CN104422774B

CN104422774B - Fluorescence immune chromatography test paper of detection people's PGII albumen and preparation method thereof

Info

Publication number: CN104422774B
Application number: CN201310370234.0A
Authority: CN
Inventors: 朱建安
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-08-22
Filing date: 2013-08-22
Publication date: 2016-07-06
Anticipated expiration: 2033-08-22
Also published as: CN104422774A

Abstract

The present invention relates to fluorescence immune chromatography test paper of detection people's PGII albumen and preparation method thereof.Described reagent paper is by double antibody sandwich method detection people's PGII albumen, described double antibody sandwich method adopts the PGII monoclonal antibody being marked with fluorescent microsphere as catching antibody, and a described PGII monoclonal antibody derives from the one in the sequence described in sequence table SEQ ID NO.1 and SEQ ID NO.2；And described double antibody sandwich method adopts the 2nd PGII monoclonal antibody as detection antibody, and described 2nd PGII monoclonal antibody derives from the another one in the sequence described in sequence table SEQ ID NO.1 and SEQ ID NO.2.The fluorescence immune chromatography test paper of the present invention is easy and simple to handle, quick, detection range width, specificity high, sensitivity is good.

Description

Fluorescence immune chromatography test paper of detection people's PGII albumen and preparation method thereof

Technical field

The invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to human pepsinogen II(PGII) epitope peptide, the PGII specific antigen prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, described antibody purposes on preparation people's PGII external diagnosis reagent case, people's PGII external diagnosis reagent case, and a kind of for fluorescence immune chromatography test paper of people PGII albumen and preparation method thereof in detection by quantitative determinand.

Background technology

Pepsinogen (PG) is pepsic inactive precursor in gastric juice.Human pepsinogen by fast extremely slow by its electrophoretic mobility, can be divided into seven kinds of isozymogens of PG1 to PG7, and can be divided into two subgroups of PGI and PGII according to distribution in its biochemical property, immunogenicity, cell derived and tissue.PG1 to PG5 immunogenicity in seven kinds of isozymogens is similar to, and is called pepsinogen I (PGI), and it is mainly secreted by the chief cell of gastric gland and mucus neck cell；PG6 to PG7 is referred to as pepsinogen I I(PGII), except the cell secretes secreting acid gland of mucosa at the bottom of by body of stomach stomach function regulating, the Brunner gland of the mucilage cell and duodenum epimere of secreting the pyloric gland of the mucus neck cell of acid gland, cardiac gland and gastric antrum also can produce PGII.Stomach is almost the exclusive source of PG, and the secretory volume in the secretion stage can change.Serum PG I and PGII reflects the secretory function of gastric mucosa different parts.PG major part after synthesis enters gastral cavity, activates into pepsin under acidic gastric juice effect, only a small amount of PG(about 1%) through gastric mucosa hair.Therefore, serum PG concentration can reflect its secretion level.PGI in normal human serum is 6 times of PGII, and when gastric mucosa generation pathological change, serum PG content also changes therewith.

Serum PG content is relevant to helicobacter pylori (HP) infection, chronic gastritis, gastric cancer etc..In different disease of stomach, serum PG I, PGII and PGI/PGII value generation respective change, it raises, reduces or constant and inconsistent, also different as the sensitivity and specificity diagnosing or treating result index.Therefore, for different disease of stomach, serum PG I, PGII and PGI/PGII value also have different criterions.

Gastric cancer is one of common malignant tumor of China, and its case fatality rate occupies first of various malignant tumor, and its early diagnosis, early treatment become the unique channel improving life in patients, reducing case fatality rate.Serum Obtained From Advance Gastric Cancer PG change has been done substantial amounts of research and has been found by Chinese scholars: when getting a cancer of the stomach; serum PG I level reduces; PGII level substantially keeps normal or raises; and then; the ratio of the level and PGI/PGII that measure serum PG I contributes to the Differential Diagnosis of gastric cancer, and too low PGI and PGI/PGII should watch out for early gastric cancer.Owing to the content of serum PG directly reflects the function of gastric mucosa, therefore Serum Obtained From Advance Gastric Cancer PGI level is decreased obviously, it was shown that patients with gastric cancer gastric mucosa secretion capacity declines.The gastric cancer of more than 80% is with atrophic gastritis, and atrophic gastritis may result in gastric mucosa chief cell and loses, thus affecting its secretory function；Serum Obtained From Advance Gastric Cancer PGI level is decreased obviously with patients with gastric cancer Mucosal atrophy, intestinal thus to secrete reduction relevant.Therefore serum PG I and PGI/PGII is decreased obviously and monitoring early gastric cancer is had important clinical meaning, can be efficiently applied to the preliminary examination of high incidence area of gastric cancer crowd.PG detects as noninvasive method, it is possible to decrease the misery that patients with gastric disease or gastric cancer high-risk group check, and easy, economical, is significant.

The optimal method of the PGII level in detection serum is immunoassay.Therefore, find and suitable there is immunogenic PGII epitope peptide, prepare specific PGII antigen and namely antibody become emphasis.

The determinand that fluorescence immune chromatography method is surveyed in blood is easy and simple to handle, quick, only needs within 10 minutes, just to complete sample detection and detection range width, and sensitivity is good, it is possible to the assisted diagnosis state of an illness, monitoring prognosis in time rapidly.Chinese patent application No.200910117820.8 discloses preparation method and the quantitative detecting method of a kind of fluorescent micro-ball immune chromatography test paper strip；Chinese patent application No.200910047352.1 discloses a kind of fluorescence immune chromatography test paper and its preparation method and application.But, there is presently no the fluorescence immune chromatography test paper for people's PGII albumen.

Summary of the invention

For solving problem existing in above-mentioned prior art, the invention provides a kind of for fluorescence immune chromatography test paper of people PGII albumen and preparation method thereof in detection by quantitative determinand.

Specifically, the invention provides:

It is a kind of that for the fluorescence immune chromatography test paper of people PGII albumen in detection by quantitative determinand, this reagent paper detects described people's PGII albumen by double antibody sandwich method, wherein:

Described double antibody sandwich method adopts the PGII monoclonal antibody being marked with fluorescent microsphere as catching antibody, and a described PGII monoclonal antibody derives from the one in people's PGII epitope peptide (1) and (2)；And

Described double antibody sandwich method adopts the 2nd PGII monoclonal antibody as detection antibody, and described 2nd PGII monoclonal antibody derives from the another one in people's PGII epitope peptide (1) and (2)；

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

(1)Lys-Lys-Phe-Lys-Ser-Ile-Arg-Glu-Thr-Tyr；

(2)Tyr-Thr-Pro-Ser-Arg-Ala-Ala-Pro-Pro-Ser-Ser-Thr-Leu-Gln-Leu-Pro-Glu-Lys。

Preferably, a described PGII monoclonal antibody is prepared from by a PGII antigen, and a PGII antigen is by making the one in described people's PGII epitope peptide (1) and (2) be prepared from carrier protein couplet；And

Described 2nd PGII monoclonal antibody is prepared from by the 2nd PGII antigen, and the 2nd PGII antigen is by making the another one in described people's PGII epitope peptide (1) and (2) be prepared from carrier protein couplet.

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

Preferably, the fluorescent material on described fluorescent microsphere is Fluorescein isothiocyanate, RB 200, Tetramethylrhodamine isothiocyanate or X-rhodamine, is wherein preferably X-rhodamine；The micro-sphere material of described fluorescent microsphere is the copolymer of polystyrene, polymethyl methacrylate or methyl methacrylate.

Preferably, the excitation wavelength of described fluorescent microsphere is 350～600nm, it is preferred to 390nm；Transmitting wavelength is 500～700nm, it is preferred to 615nm.

Preferably, described fluorescence immune chromatography test paper has base plate, and it is provided with sample pad, pad, reaction film, absorbent filter with the way of contact successively along chromatography direction when using on which floor plate, described pad is coated with the described PGII monoclonal antibody being marked with fluorescent microsphere, described reaction film includes detection zone and quality control region, described detection zone is coated with described 2nd PGII monoclonal antibody, and described quality control region is coated with the anti antibody being combined with the described PGII monoclonal antibody specificity being marked with fluorescent microsphere.

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

Preferably, described base plate does not substantially have photoluminescent property.

Preferably, described anti antibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody.

Present invention also offers and a kind of prepare described for the method for the fluorescence immune chromatography test paper of people PGII albumen in detection by quantitative determinand, it comprises the following steps:

1) the PGII monoclonal antibody being marked with fluorescent microsphere is provided；

2) provide pad, on described pad, be wherein coated the described PGII monoclonal antibody being marked with fluorescent microsphere；

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

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

Preferably, described step 1) includes:

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

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

C) fluorescent microsphere labelling the oneth PGII monoclonal antibody obtained by step b), preferably, the fluorescent microsphere that step b) is obtained and PGII monoclonal antibody mixing, with BSA-ethanolamine buffer blind, centrifugal, disperse with BSA-Tween solution, through ultrasonic Treatment, thus obtaining being marked with a PGII monoclonal antibody of fluorescent microsphere.

Preferably, in described step 2) in, the concentration that is coated of the described PGII monoclonal antibody being marked with fluorescent microsphere is 0.5～2mg/ml.

Preferably, in described step 3), described detection zone and described quality control region interval 3mm to 8mm, described 2nd PGII monoclonal antibody and described anti antibody be coated concentration respectively 0.5～2mg/ml.

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

1. people's PGII 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.

2. the PGII monoclonal antibody prepared by the present invention and polyclonal antibody can high special PGII in blood sample be combined.

3. people's PGII external diagnosis reagent case of the present invention can monitor the level of the PGII in serum effectively, and preparation method is simple, and cost is low, is suitable to large-scale production.

4. fluorescence analysis is combined by the present invention with flash chromatography immunological technique, provide a kind of for the fluorescence immune chromatography test paper of people PGII albumen in detection by quantitative determinand, with the people's PGII albumen in this detection paper determinand, easy and simple to handle, quick, only need just to complete sample detection in 10 minutes, and detection range width, specificity are high, sensitivity is good, it is possible to the assisted diagnosis state of an illness, monitoring prognosis in time rapidly.

5. the present invention is in preparing the process of fluorescence immune chromatography test paper of described people's PGII albumen, groped by substantial amounts of test, optimize the preparation condition of each side, when making to detect with the fluorescence immune chromatography test paper of the present invention, fluorescence signal-to-background ratio is greatly improved, thus improve detection sensitivity and credible result degree；In addition, the present invention carrys out the content of PGII in response sample also by the detection zone of reagent paper with the change of the fluorescence intensity ratio of quality control region, this is compared with the absolute fluorescence intensity that traditional chromatographic technique only examines or check detection zone, decrease the impact of external condition and background etc. to the full extent, further increase testing result credibility.

Accompanying drawing explanation

Fig. 1 is the testing result figure with the dependency of the testing result of known agent box of the people's PGII external diagnosis reagent case illustrating the present invention, wherein abscissa is the value of PGII concentration in the sample recorded with known agent box, unit is ng/ml, vertical coordinate is the value of PGII concentration in the sample recorded with test kit of the present invention, and unit is ng/ml.

Fig. 2 schematically shows the structural representation of the fluorescence immune chromatography test paper of one embodiment of the invention.

Detailed description of the invention

Below by way of the description of detailed description of the invention and the invention will be further described with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art's basic thought according to the present invention, various modifications may be made or improves, but without departing from the basic thought of the present invention, all within the scope of the present invention.

One, people PGII epitope peptide

People's PGII albumen specifically described herein is that to it known in the art, its aminoacid sequence be it known in the art, to find in the specialized databases such as NCBI.

The invention provides a kind of people's PGII epitope peptide (1) and (2), its aminoacid sequence respectively as shown in sequence table SEQ IDNo.1 and SEQIDNo.2, for:

(1)K-K-F-K-S-I-R-E-T-Y；With

(2)Y-T-P-S-R-A-A-P-P-S-S-T-L-Q-L-P-E-K。

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

PGII epitope peptide (1) is using people's PGII albumen (NCBI serial number (NCBIReferenceSequence)): NP_001159896.1) one section of peptide fragment containing 9 amino acid residues of the 24th to 32, N end is as antigenic determinant, and is added with Y at its C end.Adding Y at C end is to make this epitope peptide pass through BDB(Bis-diazotizedbenzidinedichloride) it is linked on carrier protein (such as hemocyanin (KLH)), thus preparing antibody as antigen.PGII epitope peptide (1) has the advantages that hydrophilic height, antigenicity are strong and are readily synthesized.

PGII epitope peptide (2) is using one section of peptide fragment containing 17 amino acid residues of people's PGII albumen (NCBI serial number (NCBIReferenceSequence): NP_001159896.1) the 229th to 245, C end as antigenic determinant, and is added with Y at its N.Adding Y at N end is to make this epitope peptide pass through BDB(Bis-diazotizedbenzidinedichloride) it is linked on carrier protein (such as hemocyanin (KLH)), thus preparing antibody as antigen.PGII epitope peptide (2) also has the feature that hydrophilic height, antigenicity are strong and are readily synthesized.

At present, the present invention studies discovery, and the PGII epitope peptide of the present invention has 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 be combined with people PGII specifically.

The preparation method useful chemical synthetic method of the PGII epitope peptide of the present invention: utilize the many automatic peptide synthesizers of American AB I431A type, by Solid phase synthesis epitope peptide.The epitope peptide (1) of the present invention and the molecular weight of (2) respectively 1299.66 and 1943.41, available mass spectrum is determined, and is measured by peptide sequence and identify synthesized epitope peptide sequence.The purity thin layer chromatography of peptide fragment and high performance liquid chromatography are evaluated, and measure the concentration of epitope peptide.

Two, PGII antigen

Present invention also offers a kind of PGII antigen, it passes through to make the one in people's PGII epitope peptide (1) and (2) of the present invention be prepared from carrier protein couplet.Specifically, the invention provides PGII antigen (1) and (2), described PGII antigen (1) is prepared from by making people's PGII epitope peptide (1) of the present invention and carrier protein couplet；Described PGII antigen (2) is prepared from by making people's PGII epitope peptide (2) of the present invention and carrier protein couplet.The PGII antigen of the present invention has immunogenicity and specificity, being a kind of immunogen, can be used to immune animal thus preparing specific PGII antibody.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 better, and with immune animal sibship farther out, not easily causes cross reaction with it as carrier protein, is therefore preferred.

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

Present invention also offers people's PGII monoclonal antibody and people's PGII polyclonal antibody, described antibody can be utilized respectively the PGII antigen (1) of the present invention and the preparation of (2) (immunogen) immune animal and obtain.Preparation method can adopt the ordinary skill in the art, specifically can referring to embodiment 2.

The PGII monoclonal antibody of the present invention and polyclonal antibody may be used for preparation people's PGII external diagnosis reagent case, and the PGII in human blood specimen can be detected by this test kit based on immunization method.

Therefore, the invention provides a kind of people's PGII external diagnosis reagent case, its people's PGII monoclonal antibody comprising the present invention or polyclonal antibody.

It is currently known the immunization experiment method that can be used for Clinical Laboratory and mainly includes following several: ELISA method, chemoluminescence 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 detect antibody, indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, catch the method that is coated surveys antibody etc..

People's PGII external diagnosis reagent case of the present invention preferably employs ELISA double antibody sandwich method to detect PGII 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 PGII external diagnosis reagent case adopts people's PGII monoclonal antibody of the present invention as coated antibody.At this, term " coated antibody " refers to the antibody in the ELISA Plate being coated in solid phase.In addition, described people's PGII external diagnosis reagent case is it is also preferred that comprise people's PGII polyclonal antibody using as binding antibody, wherein, when described binding antibody derives from the one in people's PGII epitope peptide (1) and (2) of the present invention, described coated antibody derives from the another one in described epitope peptide (1) and (2).At this, term " binding antibody " refers to the specific antibody can being combined in test kit with determined antigen and enzyme-labeled secondary antibody.Described test kit can also comprise the second antibody of enzyme labelling, and this second antibody can be goat anti-rabbit igg antibody, and described enzyme labelling can be horseradish peroxidase, alkali phosphatase etc..

In the test kit of the present invention, it is also possible to comprise the required any reagent of detection or instrument, for instance pre-coated plate, cleaning mixture, developer, stop buffer etc..

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

Present invention also offers that a kind of this reagent paper detects described people's PGII albumen by double antibody sandwich method for the fluorescence immune chromatography test paper of people PGII albumen in detection by quantitative determinand, wherein:

Described double antibody sandwich method also adopts the 2nd PGII monoclonal antibody as detection antibody, and described 2nd PGII monoclonal antibody derives from the another one in people's PGII epitope peptide (1) and (2)；

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

(1)Lys-Lys-Phe-Lys-Ser-Ile-Arg-Glu-Thr-Tyr；

(2)Tyr-Thr-Pro-Ser-Arg-Ala-Ala-Pro-Pro-Ser-Ser-Thr-Leu-Gln-Leu-Pro-Glu-Lys。

In the present invention, in double antibody sandwich method fluorescence immune chromatography test paper, " catching antibody " refers to the antibody of first specific recognition determined antigen, and it is generally coated on pad；" detection antibody " refer to another kind can the antibody of specific recognition determined antigen, its from catch the different epitope that antibody identifies on determined antigen molecule respectively, it is typically secured on the detection zone of reaction film.

In the present invention, a described PGII monoclonal antibody can be prepared from by a PGII antigen, and a PGII antigen can pass through to make the one in described people's PGII epitope peptide (1) and (2) be prepared from carrier protein couplet；And described 2nd PGII monoclonal antibody can be prepared from by the 2nd PGII antigen, the 2nd PGII antigen can pass through to make the another one in described people's PGII epitope peptide (1) and (2) be 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 better, and with immune animal sibship farther out, not easily causes cross reaction with it as carrier protein, 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, it is preferably 360nm, fluorescent material on fluorescent microsphere can be Fluorescein isothiocyanate, RB 200, Tetramethylrhodamine isothiocyanate or X-rhodamine etc., is wherein preferably X-rhodamine (being purchased from Shanghai Jing Chun company).The micro-sphere material of fluorescent microsphere can be the copolymer formed by polystyrene, polymethyl methacrylate or methyl methacrylate and other monomer copolymerization, and the example of other monomer is styrene etc..The excitation wavelength of fluorescent microsphere can be 350～600nm, it is preferred to 390nm；Launching wavelength can be 500～700nm, it is preferred to 615nm.

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

In a specific embodiment, the fluorescence immune chromatography test paper of the present invention has base plate, and it is provided with sample pad with the way of contact successively along chromatography direction when using on which floor plate, pad, reaction film, absorbent filter, described sample pad is for loading testing sample in use, described pad is coated with the described PGII monoclonal antibody being marked with fluorescent microsphere, described reaction film includes detection zone and quality control region, described detection zone is coated with described 2nd PGII monoclonal antibody, described quality control region is coated with the anti antibody being combined with the described PGII monoclonal antibody specificity being marked with fluorescent microsphere.

Preferably, the sample pad of the fluorescence immune chromatography test paper of the present invention, pad, reaction film, absorbent filter can overlap successively along chromatography direction when using and be arranged on base plate (referring to Fig. 2).On reaction film, spaced detection zone and quality control region can be, but are not limited to, form, detection zone and the quality control region preferred interval 3mm to 8mm such as line, band, block.

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

Generally, conventional chromatographic test paper assembly (reaction film, base plate etc.) has obvious fluorescence background under 550nm wavelength, and the detection of fluorescence signal is produced very big interference by this.The present invention is by adopting the base plate of substantially not fluorescent nitrocellulose filter and low Poison character under the wavelength more than 550nm, thus overcoming the defect of conventional fluorescent reagent paper.Additionally, the fluorescent material X-rhodamine used by the present invention can produce stronger fluorescence signal, thus substantially increasing fluorescence signal-to-background ratio further, enabling distinguish signal and background well, and then improve detection sensitivity.

In the present invention, the material of sample pad and pad can adopt material commonly used in the art, for instance, sample pad and pad can be glass fibre.

The anti antibody being combined with the PGII monoclonal antibody specificity being marked with fluorescent microsphere of the present invention can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, wherein it is preferably sheep anti-mouse igg monoclonal antibody, compared with polyclonal antibody, monoclonal antibody specificity is higher.

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

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

It will be appreciated by persons skilled in the art that and can according to actual needs the order of above-mentioned steps be adjusted, for instance step 3) can before step 1), or in step 1) and 2) between.

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 the condition of each step of the method for the fluorescence immune chromatography test paper for detecting people's PGII albumen of the preparation present invention, so that the fluorescence immune chromatography test paper of the present invention can obtain for people's PGII albumen meets the result that Clinical detection requires, that is, detection range width, specificity is high, sensitivity is good.

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

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

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

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

nullC) fluorescent microsphere labelling the oneth PGII monoclonal antibody obtained by step b)，Wherein，The ratio of the fluorescent microsphere of activation described in 1 μ l to 3 μ l antibody (10mg/ml)/100 μ l，The fluorescent microsphere that step b) is obtained and PGII monoclonal antibody mixing，1.5～3 hours (preferably 2 hours) are stirred under room temperature (25 DEG C)，Add 1ml Block buffer (1 (w/v) %BSA-0.05M ethanolamine)，Continue stirring 1 hour，It is centrifuged 5 to 10 minutes under 10000rpm to 15000rpm，Repeated centrifugation 3 times，Precipitate is distributed in 500 μ l wash buffers at end (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution)，Ultrasound wave (240W) processes 1 to 2 minute，It is settled to 500 μ l with described whole wash buffer.

Preferably, in the method for the invention, described step 2) including: a PGII monoclonal antibody antibody diluent (1% (w/v) BSA-0.01MPBS(pH7.2) buffer of fluorescent microsphere will be marked with) be diluted, to be diluted to 0.5～2mg/ml, it is preferably 1mg/ml, then with micropipettor even application on pad, post-drying or vacuum lyophilization.The step 2 of method in the present invention) in, the concentration that is coated of the described PGII monoclonal antibody being marked with fluorescent microsphere is 0.5～2mg/ml, it is preferred to 1mg/ml.

Preferably, described step 3) includes: described 2nd PGII monoclonal antibody and anti antibody are drawn on nitrocellulose filter (solid phase carrier) with metal spraying machine using as detection zone and quality control region, what make detection zone and quality control region is spaced apart 3mm to 8mm, the concentration respectively 0.5～2mg/ml of described 2nd PGII monoclonal antibody and described anti antibody, it is preferred to 1mg/ml.

Preferably, result detection utilizes special fluorescence detector (to be purchased from Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) quality control region and detection zone are detected, the content of the PGII in the ratio of detection zone and quality control region fluorescence intensity and testing sample is directly proportional.Adopt the ratio of detection zone and quality control region fluorescence intensity rather than directly adopt the absolute fluorescence value of detection zone can be reduced as far as the impact of reaction condition, substrate etc., and avoiding 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 the 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.

The preparation of embodiment 1:PGII epitope peptide (1) and (2).

Preparation method chemical synthesis: utilize the many automatic peptide synthesizers of American AB I431A type, is respectively synthesized PGII epitope peptide (1) and (2) by solid phase method.The purity high performance liquid chromatography of epitope peptide is evaluated, and measures the concentration of peptide fragment.The epitope peptide (1) of the present invention and the molecular weight of (2) respectively 1299.66 and 1943.41, utilizes mass spectrum to be determined, and is measured by peptide sequence and identifies synthesized peptide sequence.

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

Above-mentioned peptide fragment adopts Solid phase synthesis.The main thought of Solid phase peptide synthesis is: be first connected with the same insoluble macromolecular compound (resin) of covalent bond form by the carboxyl synthesizing the carboxyl-terminus amino acid of peptide chain; then it is combined in aminoacid on solid phase carrier as moiety using this; through sloughing amino protecting group and with excessive activated carboxyl component reaction, spreading peptide chain.Such step can repeatedly go on repeatedly, finally reaches the length of the peptide chain of required synthesis.This building-up process is as follows.

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

1. raw materials used:

HMPresin (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, is placed in the reaction chamber of the many automatic peptide synthesizers of American AB I431A type by 0.1mmol, synthesizer is automatically coupled together in a different order by specific aminoacid, and Conjugate ratio reaches 99%.React as follows:

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

The aminoacid of Fmoc protection

(2) aminoacid is connected to resin

(3) slough amino acid whose Fmoc and protect base

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

(5) coupling

Peptide-the resin of new coupling

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

(7) peptide resin:

Use TFA(trifluoroacetic acid) cut peptide chain, with EDT(2.5 volume %), thioanisole (2.5 volume %) make scavenger, at room temperature reaction 3.0 hours, remove cutting reagent, extract with ether again, respectively obtain the crude product 104mg of PGII peptide fragment (1) and the crude product 148mg of PGII peptide fragment (2).

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

Adopt high performance liquid chromatography separation purification:

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) 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

Mobile 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 PGII epitope peptide (1) of the peptide piecewise analysis result display present invention and the purity of (2) are more than 95%.

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

1. utilize mass spectrum to measure the PGII epitope peptide (1) of purification gained and the molecular weight of (2) 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: REFLEXIII, Bruker company of Germany)；

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

(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 sample of the above-mentioned purified polypeptide of 1 μ L (1) and (2) respectively, each mixes with the saturated stromal supernatant mixing equal-volume of 1 μ L, takes 1 μ L point respectively on sample target, sends in ion source and detects.

As a result, the molecular weight that molecular weight is 1301.2, PGII epitope peptides (2) recording gained PGII epitope peptide (1) is 1943.5, consistent with theoretical molecular 1299.66,1943.41, it was demonstrated that synthesis polypeptide namely for the purpose of product.

2. the sequence identifying gained PGII epitope peptide (1) and (2) respectively is measured by peptide sequence.

(1) principle: the ultimate principle of polypeptid acid sequence analysis is Edman degraded, is a circulating chemical reaction process.Including three main chemical steps: (1) coupling: the N-end residue of isothiocyanic acid benzene fat and proteins and peptides reacts, form phenylamino formyl sulfide (PTC) derivant, i.e. PTC-peptide.(2) cyclisation cracking: PTC-peptide cyclisation cracks.(3) convert: thiazole purine ketone phenylamino (ATZ) is converted into the different sulfur urine amino acid of benzene (PTH-aminoacid).Staying the peptide decreasing an amino acid residue in the solution to repeat and carry out above-mentioned course of reaction, whole sequencing procedure is all automatically carried out by sequenator now.

(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

Undertaken by instrument description.

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

(1)K-K-F-K-S-I-R-E-T-Y；With

(2)Y-T-P-S-R-A-A-P-P-S-S-T-L-Q-L-P-E-K。

This result is consistent with target section of synthesized peptide.

Embodiment 2: be connected to prepare PGII antigen (1) and (2) with carrier protein by the PGII epitope peptide (1) of embodiment 1 gained and (2) respectively, utilize gained antigen (1) and (2) immune animal respectively, thus utilizing antigen (1) to prepare specific monoclonal antibody and polyclonal antibody, and antigen (2) is utilized to prepare specific monoclonal antibody and polyclonal antibody.

1. the preparation of antigen: with BDB(Bis-diazotizedbenzidinedichloride) method by PGII peptide fragment (1) and (2) respectively with carrier protein KLH(keyhole limpet hemocyanin) be connected and prepare into PGII antigen (1) and (2).

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

In the present embodiment, the formula of PBS is: the Na of 0.2mol/L₂HPO₄81ml adds the NaH of 0.2mol/L₂PO₄19ml 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 PGII antigen (1) of above-mentioned preparation is taken and after (2) (immunogen) be sufficiently mixed with isopyknic Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively, individually immunity Balb/c mice, 50 μ g antigens/only, subcutaneous multi-point injection.Serum titer is surveyed after 4 weeks, select the good mice booster immunization again of immunoreactivity: take after antigen is sufficiently mixed with isopyknic incomplete Freund's adjuvant, antigen dose 25 μ g/ is only, subcutaneous multi-point injection, the number of times of booster immunization is 6 times, continuously booster immunization twice before merging, 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 merges, and with HAT conditioned medium (purchased from sigma company) selection cultivation.CO is put into after fusion₂Incubator is cultivated after 9～11 days for 37 DEG C, the cell clone that appearance is bigger in the hole in.Within 11 days, start to screen with indirect ELISA.The hole that primary dcreening operation is positive utilizes limiting dilution assay carry out 4 time cloningizations and cultivates (even if a large amount of schizogamy of cell after screening), afterwards amplifying cells, frozen, preparation ascites.

2.2. Balb/c mice norphytane (purchased from sigma company) 0.5ml/ is only processed, one week pneumoretroperitoneum inoculation hybridoma 2 × 10⁶Individual/only, collect ascites after 10 days.

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

The titer utilizing monoclonal antibody (2) prepared by PGII antigen (2) also utilizes identical method to be measured, and its titer 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, the PGII antigen (1) of above-mentioned for 1-2mg preparation and (2) (immunogen) are mixed with isopyknic Freund's complete adjuvant respectively-fully emulsified after individually carry out multiple spot subcutaneous injection at rabbit back.Every 4 weeks booster immunizations once, after antigen is fully emulsified with incomplete Freund's adjuvant, 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. measuring antibody titer: measure the titer of the polyclonal antibody (1) utilizing PGII antigen (1) to prepare with indirect elisa method, result display antibody titer reaches more than 1:16000.

The titer utilizing polyclonal antibody (2) prepared by PGII antigen (2) also utilizes identical method to be measured, and its titer also reaches more than 1:16000.

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

4. separate antibody purification: after ammonium sulfate precipitation, then through ProteinG(purchased from sigma company) affinity purification.

5. lyophilizing after antibody subpackage, cryopreservation.

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

Detect with ELISA.Respectively with people's PGII albumen, PGI albumen (all purchased from osmanthus, Shanghai Kanggong department) for detecting antigen coated elisa plate, the specific reaction of prepared PGII monoclonal antibody (1) and (2) and this people's PGII albumen is detected respectively by ELISA, making negative control with normal BALB/c mouse serum, PBS liquid makes blank.

Result: PGII monoclonal antibody (1) and (2) are only reacted with PGII respectively for positive (P/N > 2.1), and react for negative with PGI, illustrate that the PGII monoclonal antibody (1) of the present invention and (2) are respectively provided with specificity.

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

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

Result shows: PGII polyclonal antibody (1) and (2) are reacted for positive (P/N > 2.1) respectively with PGII, and react for negative with PGI, illustrate that the PGII polyclonal antibody (1) of the present invention and (2) are respectively provided with specificity.

Embodiment 5: utilize PGII monoclonal antibody and PGII polyclonal antibody preparation PGII external diagnosis reagent case.

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

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

1. the preparation of various buffer and 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, sample/lavation buffer solution: pH7.2 10 × PBS-Tween20

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: 2MH₂SO₄

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.

2. the preparation of pre-coated plate:

PGII monoclonal antibody (1) is dissolved in the carbonate buffer solution of 0.05M of pH=9.6, make pre-coated liquid, 100 μ l are added by 0.1 μ g/ hole in the upper every hole of ELISA Plate (being purchased from Shenzhen Jin Canhua company), put 4 DEG C to place 18-24 hour, take 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 of preservations.

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

4. the composition of test kit:

Pre-coated plate: 48/96 hole

PGII calibration object (raw material is purchased from osmanthus, Shanghai Kanggong department): 6: 6 × 1.0ml(concentration respectively 0ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml

PGII binding antibody: 1 × 10ml(dilutes through 1:4000)

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:

Each hole of pre-coated plate is separately added into blood sample to be checked and standard substance 100 μ l/ hole, is diplopore, hatch 60 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add PGII binding antibody 100 μ l/ hole, hatch 30 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry.In each hole, add enzyme connection thing 100 μ l/ hole again, hatch 30 minutes for 37 DEG C, 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 minutes for 37 DEG C.Add stop buffer 50 μ l/ hole and terminate reaction, join detector (model RT-6000 is purchased from Lei Du company) with enzyme and detect absorbance with dual wavelength (450nm, 620nm).

6. result judges:

1. the test kit utilizing the present invention carries out serum PG II detection

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

Concentration ng/ml	0	2	5	10	25	50
							Mean OD value	0.058	0.158	0.304	0.550	1.022	1.789

With standard concentration and corresponding absorbance drawing standard curve, the R of standard curve²=0.992。

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

20 example gastric ulcer patients, 30 example atrophic gastritis patients, 34 example Patients with Gastric Cancer and 70 example healthy persons are carried out serum PG II detection in a manner described, and testing result is in Table 2.

Table 2: each group sample PGII concentration compares

Table 2, can be corresponding with Clinical detection result it is shown that the detected value difference between each group is inconspicuous.

2. the correlation analysis of the testing result of the ELISA kit of the testing result of test kit of the present invention and known detection PGII

Utilize DRG test kitHumanPepsinogenIIELISA, purchased from ALPCODiagnostics company) and the test kit of the present invention detect same 50 part blood sample (including patients w ith peptic ulcer disease blood sample, patients with atrophic gastritis blood sample and gastric cancer blood sample of patient) respectively.With the measured value of DRG test kit be abscissa, with the measured value of test kit of the present invention for vertical coordinate, set up linear equation calculate correlation coefficient.

The result of two kinds of test kits is carried out paired t-test, it is judged that whether there were significant differences for both quantitative results.

As it is shown in figure 1, the coefficient R of the measured value of test kit of the present invention and DRG test kit²=0.997, dependent equation is y=1.0159x-0.488, and wherein y represents the measured value of test kit of the present invention, and x represents the measured value of DRG test kit.As shown in Figure 1, the measured value of test kit of the present invention is good with the dependency of the measured value of DRG test kit.

The measured value paired t-test of two kinds of test kits is carried out statistical procedures, obtains | t |=1.626, v=n-1=49, look into t dividing value table and obtain P 0.05.This result shows that the testing result of two kinds of test kits is without significant difference, illustrates that the measured value of test kit of the present invention is good with the dependency of the measured value of DRG test kit, thus the test kit of the present invention is with a high credibility, and can be further used for PGI/PGII joint-detection.

Embodiment six: for detecting the preparation of the fluorescence immune chromatography test paper of people PGII 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, it is centrifuged 10 minutes with 12000rpm, removing supernatant, be distributed to by precipitate in distilled water or the first wash buffer (the MES aqueous solution of 0.1M) of 500 μ l, ultrasound wave (240W) processes 2 minutes, repeat above procedure three times, adding carbodiimide (purchased from Shanghai Jing Chun company) 50mg, stirring 15 minutes, thus activating described fluorescent microsphere.

1.2, with the fluorescent microsphere traget antibody activated:

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

2, it is coated pad

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

Two, the preparation of reaction film

PGII monoclonal antibody (2) and sheep anti-mouse igg monoclonal antibody (purchased from Beijing company of Zhong Shan Golden Bridge) according to embodiment 2 preparation are diluted to 1mg/ml respectively with the PBS of 50mMpH7.2, the detection line of metal spraying machine (purchased from Hangzhou Feng Hang company) and nature controlling line spacing parameter are set to 6mm, package amount is respectively set to 1.0 μ l/cm, drawing PGII monoclonal antibody (2) and sheep anti-mouse igg monoclonal antibody on nitrocellulose filter with metal spraying machine, room temperature dries standby.

Three, the assembling of reagent paper and cutting

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

Four, the preparation of PGII 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 on the sample pad of test strips and the position of reaction film and has well and observation window.Detection card loads in aluminium foil bag after assembling, and adds desiccant sealing and preserves, can preserve more than 1 year when drying at room temperature.

Five, the detection of sample

PGII standard substance (purchased from osmanthus, Shanghai Kanggong department) sample diluting liquid (1% (w/v) BSA-0.01MPBS(pH7.2) buffer) it is configured to the calibration object of following series concentration: 50ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2ng/ml, 1ng/ml, 0ng/ml, the 50 above calibration objects of μ l are added drop-wise on well respectively, with fluorescence detector (purchased from Anqun Bioengineering Co., Ltd., Shenzhen after 10 minutes, model AQ-3000) detection, fluorescence can be collected on detection line and nature controlling line position.With sample concentration for abscissa, the ratio of the fluorescence intensity at detection line and nature controlling line place is that vertical coordinate draws calibration curve, R²It is 0.994.Nature controlling line does corresponding correction for reagent paper Effective judgement and to inspection line signals, as band does not occur in nature controlling line, then illustrates that reagent paper lost efficacy.

Taking the blood sample to be checked of 50 μ l, be added drop-wise on well, detect with fluorescence detector after 10 minutes, if band occurs in detection line, illustrate containing PGII in sample, its concentration can obtain according to calibration curve.

Six, PGII 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, and through data fitting and statistical analysis, reagent paper linear detection range of the present invention is 0.1ng/ml-50ng/ml.

2) minimum detectability: PGII null value blood sample (without PGII composition) (purchased from Central Plains, Shenzhen company) is divided into 20 parts and detects, calculates mean concentration and 2 times of standard deviation sums, obtains reagent paper lowest detection of the present invention and be limited to 0.05ng/ml.

3) precision: detect the blood sample of PGII concentration respectively 40ng/ml, 15ng/ml, 2ng/ml, duplicate detection 10 times with the PGII fluorescence immunoassay reagent paper of the present invention respectively, carry out withinrun precision mensuration.The sample of above-mentioned 3 concentration is measured by every day, 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 PGII, sensitivity are good.

Claims

1., for a fluorescence immune chromatography test paper for people's PGII albumen in detection by quantitative determinand, this reagent paper detects described people's PGII albumen by double antibody sandwich method, wherein:

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

(1)Lys-Lys-Phe-Lys-Ser-Ile-Arg-Glu-Thr-Tyr；

(2)Tyr-Thr-Pro-Ser-Arg-Ala-Ala-Pro-Pro-Ser-Ser-Thr-Leu-Gln-Leu-Pro-Glu-Lys。

2. fluorescence immune chromatography test paper according to claim 1, a wherein said PGII monoclonal antibody is prepared from by a PGII antigen, and a PGII antigen is by making the one in described people's PGII epitope peptide (1) and (2) be prepared from carrier protein couplet；And

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

4. fluorescence immune chromatography test paper according to claim 1, the fluorescent material on wherein said fluorescent microsphere is Fluorescein isothiocyanate, RB 200, Tetramethylrhodamine isothiocyanate or X-rhodamine；The micro-sphere material of described fluorescent microsphere is the copolymer of polystyrene, polymethyl methacrylate or methyl methacrylate.

5. fluorescence immune chromatography test paper according to claim 4, the fluorescent material on wherein said fluorescent microsphere is X-rhodamine.

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

7. fluorescence immune chromatography test paper according to claim 6, the excitation wavelength of wherein said fluorescent microsphere is 390nm；Transmitting wavelength is 615nm.

8. fluorescence immune chromatography test paper according to claim 1, wherein said reagent paper has base plate, and it is provided with sample pad with the way of contact successively along chromatography direction when using on which floor plate, pad, reaction film, absorbent filter, described pad is coated with the described PGII monoclonal antibody being marked with fluorescent microsphere, described reaction film includes detection zone and quality control region, described detection zone is coated with described 2nd PGII monoclonal antibody, described quality control region is coated with the anti antibody being combined with the described PGII monoclonal antibody specificity being marked with fluorescent microsphere.

9. fluorescence immune chromatography test paper according to claim 8, wherein said reaction film is substantially not fluorescent nitrocellulose filter under the wavelength more than 550nm.

10. fluorescence immune chromatography test paper according to claim 8, wherein said base plate does not substantially have photoluminescent property.

11. fluorescence immune chromatography test paper according to claim 8, wherein said anti antibody is sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody.

12. fluorescence immune chromatography test paper according to claim 11, wherein said anti antibody is sheep anti-mouse igg monoclonal antibody.

13. the method preparing fluorescence immune chromatography test paper according to any one in claim 1 to 12, it comprises the following steps:

14. method according to claim 13, wherein said step 1) including:

A) carbodiimide activation fluorescent microsphere is used；

B) fluorescent microsphere of the activation that washing step a) obtains；

C) fluorescent microsphere labelling the oneth PGII monoclonal antibody that step b) obtains is used.

15. method according to claim 14, wherein said step 1) including:

A) aqueous dispersions of fluorescent microsphere or MES buffer dispersion liquid are mixed through ultrasonic Treatment and with carbodiimide, thus activating described fluorescent microsphere；

B) by step A) fluorescent microsphere N-hydroxy thiosuccinimide-citrate buffer solution washing of the activation that obtains, dispersion, and through ultrasonic Treatment；

C) by step B) fluorescent microsphere that obtains and PGII monoclonal antibody mixing, with BSA-ethanolamine buffer blind, centrifugal, disperse with BSA-Tween solution, through ultrasonic Treatment, thus obtaining being marked with a PGII monoclonal antibody of fluorescent microsphere.

16. method according to claim 13, wherein in described step 2) in, the concentration that is coated of the described PGII monoclonal antibody being marked with fluorescent microsphere is 0.5～2mg/ml.

17. method according to claim 13, wherein in described step 3) in, described detection zone and described quality control region interval 3mm to 8mm, described 2nd PGII monoclonal antibody and described anti antibody be coated concentration respectively 0.5～2mg/ml.