 1, wherein the test paper has bottom plate, and on which floor plate It is provided with successively with the way of contact along chromatography direction when using：Sample pad, pad, reaction film, absorbent filter, the combination Pad is provided with the first HSP90 α -2 monoclonal antibodies that described mark has, and the reaction film includes detection zone and matter Area is controlled, the detection zone is coated with the 2nd HSP90 α -2 monoclonal antibodies, and the quality control region is coated with can be with described mark The antiantibody that the first HSP90 α -2 monoclonal antibody specificities that note has fluorescent microsphere are combined.

7. fluorescence immune chromatography test paper according to claim 6, wherein the reaction film is under the wavelength more than 550nm Substantially not fluorescent nitrocellulose filter.

8. fluorescence immune chromatography test paper according to claim 6, wherein the bottom plate does not have photoluminescent property substantially.

9. fluorescence immune chromatography test paper according to claim 6, wherein the antiantibody is sheep anti-mouse igg monoclonal antibody Or rabbit anti-mouse igg monoclonal antibody.

10. fluorescence immune chromatography test paper according to claim 4, wherein the fluorescent material on the fluorescent microsphere is sieve X- It is red bright.

11. fluorescence immune chromatography test paper according to claim 5, wherein the excitation wavelength of the fluorescent microsphere is 390nm； Launch wavelength is 615nm.

12. fluorescence immune chromatography test paper according to claim 9, wherein the antiantibody is anti-for sheep anti-mouse igg monoclonal Body.

13. a kind of method of the fluorescence immune chromatography test paper prepared according to any one in claim 1 to 12, it includes Following steps：

2) pad is provided, wherein being coated with the first HSP90 α -2 Dan Ke that described mark has on the pad Grand antibody；

3) reaction film is provided, wherein fixing the 2nd HSP90 α -2 Dan Ke along chromatography direction interval when using on the reaction film Grand antibody and antiantibody, to form detection zone and quality control region respectively；With

4) sample pad, the pad, the reaction are set with the way of contact successively along chromatography direction when using on bottom plate Film, absorbent filter, so that the fluorescence immune chromatography test paper is made.

14. method according to claim 13, wherein the step 1) include：

A) carbodiimide activation fluorescent microsphere is used；

B) fluorescent microsphere for the activation that washing step a) is obtained；

C) HSP90 α -2 monoclonal antibodies of fluorescent microsphere mark the first that step b) is obtained are used.

15. method according to claim 14, wherein the step 1) include：

A) aqueous dispersions of fluorescent microsphere or MES buffer solutions dispersion liquid are mixed through ultrasonication and with carbodiimide, so that Activate the fluorescent microsphere；

B) by step A) fluorescent microsphere of activation that is obtained washs with N- hydroxy thiosuccinimides-citrate buffer solution, It is scattered, and through ultrasonication；

C) by step B) fluorescent microsphere that is obtained mixes with the first HSP90 α -2 monoclonal antibodies, with BSA- monoethanolamine buffer solutions Closing, centrifugation is scattered with BSA- Tween solutions, through ultrasonication, so as to obtain being marked with the first HSP90 of fluorescent microsphere α -2 monoclonal antibodies.

16. method according to claim 13, wherein in the step 2) in, described mark have first The coating concentration of HSP90 α -2 monoclonal antibodies is 0.5~2mg/ml.

17. method according to claim 13, wherein in the step 3) in, the detection zone and the quality control region interval 3mm to 8mm.

18. the method according to claim 13 or 17, wherein in the step 3) in, the 2nd HSP90 α -2 monoclonals The coating concentration of antibody and the antiantibody is respectively 0.5~2mg/ml.