CN104422775B

CN104422775B - Fluorescence immune chromatography test paper and the method for making of joint-detection people PGI albumen and people PGII albumen

Info

Publication number: CN104422775B
Application number: CN201310370383.7A
Authority: CN
Inventors: 朱建安
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-08-22
Filing date: 2013-08-22
Publication date: 2016-05-18
Anticipated expiration: 2033-08-22
Also published as: CN104422775A

Abstract

The present invention relates to fluorescence immune chromatography test paper and the method for making of joint-detection people PGI albumen and people PGII albumen. Described test paper detects people PGI albumen and people PGII albumen by double antibody sandwich method, and the capture antibody that described double antibody sandwich method adopts is respectively a PGI monoclonal antibody of the one deriving from sequence table sequence 1 and sequence 2 and derives from a PGII monoclonal antibody of the one in sequence table sequence 3 and sequence 4 sequences; And the detection antibody that described double antibody sandwich method adopts is respectively the 2nd PGI monoclonal antibody of the another one deriving from sequence table sequence 1 and sequence 2 and derives from the 2nd PGII monoclonal antibody of the another one in sequence table sequence 3 and sequence 4. Fluorescence immune chromatography test paper of the present invention is easy and simple to handle, quick, detection range is wide, specificity is high, sensitivity is good.

Description

Fluorescence immune chromatography test paper and the method for making of joint-detection people PGI albumen and people PGII albumen

Technical field

The invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to human pepsinogen I(PGI) epitope peptide,The PGI specific antigen of preparing with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, described antibody are in systemPurposes, people PGI external diagnosis reagent case and human pepsinogen II(PGII on standby people PGI external diagnosis reagent case) anti-Former epitope peptide, the PGII specific antigen of preparing with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, instituteState purposes, the people PGII external diagnosis reagent case of antibody on preparation people PGII external diagnosis reagent case, the invention still further relates to useFluorescence immune chromatography test paper of people PGI albumen and people PGII albumen and preparation method thereof in the quantitative detection of associating determinand.

Background technology

Propepsin (PG) is pepsic inactive precursor in gastric juice. Human pepsinogen is by its electrophoretic mobilityCan be divided into seven kinds of isozymogens of PG1 to PG7 by fast to slow, and according to its biochemical property, immunogenicity, cell derived andIn tissue, distribution can be divided into PGI and two subgroups of PGII. PG1 to PG5 immunogenicity in seven kinds of isozymogens is approximate, is called stomachPepsinogen I(PGI), it is mainly secreted by chief cell and the mucus neck cell of gastric gland; PG6 to PG7 is called as propepsinII(PGII),, except the chief cell secretion of secreting acid gland by mucous membrane at the bottom of body of stomach and stomach, secrete mucus neck cell, the cardiac gland of acid glandAlso can produce PGII with the mucilage cell of pyloric gland and the Brunner gland of duodenum epimere of stomach hole. Stomach is almost PGUnique source, and can change in the secretory volume in secretion stage. Serum PG I and PGII have reflected gastric mucosa different partsSecreting function. PG major part after synthetic enters gastral cavity, under acidic gastric juice effect, activates into pepsin, only has a small amount of PG(approximately 1%) sees through stomach lining hair. Therefore, serum PG concentration can reflect its secretion level. PGI in normal human serum is PGII6 times, in the time of stomach lining generation pathological change, serum PG content also changes thereupon.

Serum PG content is relevant to helicobacter pylori (HP) infection, chronic gastritis, cancer of the stomach etc. In different disease of stomach,The value generation respective change of serum PG I, PGII and PGI/PGII, its raise, reduce or constant and inconsistent, as diagnosis orThe sensitivity for the treatment of results index and specificity are also different. Therefore, for different disease of stomach, serum PG I, PGII andThe value of PGI/PGII also has different criterions.

PGI is the pointer that detects oxyntic gland (fundus gland) cell function, and gastric acid secretion increases, and PGI raises; Hydrochloric acid in gastric juice dividesSecrete and reduce or the atrophy of gastric mucosa body of gland, PGI reduces. For example,, superficial gastritis and the helicobacter pylori of gastroxia(HP), in the gastritis infecting, the secretion of PGI can increase; And in chronic serious atrophic gastritis, in the time that chief cell reduces, PGI containsAmount declines. The PGI of high concentration is also as the subclinical indication of duodenal ulcer and complication danger, and can be used asObserve an index of HP infection (helicobacter pylori) eradication therapy curative effect.

Cancer of the stomach is one of common malignant tumour of China, and its case fatality rate occupies first of various malignant tumours, its early diagnosis, earlyThe unique channel that phase treatment becomes and improves life in patients, reduces case fatality rate. Chinese scholars becomes Serum Obtained From Advance Gastric Cancer PGHaving turned a large amount of research into finds: in the time getting a cancer of the stomach, serum PG I level reduces, and PGII level substantially keeps normal or raises, and entersAnd, measure the antidiastole that the level of serum PG I and the ratio of PGI/PGII contribute to cancer of the stomach, too low PGI and PGI/PGIIShould watch out for early carcinoma of stomach. Because the content of serum PG directly reflects the function of stomach lining, therefore Serum Obtained From Advance Gastric Cancer PGI level is brightAobvious decline, shows that patients with gastric cancer stomach lining secretion capacity declines. More than 80% cancer of the stomach is with atrophic gastritis, and atrophic stomachInflammation can cause stomach lining chief cell to be lost, thereby affects its secreting function; Serum Obtained From Advance Gastric Cancer PGI level obviously declines and cancer of the stomachThereby Stomach in Patients mucosal atrophy, intestines secretion reduce relevant. Therefore serum PG I and PGI/PGII obviously decline to monitoring early stage stomachCancer has important clinical meaning, can effectively be applied to high incidence area of gastric cancer crowd's preliminary examination. PG detects as NoninvasiveMethod, can reduce the misery that patients with gastric disease or cancer of the stomach people at highest risk check, and easy, economical, is significant.

PGI in detection serum and the optimal method of PGII level are immunoassays. Therefore, find suitable havingImmunogenic PGI epitope peptide, prepare specific PGI antigen and antibody and find the suitable immunogenicity that hasPGII epitope peptide, prepare specific PGII antigen and antibody become emphasis.

Determinand in fluorescence immune chromatography method survey blood is easy and simple to handle, quick, only needs just can complete for 10 minutes sampleDetect, and detection range is wide, and sensitivity is good, can be rapidly the assisted diagnosis state of an illness in time, monitoring prognosis. Chinese patent applicationNo.200910117820.8 discloses a kind of preparation method and quantitative detecting method of fluorescent micro-ball immune chromatography test paper strip; InState patent application No.200910047352.1 discloses a kind of fluorescence immune chromatography test paper and its preparation method and application. But,Also there is no at present the fluorescence immune chromatography test paper of joint-detection people PGI albumen and people PGII albumen.

Summary of the invention

For solving existing problem in above-mentioned prior art, the invention provides a kind of for joint-detection people PGI eggFluorescence immune chromatography test paper and the method for making of white and people PGII albumen.

Particularly, the invention provides:

For combining a fluorescence immune chromatography test paper for quantitative detection determinand people PGI albumen and people PGII albumen,This test paper detects respectively described people PGI albumen and described people PGII albumen by double antibody sandwich method, wherein:

(a), for described people PGI albumen, described double antibody sandwich method adopts a PGI Dan Ke who is marked with fluorescent microsphereGrand antibody is as capture antibody, and a described PGI monoclonal antibody derives from one in people PGI epitope peptide (1) and (2)Person; And

Described double antibody sandwich method adopts the 2nd PGI monoclonal antibody as detecting antibody, and described the 2nd PGI monoclonal is anti-Body derives from the another one in people PGI epitope peptide (1) and (2);

Described people PGI epitope peptide (1) and (2) are respectively:

(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；

(2) Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln; And

(b), for described people PGII albumen, the PGII that described double antibody sandwich method employing is marked with fluorescent microsphere is mono-Clonal antibody is as capture antibody, and a described PGII monoclonal antibody derives from people PGII epitope peptide (3) and (4)One; And

Described double antibody sandwich method adopts the 2nd PGII monoclonal antibody as detecting antibody, described the 2nd PGII monoclonalThe another one of antibody sources in people PGII epitope peptide (3) and (4);

Described people PGII epitope peptide (3) and (4) are respectively:

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

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

Preferably, a described PGI monoclonal antibody is prepared from by a PGI antigen, and a PGI antigen isBy one in described people PGI epitope peptide (1) and (2) and carrier protein couplet are prepared from; And

Described the 2nd PGI monoclonal antibody is prepared from by the 2nd PGI antigen, and the 2nd PGI antigen is by makingAnother one in described people PGI epitope peptide (1) and (2) and carrier protein couplet are prepared from.

Preferably, a described PGII monoclonal antibody is prepared from by a PGII antigen, and a PGII is anti-Former is by one and carrier protein couplet in described people PGII epitope peptide (3) and (4) are prepared from; And

Described the 2nd PGII monoclonal antibody is prepared from by the 2nd PGII antigen, and the 2nd PGII antigen is to pass throughAnother one in described people PGII epitope peptide (3) and (4) and carrier protein couplet are prepared from.

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

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

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

Preferably, described test paper has base plate, and on this base plate along use time chromatography direction successively with the side of contactFormula is provided with: sample pad, pad, reaction film, absorbent filter, described pad is coated with the described fluorescent microsphere that is marked withThe one PGI monoclonal antibody and a described PGII monoclonal antibody that is marked with fluorescent microsphere, described reaction film comprisesOne detection zone, the second detection zone and Quality Control district, described the first detection zone is coated with described the 2nd PGI monoclonal antibody and describedOne in two PGII monoclonal antibodies, described the second detection zone is coated with described the 2nd PGI monoclonal antibody and described secondAnother one in PGII monoclonal antibody, described Quality Control district is coated with can be mono-with a described PGI who is marked with fluorescent microsphereThe antiantibody of clonal antibody and the described equal specific binding of a PGII monoclonal antibody that is marked with fluorescent microsphere.

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

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

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

The present invention also provide a kind of prepare described for combining quantitative detection determinand people PGI albumen and people PGIIThe method of the fluorescence immune chromatography test paper of albumen, it comprises the following steps:

1) provide a PGI monoclonal antibody that is marked with fluorescent microsphere and a PGII Dan Ke who is marked with fluorescent microsphereGrand antibody;

2) provide pad, wherein a coated described PGI Dan Ke who is marked with fluorescent microsphere on described padGrand antibody and a described PGII monoclonal antibody that is marked with fluorescent microsphere;

3) provide reaction film, wherein on described reaction film along use time chromatography direction interval arrange the first detection zone,The second detection zone and Quality Control district, described the first detection zone is fixed with the 2nd PGI monoclonal antibody and the 2nd PGII monoclonal antibodyIn one, described the second detection zone is fixed with the another one in the 2nd PGI monoclonal antibody and the 2nd PGII monoclonal antibody,Described Quality Control district is fixed with antiantibody; With

4) on base plate the chromatography direction when using successively with the way of contact arrange sample pad, described pad, described inReaction film, absorbent filter, thus described fluorescence immune chromatography test paper made.

Preferably, described step 1) comprises:

A) activate fluorescent microsphere with carbodiimide, preferably, by the aqueous dispersions of fluorescent microsphere or MES buffer solution dispersion liquidProcess and mix with carbodiimide through ultrasonic wave, thereby activating described fluorescent microsphere;

The fluorescent microsphere of the activation that b) washing step a) obtains, preferably, the fluorescence of the activation that step a) is obtainedThe washing of N-hydroxy thiosuccinimide for microballoon-citrate buffer solution, dispersion, and through ultrasonic wave processing;

C) fluorescent microsphere difference mark the one PGI monoclonal antibody and the PGII monoclonal that obtain by step b) are anti-Body, preferably, the fluorescent microsphere that step b) is obtained respectively with a PGI monoclonal antibody and a PGII monoclonal antibodyMix, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution, through ultrasonic wave processing, thereby respectivelyTo the PGI monoclonal antibody and a PGII monoclonal antibody that is marked with fluorescent microsphere that are marked with fluorescent microsphere.

Preferably, in described step 2) in, a described PGI monoclonal antibody and described that is marked with fluorescent microsphereThe coated concentration that is marked with a PGII monoclonal antibody of fluorescent microsphere is respectively 0.5～2mg/ml.

Preferably, in described step 3), between described the first detection zone and described the second detection zone, be spaced apart 3mm,Between described the second detection zone and described Quality Control district, be spaced apart 5mm, described the 2nd PGI monoclonal antibody, described the 2nd PGIIThe coated concentration of monoclonal antibody and described antiantibody is respectively 0.5～2mg/ml.

The present invention compared with prior art has the following advantages and good effect:

1. people PGI epitope peptide of the present invention has good antigenicity, antigen (immunogene) immunity of preparing with itAnimal can produce monoclonal antibody and the polyclonal antibody of high degree of specificity.

2. the PGI monoclonal antibody of preparing with the present invention and polyclonal antibody can high special with blood sample inPGI combination.

3. people PGI external diagnosis reagent case of the present invention can be monitored the level of the PGI in serum effectively.

4. people PGII epitope peptide of the present invention has good antigenicity, antigen (immunogene) immunity of preparing with itAnimal can produce monoclonal antibody and the polyclonal antibody of high degree of specificity.

5. the PGII monoclonal antibody of preparing with the present invention and polyclonal antibody can high special with blood sample inPGII combination.

6. people PGII external diagnosis reagent case of the present invention can be monitored the level of the PGII in serum effectively.

7. the present invention combines fluorescence analysis with flash chromatography immunological technique, provides a kind of for combining quantitativelyThe fluorescence immune chromatography test paper of people PGI albumen and people PGII albumen in detection determinand, with the people in this detection paper determinandPGI albumen and people PGII albumen, easy and simple to handle, quick, only need just can complete sample detection in 10 minutes, and detection range is wide,Specificity is high, sensitivity good, can be rapidly the assisted diagnosis state of an illness in time, monitoring prognosis.

8. the present invention is in the mistake of the fluorescence immune chromatography test paper of the described joint-detection people PGI albumen of preparation and people PGII albumenCheng Zhong, gropes by a large amount of tests, has optimized the preparation condition of each side, makes to use fluorescence immune chromatography test paper of the present inventionWhile detection, fluorescence signal-to-background ratio improves greatly, thereby has improved detection sensitivity and credible result degree; In addition, the present invention alsoCarry out the content of PGI and PGII in response sample by the variation of the detection zone of test paper and the fluorescence intensity ratio in Quality Control district, this withTraditional chromatographic technique is only examined or check the absolute fluorescence intensity of detection zone and is compared, and has reduced to the full extent external condition and background etc.Impact, 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.

Fig. 2 illustrates the testing result of people PGI external diagnosis reagent case of the present invention and the ELISA of known detection PGIThe figure of the correlation of the testing result of kit, wherein abscissa is the value of PGI concentration in the sample recording with known agent box,Unit is ng/ml, and ordinate is the value of PGI concentration in the sample recording with kit of the present invention, and unit is ng/ml.

Fig. 3 illustrates the testing result of people PGII external diagnosis reagent case of the present invention and known detection PGIIThe figure of the correlation of the testing result of ELISA kit, wherein abscissa is that in the sample recording with known agent box, PGII is denseThe value of degree, unit is ng/ml, and ordinate is the value of PGII concentration in the sample recording with kit of the present invention, and unit is ng/ml。

Detailed description of the invention

The below description by detailed description of the invention the invention will be further described with reference to accompanying drawing, but this is not rightRestriction of the present invention, those skilled in the art, according to basic thought of the present invention, can make various amendments or improvement, but onlyOtherwise depart from basic thought of the present invention, all within the scope of the present invention.

One, people PGI epitope peptide

Described people PGI albumen is known in the art herein, and its amino acid sequence is known in the art, Ke YiIn the specialized databases such as NCBI, find.

The invention provides a kind of people PGI epitope peptide (1) and (2), its amino acid sequence is respectively as sequence table SEQShown in IDNo.1 and SEQIDNo.2, for:

(1) Y-K-V-P-L-I-R-K-K-S-L-R-R; With

(2)Y-K-N-F-T-V-F-D-R-A-N-N-Q。

The present inventor gropes through a large amount of theoretical research and experiments, and screening obtains two and has good antigenThe epitope peptide of property.

PGI epitope peptide (1) with one section of the 19th to 30, people PGI albumen (PG3) N end containing 12 amino acid residuesPeptide section is as antigenic determinant, and is added with Y at its N end. Adding Y at N end is in order to make this epitope peptide pass through BDB(Bis-diazotizedbenzidinedichloride) be linked to carrier protein (for example hemocyanin (KLH)) above, therebyCarry out Dispersal risk as antigen. PGI epitope peptide (1) has that hydrophily is high, antigenicity is strong and be easy to synthetic feature.

PGI epitope peptide (2) holds one section of the 372nd to 381 to contain 10 amino acid residues with people PGI albumen (PG3) CPeptide section as antigenic determinant, and be added with 3 amino acid residue: Y, K, N at its N end. The PGI antigen table forming like thisPosition peptide (2) also has that hydrophily is high, antigenicity is strong and be easy to synthetic feature.

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

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

The preparation method of PGI epitope peptide of the present invention can be by chemical synthesis: utilize American AB I431A type polypeptide fromMoving synthesizer, by solid phase method synthetic antigen epitope peptide. The molecular weight of epitope peptide of the present invention (1) and (2) is respectively1657.24 and 1616.93, available mass spectrum is determined, and is measured the epitope peptide order of qualification synthesized by peptide sequenceRow. The purity of peptide section is evaluated with thin-layer chromatography and high performance liquid chromatography, and measures the concentration of epitope peptide.

Two, PGI antigen

The present invention also provides a kind of PGI antigen, and it is by making in people PGI epitope peptide of the present invention (1) and (2)One and carrier protein couplet are prepared from. Particularly, the invention provides PGI antigen (1) and (2), described PGI antigen(1) by people PGI epitope peptide of the present invention (1) and carrier protein couplet are prepared from; Described PGI antigen (2) passes throughPeople PGI epitope peptide of the present invention (2) and carrier protein couplet are prepared from. PGI antigen of the present invention has immunogeneProperty and specificity, be a kind of immunogene, prepares specific PGI antibody thereby can be used to immune animal. In the present invention, availableThe example of carrier protein comprise KLH(keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc. Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better, and far away with immune animal affiliation,Being difficult for causing cross reaction with it as carrier protein, is therefore preferred.

Three, PGI monoclonal antibody, PGI polyclonal antibody and people PGI external diagnosis reagent case

The present invention also provides people PGI monoclonal antibody and people PGI polyclonal antibody, and described antibody can utilize respectively thisThe PGI antigen (1) of invention and (2) (immunogene) immune animal are prepared and obtain. Preparation method can adopt the ordinary skill in the art,Specifically can be referring to embodiment 2.

PGI monoclonal antibody of the present invention and polyclonal antibody can be for the preparation of people PGI external diagnosis reagent case, this examinationsAgent box can detect the PGI in blood preparation based on immunization method.

Therefore, the invention provides a kind of people PGI external diagnosis reagent case, it is anti-that it comprises people PGI monoclonal of the present inventionBody or polyclonal antibody.

The known immunization experiment method that can be used for clinical examination mainly comprises following several at present: ELISA method, chemiluminescenceMethod, fluorescent chromatographic method, colloid gold immune determination method etc.

And ELISA method comprises following several types: double antibody sandwich method detectable antigens, dual-antigen sandwich method detect antibody,Indirect method is surveyed antibody, competition law is surveyed antibody, competition law survey antigen, caught coated method survey antibody etc.

People PGI external diagnosis reagent case of the present invention preferably adopts ELISA double antibody sandwich method to detect PGI albumen. ShouldKit can comprise SA and/or necessary instrument and the reagent etc. of coated antibody, binding antibody, enzyme labeling.

Preferably, described people PGI external diagnosis reagent case adopts people PGI monoclonal antibody of the present invention as coated anti-Body. At this, term " coated antibody " refers to the antibody on the ELISA Plate that is coated in solid phase. In addition described people PGI in-vitro diagnosis examination,Agent box also preferably comprises people PGI polyclonal antibody using as binding antibody, wherein, and when described binding antibody derives from of the present inventionWhen one in people PGI epitope peptide (1) and (2), described coated antibody derives from described epitope peptide (1) and (2)Another one. At this, term " binding antibody " refers to the spy that can be combined with determined antigen and enzyme-labeled secondary antibody in kitHeterogenetic antibody. Described kit can also comprise the SA of enzyme labeling, and this SA can be goat anti-rabbit igg antibody,Described enzyme labeling can be horseradish peroxidase, alkaline phosphatase etc.

In kit of the present invention, can also required any reagent or the instrument of inclusion test, for example pre-coated plate, washWash liquid, developer, stop buffer etc.

Four, people PGII epitope peptide

Described people PGII albumen is known in the art herein, and its amino acid sequence is known in the art, canIn the specialized databases such as NCBI, find.

The invention provides a kind of people PGII epitope peptide (3) and (4), its amino acid sequence is respectively as sequence table SEQShown in IDNo.3 and SEQIDNo.4, for:

(3) K-K-F-K-S-I-R-E-T-Y; With

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

The present inventor gropes through a large amount of theoretical research and experiments, and final screening obtains two kinds and has goodAntigenic epitope peptide.

PGII epitope peptide (3) is with people PGII albumen (NCBI sequence number (NCBIReferenceSequence)):NP_001159896.1) one section of peptide section containing 9 amino acid residues of the 24th to 32, N end is as antigenic determinant, andIts C end is added with Y. Adding Y at C end is in order to make this epitope peptide pass through BDB(Bis-diazotizedbenzidineDichloride) be linked to carrier protein (for example hemocyanin (KLH)) above, thereby carry out Dispersal risk as antigen. PGII is anti-Former epitope peptide (3) has that hydrophily is high, antigenicity is strong and be easy to synthetic feature.

PGII epitope peptide (4) is with people PGII albumen (NCBI sequence number (NCBIReferenceSequence): NP_001159896.1) one section of peptide section containing 17 amino acid residues of the 229th to 245, C end is as antigenic determinant, andIts N is added with Y. Adding Y at N end is in order to make this epitope peptide pass through BDB(Bis-diazotizedbenzidineDichloride) be linked to carrier protein (for example hemocyanin (KLH)) above, thereby carry out Dispersal risk as antigen. PGII is anti-Former epitope peptide (4) also has that hydrophily is high, antigenicity is strong and be easy to synthetic feature.

At present, the present invention studies discovery, and 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 immunogene stimulating animal; 3.The antibody of preparing with epitope peptide can be combined with people PGII specifically.

The preparation method of PGII epitope peptide of the present invention can be by chemical synthesis: utilize American AB I431A type polypeptideAutomatic synthesizer, by solid phase method synthetic antigen epitope peptide. The molecular weight of epitope peptide of the present invention (3) and (4) is respectively1299.66 and 1943.41, available mass spectrum is determined, and is measured the epitope peptide order of qualification synthesized by peptide sequenceRow. The purity of peptide section is evaluated with thin-layer chromatography and high performance liquid chromatography, and measures the concentration of epitope peptide.

Five, PGII antigen

The present invention also provides a kind of PGII antigen, and it is by making in people PGII epitope peptide of the present invention (3) and (4)One and carrier protein couplet be prepared from. Particularly, the invention provides PGII antigen (3) and (4), described PGII is anti-Former (3) are by being prepared from people PGII epitope peptide of the present invention (3) and carrier protein couplet; Described PGII antigen (4)By people PGII epitope peptide of the present invention (4) and carrier protein couplet are prepared from. PGII antigen of the present invention hasImmunogenicity and specificity, be a kind of immunogene, prepares specific PGII antibody thereby can be used to immune animal. In the present inventionIn, the example of available carrier protein comprises KLH(keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc.Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better, and closes with immune animal relationshipBeing far away, being difficult for causing cross reaction with it as carrier protein, is therefore preferred.

Six, PGII monoclonal antibody, PGII polyclonal antibody and people PGII external diagnosis reagent case

The present invention also provides people PGII monoclonal antibody and people PGII polyclonal antibody, and described antibody can utilize respectivelyPGII antigen of the present invention (3) and (4) (immunogene) immune animal are prepared and obtain. Preparation method can adopt the conventional skill of this areaArt, specifically can be referring to embodiment 2.

PGII monoclonal antibody of the present invention and polyclonal antibody can, for the preparation of people PGII external diagnosis reagent case, be somebody's turn to doKit can detect the PGII in human blood sample based on immunization method.

Therefore, the invention provides a kind of people PGII external diagnosis reagent case, it comprises people PGII monoclonal of the present inventionAntibody or polyclonal antibody.

People PGII external diagnosis reagent case of the present invention preferably adopts ELISA double antibody sandwich method to detect PGII albumen.This kit can comprise SA and/or necessary instrument and the reagent etc. of coated antibody, binding antibody, enzyme labeling.

Preferably, described people PGII external diagnosis reagent case adopts people PGII monoclonal antibody of the present invention as coatedAntibody. At this, term " coated antibody " refers to the antibody on the ELISA Plate that is coated in solid phase. In addition external the examining of described people PGII,Disconnected kit also preferably comprises people PGII polyclonal antibody using as binding antibody, wherein, and when described binding antibody derives from thisInvention people PGII epitope peptide (3) and (4) in one time, described coated antibody derives from described epitope peptide (3)(4) another one in. At this, term " binding antibody " refers in kit and can tie with determined antigen and enzyme-labeled secondary antibodyThe specific antibody closing. Described kit can also comprise the SA of enzyme labeling, and this SA can be goat anti-rabbit iggAntibody, described enzyme labeling can be horseradish peroxidase, alkaline phosphatase etc.

Seven, for combining the fluorescence immune chromatography test paper of quantitative detection people's PGI albumen and people PGII albumen

It is a kind of for combining the fluorescence of quantitative detection determinand people PGI albumen and people PGII albumen that the present invention also providesImmune chromatography test paper, this test paper detects respectively described people PGI albumen and described people PGII albumen by double antibody sandwich method, itsIn:

Described double antibody sandwich method also adopts the 2nd PGI monoclonal antibody as detecting antibody, described the 2nd PGI monoclonalThe another one of antibody sources in people PGI epitope peptide (1) and (2);

Described people PGI epitope peptide (1) and (2) are respectively:

(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；

(2) Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln; And

(b), for described people PGII albumen, described double antibody sandwich method also adopts a PGII who is marked with fluorescent microsphereMonoclonal antibody is as capture antibody, and a described PGII monoclonal antibody derives from people PGII epitope peptide (3) and (4)One; And

Described people PGII epitope peptide (3) and (4) are respectively:

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

(4)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, " capture antibody " refers to can be first specialThe antibody of opposite sex identification determined antigen, it is coated on pad conventionally; " detection antibody " refers to that another kind can specificity knowledgeThe antibody of other determined antigen, it identifies respectively the different epitope on determined antigen molecule from capture antibody, and it is conventionally solidFix on the detection zone of reaction film.

In the present invention, a described PGI monoclonal antibody can be prepared from by a PGI antigen, and a PGI is anti-Former can be by one and carrier protein couplet in described people PGI epitope peptide (1) and (2) be prepared from; And instituteStating the 2nd PGI monoclonal antibody can be prepared from by the 2nd PGI antigen, and the 2nd PGI antigen can be by making described people PGIAnother one and carrier protein couplet in epitope peptide (1) and (2) are prepared from.

In the present invention, a described PGII monoclonal antibody can be prepared from by a PGII antigen, and this is first years oldPGII antigen can be by being prepared from one and carrier protein couplet in described people PGII epitope peptide (3) and (4);And described the 2nd PGII monoclonal antibody can be prepared from by the 2nd PGII antigen, and the 2nd PGII antigen can be by makingAnother one and carrier protein couplet in described people PGII epitope peptide (3) and (4) are prepared from.

In the present invention, the example of available carrier protein comprises KLH(keyhole limpet hemocyanin), bovine serum albumin(BSA)(BSA), ovalbumin OVA etc. Due to KLH(keyhole limpet hemocyanin) immunogenicity is strong, and binding site is many, and immune effect is better,And far away with immune animal affiliation, be difficult for causing cross reaction with it as carrier protein, be therefore preferred.

Preferably, in fluorescence immune chromatography test paper of the present invention, the particle diameter of fluorescent microsphere used is 320nm to 400nm,Be preferably 360nm, the fluorescent material on fluorescent microsphere can be fluorescein isothiocynate, RB 200, the different sulphur cyanogen of tetramethylAcid rhodamine or X-rhodamine etc., be wherein preferably X-rhodamine (can purchased from Shanghai Jing Chun company). The microballoon material of fluorescent microsphereMaterial can be the copolymerization being formed by polystyrene, polymethyl methacrylate or methyl methacrylate and other monomer copolymerizationThing, the example of other monomer is styrene etc. The excitation wavelength of fluorescent microsphere can be 350～600nm, is preferably 390nm; Send outEjected wave length can be 500～700nm, is preferably 615nm.

In the present invention, maximum excitation wavelength and the emission wavelength difference of fluorescent microsphere are larger, illustrate that fluorescent microsphere hasLarge Stokes (Stokes) displacement, like this, the ambient interferences of fluorescent test paper is lower, does immunochromatography label with this microballoonThere is stronger advantage.

In a specific embodiment, fluorescence immune chromatography test paper of the present invention has base plate, and at this base plateChromatography direction when upper edge is used is provided with the way of contact successively: sample pad, pad, reaction film, absorbent filter; Described sampleProduct pad is for loading in use testing sample; It is mono-that described pad is coated with a described PGI who is marked with fluorescent microsphereClonal antibody and a described PGII monoclonal antibody that is marked with fluorescent microsphere, it will be appreciated by those skilled in the art thatThat described two kinds of antibody can be coated in the same position of pad; Described reaction film comprises that the first detection zone, second detectsDistrict and Quality Control district, described the first detection zone is coated with described the 2nd PGI monoclonal antibody and described the 2nd PGII monoclonal antibodyIn one, described the second detection zone is coated with in described the 2nd PGI monoclonal antibody and described the 2nd PGII monoclonal antibodyAnother one, described Quality Control district be coated with can with a described PGI monoclonal antibody and described that is marked with fluorescent microsphereBe marked with the antiantibody of a PGII monoclonal antibody specificity combination of fluorescent microsphere.

Preferably, the sample pad of fluorescence immune chromatography test paper of the present invention, pad, reaction film, absorbent filter can edgeChromatography direction when use overlaps successively and is arranged on (referring to Fig. 1) on base plate. Spaced the first detection zone, on reaction filmTwo detection zones and Quality Control district can be, but be not limited to the forms such as line, band, piece. The first detection zone, the second detection zone and Quality Control districtCan be on described reaction film chromatography direction interval when using arrange, the interval between the first detection zone and the second detection zoneBe preferably 3mm, the interval between the second detection zone and Quality Control district is preferably 5mm. It will be appreciated by persons skilled in the art that canSo that to the first detection zone and the second detection zone, the position relationship on reaction film carries out multiple change, if detect time between the twoFluorescence signal do not interfere with each other. For example, in chromatography direction in use, the first detection zone and the second detection zone can divideNot with Quality Control interval every identical distance, meanwhile, in the orthogonal direction of chromatography direction in test paper plane during with use, firstDetection zone and the second detection zone be each other at a distance of certain distance, and while making to detect, fluorescence signal does not between the two interfere with each other.

In the present invention, reaction film is preferably under the wavelength that is greater than 550nm not fluorescent celluloid substantiallyFilm. In addition, base plate does not preferably have photoluminescent property substantially.

Conventionally, conventional chromatographic test paper assembly (reaction film, base plate etc.) has the obvious fluorescence back of the body under 550nm wavelengthScape, this detection to fluorescence signal produces very large interference. The present invention does not send out under the wavelength that is greater than 550nm substantially by adoptingThe nitrocellulose filter of fluorescence and the base plate of low photoluminescent property, thus overcome the defect of conventional fluorescent test paper. In addition the present invention,Fluorescent material X-rhodamine used can produce stronger fluorescence signal, thereby has further greatly improved fluorescence signal-to-background ratio, makesObtain and can distinguish well signal and background, and then improve detection sensitivity.

In the present invention, the material of sample pad and pad can adopt the normally used material in this area, for example, and sample padWith pad can be glass fibre.

Of the present invention can be marked with a PGI monoclonal antibody of fluorescent microsphere and be marked with fluorescent microsphereThe antiantibody of the one PGII monoclonal antibody specificity combination can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg Dan KeGrand antibody, is wherein preferably sheep anti-mouse igg monoclonal antibody, and compared with polyclonal antibody, monoclonal antibody specificity is higher.

In a specific embodiment, fluorescence immune chromatography test paper of the present invention in use, drips in sample padAdd sample liquid (as the blood sample that contains PGI and PGII), under capillarity, sample liquid moves to absorbent filter one end, at knotClose pad place, PGI and a described PGI monoclonal antibody that is marked with fluorescent microsphere form immune complex 1, PGII with described inBe marked with fluorescent microsphere the one PGII monoclonal antibody form immune complex 2, the immune complex forming is furtherMobile, on detection line separately, immune complex 1 is combined formation double-antibody sandwich with described the 2nd PGI monoclonal antibodyImmune complex 1, immune complex 2 is combined with described the 2nd PGII monoclonal antibody and is formed the immune complex of double-antibody sandwich2, and do not form a PGI monoclonal antibody that is marked with fluorescent microsphere of immune complex and be marked with first of fluorescent microspherePGII monoclonal antibody respectively the antiantibody on nature controlling line be combined. This process need 10 minutes to 15 minutes, afterwards, with glimmeringOptical detector detects, if band does not appear in nature controlling line place, illustrates that test paper lost efficacy; If there is band in nature controlling line place,And band does not appear in the first detection line or the second detection line place, in interpret sample, do not contain people PGI albumen or people PGII albumen; AsThere is band at nature controlling line place in fruit, and occurs band on the first detection line and/or the second detection line, in interpret sample, containsPeople PGI albumen and/or people PGII albumen.

In yet another aspect, the invention provides a kind of for the preparation of associating quantitatively detect in determinand people PGI albumen andThe method of the fluorescence immune chromatography test paper of people PGII albumen, it comprises the following steps:

It will be appreciated by persons skilled in the art that can in the same position of pad, be coated with described be marked with glimmeringThe one PGI monoclonal antibody of light microballoon and a described PGII monoclonal antibody that is marked with fluorescent microsphere. For example, can be byBeing marked with a PGI monoclonal antibody of fluorescent microsphere and a described PGII monoclonal antibody that is marked with fluorescent microsphere dividesBe not coated in the same position of pad, or can will be marked with a PGI monoclonal antibody and described of fluorescent microsphereA PGII monoclonal antibody mixing that is marked with fluorescent microsphere is coated on pad again.

In addition, it will be appreciated by persons skilled in the art that and can carry out the order of above-mentioned steps according to actual needsAdjust, for example step 3) can be before step 1), or in step 1) and 2) between.

Method of the present invention can also comprise the step 5) that the fluorescence immune chromatography test paper of making is cut into proper width.

The present inventor gropes by a large amount of tests, has optimized preparation of the present invention for joint-detection people PGIThe condition of each step of the method for the fluorescence immune chromatography test paper of albumen and people PGII albumen, thus make fluorescence of the present inventionImmune chromatography test paper can obtain the result that meets Clinical detection requirement for people PGI albumen and people PGII albumen,, detects model that isEnclose wide, specificity is high, sensitivity good.

Therefore, preferably, in the method for the invention, described step 1) comprises:

C) fluorescent microsphere difference mark the one PGI monoclonal antibody and the PGII monoclonal that obtain by step b) are anti-Body, preferably, the fluorescent microsphere that step b) is obtained respectively with a PGI monoclonal antibody and a PGII monoclonal antibodyMix, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution, through ultrasonic wave processing, thereby markedNote has a PGI monoclonal antibody of fluorescent microsphere.

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

A) activate fluorescent microsphere with carbodiimide, wherein, get the fluorescent microsphere aqueous dispersions of 1 (w/v) %, 10000rpm extremelyCentrifugal 5 to 10 minutes of 15000rpm low temperature (for example 10 DEG C), removes supernatant, sediment is distributed to distilled water or the previous cleaning of 500 μ lIn buffer solution (the MES aqueous solution of 0.1M), ultrasonic wave (240W) is processed 1 to 2 minute, repeats above process three times, adds carbon twoImines 10mg to 50mg, stirs 10～15 minutes, thereby activates described fluorescent microsphere;

The fluorescent microsphere of the activation that b) washing step a) obtains, wherein, the fluorescence of the activation that step a) is obtained is micro-Ball under 1000rpm to 15000rpm centrifugal 5 to 10 minutes, by sediment be distributed to 1ml coupling buffer (20～100mM'sN-hydroxy thiosuccinimide-citrate buffer solution) in, ultrasonic wave (240W) is processed 1 to 2 minute, repeats above process threeInferior;

C) fluorescent microsphere difference mark the one PGI monoclonal antibody and the PGII monoclonal that obtain by step b) are anti-Body, wherein, respectively according to the ratio of the fluorescent microsphere activating described in 1 μ l to 3 μ l antibody (10mg/ml)/100 μ l, by step b)The fluorescent microsphere obtaining mixes with a PGI monoclonal antibody and a PGII monoclonal antibody respectively, under room temperature (25 DEG C)Stir 1.5～3 hours (preferably 2 hours), add 1ml sealing buffer solution (1 (w/v) %BSA-0.05M monoethanolamine), continue to stir 1Hour, under 10000rpm to 15000rpm centrifugal 5 to 10 minutes, repeated centrifugation 3 times, was distributed to 500 μ l by sediment and washes eventuallyIn buffer solution (0.5 (w/v) %BSA-0.11 (v/v) % Tween solution), ultrasonic wave (240W) is processed 1 to 2 minute, uses described endWash buffer is settled to 500 μ l.

Preferably, in the method for the invention, described step 2) comprising: will be marked with a PGI of fluorescent microsphere respectivelyAntibody diluent for monoclonal antibody (1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) dilute, to be diluted to 0.5～2mg/ml, is preferably 1mg/ml, is then evenly sprayed on respectively on pad with micropipettor, and oven dry or vacuum are cold afterwardsFreeze-drying is dry. In the step 2 of method of the present invention) in, a described PGI monoclonal antibody that is marked with fluorescent microsphere and described inThe coated concentration of a PGII monoclonal antibody that is marked with fluorescent microsphere be respectively 0.5～2mg/ml, be preferably 1mg/ml.

Preferably, described step 3) comprises: with metal spraying machine by mono-to described the 2nd PGI monoclonal antibody, described the 2nd PGIIClonal antibody and described antiantibody draw nitrocellulose filter (solid phase carrier) upper using as the first detection zone, the second detection zone andQuality Control district, makes the 3mm that is spaced apart between the first detection zone and the second detection zone, and makes between the second detection zone and Quality Control districtBe spaced apart 5mm, the concentration of described the 2nd PGI monoclonal antibody, described the 2nd PGII monoclonal antibody and described antiantibody respectivelyBe 0.5～2mg/ml, be preferably 1mg/ml.

Preferably, result detect utilize special fluorescence detector (can be purchased from Anqun Bioengineering Co., Ltd., Shenzhen,Model AQ-3000) Quality Control district, the first detection zone and the second detection zone are detected, the first detection zone and Quality Control district fluorescence are strongThe content of PGI in ratio and the testing sample of degree be directly proportional (situation of coated the 2nd PGI monoclonal antibody of the first detection zone),The content of PGII in ratio and the testing sample of the second detection zone and Quality Control district fluorescence intensity is directly proportional, and (the second detection zone is coatedThe situation of the 2nd PGII monoclonal antibody). Adopt the ratio of detection zone and Quality Control district fluorescence intensity instead of directly adopt detectionThe absolute fluorescent value in district can reduce the impact of reaction condition, matrix etc. as much as possible, and avoids ambient interferences as far as possible.

Further explain and describe content of the present invention by the mode of example below, but these examples should not be understood toTo the restriction of protection scope of the present invention.

Embodiment

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

The preparation of embodiment 1:PGI epitope peptide (1) and (2) and PGII epitope peptide (3) and (4).

Preparation method's chemical synthesis: utilize American AB I431A type polypeptide automatic synthesizer, close respectively by solid phase methodBecome PGI epitope peptide (1) and (2) and PGII epitope peptide (3) and (4). The purity high efficiency liquid phase of epitope peptideChromatogram is evaluated, and measures the concentration of peptide section. The molecular weight of PGI epitope peptide of the present invention (1) and (2) is respectively1657.24,1616.93, the molecular weight of PGII epitope peptide (3) and (4) is respectively 1299.66 and 1943.41, utilizes mass spectrumDetermine, measure the peptide sequence of qualification synthesized by peptide sequence.

One, PGI epitope peptide (1) and (2) and PGII epitope peptide (3) and (4) is synthetic

Above-mentioned peptide section adopts solid phase method synthetic. The synthetic main thought of solid-phase peptide is: first will will synthesize the carboxyl of peptide chainThe carboxyl of end amino acid is connected with the same insoluble macromolecular compound of covalent bond form (resin), then ties with thisBe combined in amino acid on solid phase carrier as amino component, through deaminate protecting group anti-with excessive activated carboxyl componentShould, spreading peptide chain. Such step can repeatedly go on repeatedly, finally reaches the length of required synthetic peptide chain.This building-up process is as follows.

The concrete system separately of PGI epitope peptide of the present invention (1) and (2) and PGII epitope peptide (3) and (4)Standby step is as follows:

1. raw materials used:

HMPresin (P-hydroxymethyl phenoxy methyl poly vinyl, can purchased from sigma company)

Fmoc-AA (amino acid of 9-fluorenyl methoxy carbonyl acyl group protection, can purchased from Merck company)

NMP(nitrogen methyl pyrrolidone, can be purchased from sigma company)

DCM(carrene, can be purchased from Central Plains chemical company)

MeoH(methyl alcohol, can be purchased from Central Plains chemical company)

Piperidine(piperidines, can be purchased from sigma company)

DMAP(dimethyl aminopyridine, can be purchased from sigma company)

HOBT(hydroxybenzotriazole, can be purchased from sigma company)

DCC(dicyclohexylcarbodiimide, can be purchased from sigma company)

TFA(trifluoroacetic acid, can be purchased from sigma company)

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

Thio phenyl methyl ether, can be purchased from Guangzhou Wei Bai Chemical Co., Ltd.

Crystallization phenol, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group

Acetonitrile, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group

2. use instrument:

Polypeptide automatic synthesizer, model 431A, can be purchased from ABI company

Rotary Evaporators, model R-201, can be purchased from Shanghai Shen Shun company

High performance liquid chromatograph, Waters600, can be purchased from Waters company of the U.S.

Freeze drier, model VFD-2000, can be purchased from Beijing rich doctor Kanggong department

3. synthetic method and process:

Take HMP resin 100mg, replacing equivalent is 1.0meq, is placed in American AB I431A type polypeptide certainly by 0.1mmolIn the reaction chamber of moving synthesizer, by synthesizer automatically by specific amino acid by different being linked in sequence, coupling rate reaches99%. React as follows:

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

The amino acid of Fmoc protection

(2) connect amino acid to resin

(3) the Fmoc protecting group of deaminate acid

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

(5) coupling

Peptide-the resin of new coupling

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

(7) peptide resin:

Resin transfer, to beaker, is used to TFA(trifluoroacetic acid) cutting peptide chain, with EDT(2.5 volume %), thio phenyl methyl ether(2.5 volume %) makes scavenger, at room temperature reacts 3.0 hours, removes cutting reagent, then uses extracted with diethyl ether, obtains respectively PGICrude product 104mg and the PGII peptide section of the crude product 124.3mg of peptide section (1), the crude product 119.8mg of PGI peptide section (2), PGII peptide section (3)(4) crude product 148mg.

Two, the purifying of PGI epitope peptide (1) and (2) and PGII epitope peptide (3) and (4) crude product:

Adopt high performance liquid chromatography separation and purification:

Condition: chromatographic column: C810 × 100mm, can be purchased from Waters company of the U.S.

Chromatograph: Waters600, Waters company of the U.S.

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

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

Detect wavelength: 214nm

Flow velocity: 4ml/ minute

Gradient: 20-60%B, 30 minutes

HPLC(high performance liquid chromatography) analyze

Chromatographic column: C184.6 × 150mm, can be purchased from Waters company of the U.S.

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

B:0.1%TFA(trifluoroacetic acid) in acetonitrile

Detect wavelength: 214nm

Flow velocity: 1ml/ minute

Gradient: 0-60%B, 30 minutes

Peptide piecewise analysis result show PGI epitope peptide of the present invention (1) and (2) and PGII epitope peptide (3) and(4) purity is more than 95%.

Three, the qualification of PGI epitope peptide (1) and (2) and PGII epitope peptide (3) and (4)

1. utilize mass spectrum to measure respectively PGI epitope peptide (1) and (2) and the PGII epitope peptide of purifying gainedAnd the molecular weight of (4) (3).

(1) reagent raw material

TFA(trifluoroacetic acid, can be purchased from sigma company)

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

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

(2) instrument

Ground substance assistant laser desorption ionization time of-flight mass spectrometer MALDI-TOF-MS(model: REFLEXIII, GermanyBruker company);

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

(4) instrument testing conditions: reflection detection mode; Flight pipe range 3m; Nitrogen laser: wavelength 337nm, accelerating potential20KV; Reflected voltage 23KV.

(5) operating procedure: get respectively the above-mentioned purified polypeptide of 1 μ L (1) and (2) and PGII epitope peptide (3) and (4)Sample, mixes with the saturated stromal supernatant mixing equal-volume of 1 μ L separately, gets respectively 1 μ L point on sample target, sends into ion gunIn detect.

As a result, the molecular weight that records gained PGI epitope peptide (1) is the molecule of 1658.5, PGI epitope peptide (2)Amount is 1618.4, consistent with theoretical molecular 1657.24,1616.93, and records the molecular weight of PGII epitope peptide (3)The molecular weight that is 1301.2, PGII epitope peptide (4) is 1943.5, consistent with theoretical molecular 1299.66,1943.41, cardBright synthetic polypeptide is object product.

2. measure and identify respectively gained PGI epitope peptide (1) and (2) and PGII epitope peptide by peptide sequenceAnd the sequence of (4) (3).

(1) principle: the general principle of polypeptid acid sequence analysis is Edman degraded, is that a circulating chemistry is anti-Answer process. Comprise three main chemical steps: (1) coupling: the N-end residue of isothiocyanic acid benzene fat and proteins and peptides is anti-Should, form phenylamino formyl sulfide (PTC) derivative, i.e. PTC-peptide. (2) cyclisation cracking: PTC-peptide cyclisation cracking. (3) transform: thiopheneAzoles purine ketone phenylamino (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH-amino acid). The amino acid of having stayed minimizing in solutionThe peptide of residue repeats above-mentioned course of reaction again, and whole order-checking process is all automatically to carry out by sequenator now.

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

(3) reagent raw material

Phenyl isothiocyanate PITC, can be purchased from sigma company

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

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

TFA(trifluoroacetic acid, can be purchased from sigma company)

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

Chlorobutane, can be purchased from sigma company

Acetonitrile, can be purchased from Chemical Reagent Co., Ltd., Sinopharm Group

(4) measure

Undertaken by instrument description.

Result: through qualification, the sequence of gained PGI epitope peptide (1) and (2) is respectively:

(1) Y-K-V-P-L-I-R-K-K-S-L-R-R; With

(2)Y-K-N-F-T-V-F-D-R-A-N-N-Q。

The sequence of gained PGII epitope peptide (3) and (4) is respectively:

(3) K-K-F-K-S-I-R-E-T-Y; With

(4)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: by the PGI epitope peptide (1) of embodiment 1 gained and (2) and PGII epitope peptide (3) and(4) be connected to prepare respectively PGI antigen (1), PGI antigen (2), PGII antigen (3) and PGII antigen (4), profit with carrier proteinWith gained antigen (1), (2), (3) and (4) immune animal respectively, thereby utilize separately respectively antigen (1), (2), (3) and (4) systemStandby specific monoclonal antibody and polyclonal antibody.

1. the preparation of antigen: with BDB(Bis-diazotizedbenzidinedichloride) method by PGI peptide section (1),PGI peptide section (2), PGII peptide section (3) and PGII peptide section (4) respectively with carrier protein KLH(keyhole limpet hemocyanin) be connected and be prepared intoPGI antigen (1), PGI antigen (2), PGII antigen (3) and PGII antigen (4).

Get PGI peptide section (1) or PGI peptide section (2) or PGII peptide section (3) or PGII peptide section (4) 10.0mg, use 1ml0.1MPBS buffer solution (pH7.4) dissolves; KLH10mg, with 0.2M borate buffer solution (pH9.0) 20ml dissolving; Then both are mixedClose, be cooled to 0 DEG C, get BDBCl₂110 μ L, react 1.5h under room temperature, packing after dialysed overnight ,-20 DEG C of preservations.

In the present embodiment, the formula of PBS buffer solution is: the Na of 0.2mol/L₂HPO₄81ml adds 0.2mol/L'sNaH₂PO₄19ml mixes.

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

2. immune animal is prepared monoclonal antibody:

2.1. get PGI antigen (1), PGI antigen (2), PGII antigen (3) and PGII antigen (the 4) (immunity of above-mentioned preparationFormer) fully mix with isopyknic Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively after, respectively separately immunityBalb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection. Survey serum titer after 4 weeks, select mouse that immunoreactivity is good againBooster immunization: after getting antigen and fully mixing with isopyknic incomplete Freund's adjuvant, antigen dose 25 μ g/ only, subcutaneous multiple spot notePenetrate, the number of times of booster immunization is 6 times, continuous booster immunization twice before merging, and extracting spleen cell and Sp2/0 myeloma cell press afterwardsConventional method 50%PEG(MW4000) (purchased from Central Plains chemical company) mediation merge, and with HAT conditioned medium (purchased fromSigma company) select to cultivate. After fusion, put into CO₂In incubator, cultivate after 9～11 days for 37 DEG C, in hole, occur larger cellClone. Within 11 days, start to screen with indirect ELISA. Utilize limiting dilution assay to carry out 4 time cloningization trainings to the hole of the primary dcreening operation positiveSupport (even a large amount of schizogamies of cell after screening), afterwards amplifying cells, frozen, preparation ascites.

2.2. by Balb/c for mouse norphytane (purchased from sigma company) 0.5ml/ only process, the inoculation of one week pneumoretroperitoneum is assortedHand over oncocyte 2 × 10⁶Individual/only, after 10 days, to collect ascites.

2.3. measure antibody titer: measure the monoclonal antibody (1) of utilizing PGI antigen (1) to prepare with indirect ELISA methodTire, more than result shows tiring and reaching 1:32000 of monoclonal antibody.

Utilize monoclonal antibody (2) prepared by PGI antigen (2), the monoclonal antibody (3) of utilizing PGII antigen (3) to prepareAnd utilizing tiring of monoclonal antibody (4) prepared by PGII antigen (4) also to utilize identical method to measure, it is tired alsoMore than all reaching 1:32000.

3. immune animal is prepared polyclonal antibody:

3.1. select NZw that three monthly ages, body weight are about about 2kg as immune animal. In fundamental immunity,By the PGI antigen (1) of the above-mentioned preparation of 1-2mg, PGI antigen (2), PGII antigen (3) and PGII antigen (4) (immunogene) respectively withIsopyknic Freund's complete adjuvant mixes-fully emulsifiedly carry out multiple spot hypodermic injection at rabbit back separately respectively afterwards. Every 4 weeksBooster immunization once, after antigen and incomplete Freund's adjuvant are fully emulsified, with 100 μ g/ only in back multiple spot hypodermic injection. LastArteria carotis bloodletting in the 10th day after booster immunization, separation of serum.

3.2. measure antibody titer: measure and utilize polyclonal antibody (1) prepared by PGI antigen (1) with indirect elisa methodTire, more than result shows that antibody titer reaches 1:16000.

Utilize polyclonal antibody (2) prepared by PGI antigen (2), the polyclonal antibody (3) that utilizes PGII antigen (3) to prepareAnd utilizing tiring of polyclonal antibody (4) prepared by PGII antigen (4) also to utilize identical method to measure, it is tired alsoMore than all reaching 1:16000.

3.3. get blood and separation of serum: arteria carotis intubate is got blood, separation of serum.

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

5. freeze-drying after antibody packing, low temperature is preserved.

Embodiment 3: the specificity mirror of people PGI monoclonal antibody (1) and (2) and people PGII monoclonal antibody (3) and (4)Fixed

Detect with ELISA. Anti-as detecting taking people PGI albumen and PGII albumen (all purchased from osmanthus, Shanghai Kanggong department) respectivelyPrimordial covering elisa plate, detects respectively prepared PGI monoclonal antibody (1) and the specific reaction of (2) by ELISA, andPrepared PGII monoclonal antibody (3) and the specific reaction of (4), make negative control, PBS with normal BALB/c mouse serumLiquid is made blank.

Result: positive (P/N > 2.1) only reacted respectively in PGI monoclonal antibody (1) and (2) with PGI, and reacts with PGIINegative, illustrate that PGI monoclonal antibody of the present invention (1) and (2) have respectively specificity.

That only react with PGII respectively PGII monoclonal antibody (3) and (4) is positive (P/N > 2.1), be the moon and react with PGIProperty, illustrate that PGII monoclonal antibody of the present invention (3) and (4) have respectively specificity.

Embodiment 4: the specificity mirror of people PGI polyclonal antibody (1) and (2) and people PGII polyclonal antibody (3) and (4)Fixed

Utilize the method identical with above-mentioned qualification monoclonal antibody specificity to identify.

Result shows: positive (P/N > 2.1) reacted respectively in PGI polyclonal antibody (1) and (2) with PGI, and anti-with PGIIShould be negative, illustrate that PGI polyclonal antibody of the present invention (1) and (2) have respectively specificity.

That react with PGII respectively PGII polyclonal antibody (3) and (4) is positive (P/N > 2.1), and react with PGI for cloudyProperty, illustrate that PGII polyclonal antibody of the present invention (3) and (4) have respectively specificity.

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

In the present embodiment, using the monoclonal antibody (1) of utilizing PGI epitope peptide (1) to prepare in embodiment 2 as thisCoated antibody in kit; Using the polyclonal antibody (2) that utilizes PGI epitope peptide (2) to prepare in embodiment 2 as combinationAntibody.

The preparation of PGI external diagnosis reagent case and operation are as follows:

1. the preparation of various buffer solutions and reagent:

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

Na₂CO₃: 16.0 grams

NaHCO₃: 29.0 grams

Distill water-soluble to 1000ml

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

Na₂HPO₄·12H₂O:58 gram

KH₂PO₄: 4 grams

NaCl:100 gram

KCl:4 gram

Distill water-soluble to 1000ml

Add Tween20:20ml

C, enzyme labeling thing dilution:

10×PBS-Tween20：10ml

FCS(calf serum): 20ml

Distill water-soluble to 1000ml

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

Biological preservative (can 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

TMB2HCl:2 gram

Distill water-soluble to 1000ml

F, stop buffer: 2MH₂SO₄

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

Distilled water: 177.3ml

Timing slowly splashes into the concentrated sulfuric acid in distilled water, and limit edged shakes up.

2. the preparation of pre-coated plate:

PGI monoclonal antibody (1) is dissolved in the carbonate buffer solution of 0.05M of pH=9.6, makes pre-coated liquid, at enzymeThe upper every hole of target (can purchased from Shenzhen Jin Canhua company) adds 100 μ l by 0.1 μ g/ hole, puts 4 DEG C and places 18-24 hour, take out,Get rid of coating buffer, washing packs into and in aluminide-coating bag, vacuumizes sealing, and is placed in 4 DEG C of guarantors after BSA sealing 16 hours, dried overnightDeposit.

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

4. the composition of kit:

Pre-coated plate: 48/96 hole

PGI calibration object (raw material is purchased from osmanthus, Shanghai Kanggong department): 6: 6 × 1.0ml(concentration is respectively 0ng/ml, 20ng/ml、40ng/ml、80ng/ml、160ng/ml、320ng/ml）

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

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

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

Developer A:1 × 6.0ml

Developer B:1 × 6.0ml

Stop buffer: 1 × 6.0ml

5. the operating procedure of kit:

In each hole of pre-coated plate, add respectively blood sample to be checked and standard items 100 μ l/ holes, be diplopore, incubate for 37 DEG CEducate 60 minutes, with 1 × lavation buffer solution washing 5 times, pat dry. In each hole, add PGI binding antibody 100 μ l/ holes, hatch for 37 DEG C30 minutes, with 1 × lavation buffer solution washing 5 times, pat dry. In each hole, add again enzyme connection thing 100 μ l/ holes, hatch 30 points for 37 DEG CClock, with 1 × lavation buffer solution washing 5 times, pats dry. Add developer A, B liquid, the each 50 μ l in every hole, mix, and hatch 15 points for 37 DEG CClock. Add stop buffer 50 μ l/ hole cessation reactions, join detector (model RT-6000, can purchased from Lei Du company) dual wavelength with enzyme(450nm, 620nm) detects absorbance.

6. result is judged:

1. utilize kit of the present invention to carry out serum PG I detection

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

Concentration ng/ml	0	20	40	80	160	320
							Average OD value	0.068	0.143	0.320	0.585	1.023	1.867

With standard items concentration and corresponding absorbance drawing standard curve, the R of calibration curve²=0.997。

Calculate the PGI concentration results in the sample detecting according to calibration curve.

Press upper to 28 routine superficial gastritis patients, 30 routine atrophic gastritis patients, 34 routine Patients with Gastric Cancer and 70 routine healthy personsThe mode of stating is carried out serum PG I detection, and testing result is in table 2.

Table 2: four groups of sample PGI concentration ratios

Table 2 result shows, the difference between each group has statistical significance (P < 0.01), can be corresponding with Clinical detection result.

2. the testing result of the testing result of kit of the present invention and the ELISA kit of known detection PGI is relevantProperty is analyzed

Utilize DRG kit (HumanPepsinogenIELISA, purchased from ALPCODiagnostics public affairsDepartment) and kit of the present invention detect respectively 50 parts of same blood samples (comprising superficial gastritis blood sample of patient, atrophic stomachScorching blood sample of patient and patients with gastric cancer blood sample). Taking the measured value of DRG kit as abscissa, with the measured value of kit of the present inventionFor ordinate, set up linear equation and calculate coefficient correlation.

The result of two kinds of kits is carried out to paired t-test, and whether there were significant differences to judge both quantitative results.

As shown in Figure 2, the coefficient R of the measured value of kit of the present invention and DRG kit²=0.997, dependent equationFor y=0.9968x+0.0144, wherein y represents the measured value of kit of the present invention, and x represents the measured value of DRG kit. By Fig. 2Known, the correlation of the measured value of the measured value of kit of the present invention and DRG kit is good.

The measured value of two kinds of kits is carried out to statistical procedures with paired t-test, obtains | t|=0.996, 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 kits is without significant difference, and kit of the present invention is describedThe correlation of the measured value of measured value and DRG kit is good, thereby kit of the present invention is with a high credibility, and can be furtherFor PGI/PGII joint-detection.

Embodiment 6: utilize PGII monoclonal antibody and PGII polyclonal antibody to prepare PGII external diagnosis reagent case.

In the present embodiment, using the monoclonal antibody (3) of utilizing PGII epitope peptide (3) to prepare in embodiment 2 asCoated antibody in this kit; Using the polyclonal antibody (4) that utilizes PGII epitope peptide (4) to prepare in embodiment 2 asBinding antibody.

The preparation of PGII external diagnosis reagent case and operation are as follows:

1. the preparation of various buffer solutions and reagent:

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

Na₂CO₃: 16.0 grams

NaHCO₃: 29.0 grams

Distill water-soluble to 1000ml

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

Na₂HPO₄·12H₂O:58 gram

KH₂PO₄: 4 grams

NaCl:100 gram

KCl:4 gram

Distill water-soluble to 1000ml

Add Tween20:20ml

C, enzyme labeling thing dilution:

10×PBS-Tween20：10ml

FCS(calf serum): 20ml

Distill water-soluble to 1000ml

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

Biological preservative (can 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

TMB2HCl:2 gram

Distill water-soluble to 1000ml

F, stop buffer: 2MH₂SO₄

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

Distilled water: 177.3ml

2. the preparation of pre-coated plate:

PGII monoclonal antibody (3) is dissolved in the carbonate buffer solution of 0.05M of pH=9.6, makes pre-coated liquid,The upper every hole of ELISA Plate (can purchased from Shenzhen Jin Canhua company) adds 100 μ l by 0.1 μ g/ hole, puts 4 DEG C and places 18-24 hour, getsGo out, get rid of coating buffer, washing packs into and in aluminide-coating bag, vacuumizes sealing, and is placed in 4 DEG C after BSA sealing 16 hours, dried overnightPreserve.

3. (goat anti-rabbit igg of horseradish peroxidase-labeled is anti-for binding antibody (PGII polyclonal antibody (4)) and enzyme connection thingBody (purchased from Beijing company of Zhong Shan Golden Bridge)) dilution ratio by square formation titration experiments determine.

4. the composition of kit:

Pre-coated plate: 48/96 hole

PGII calibration object (raw material is purchased from osmanthus, Shanghai Kanggong department): 6: 6 × 1.0ml(concentration is 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 kit:

In each hole of pre-coated plate, add respectively blood sample to be checked and standard items 100 μ l/ holes, be diplopore, incubate for 37 DEG CEducate 60 minutes, with 1 × lavation buffer solution washing 5 times, pat dry. In each hole, add PGII binding antibody 100 μ l/ holes, incubate for 37 DEG CEducate 30 minutes, with 1 × lavation buffer solution washing 5 times, pat dry. In each hole, add again enzyme connection thing 100 μ l/ holes, hatch 30 for 37 DEG CMinute, with 1 × lavation buffer solution washing 5 times, pat dry. Add developer A, B liquid, the each 50 μ l in every hole, mix, and hatch 15 points for 37 DEG CClock. Add stop buffer 50 μ l/ hole cessation reactions, join detector (model RT-6000, can purchased from Lei Du company) dual wavelength with enzyme(450nm, 620nm) detects absorbance.

6. result is judged:

1. utilize kit of the present invention to carry out serum PG II detection

Table 3: standard items concentration and corresponding mean light absorbency (OD) value

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

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

Calculate the PGII concentration results in the sample detecting according to calibration curve.

20 routine gastric ulcer patients, 30 routine atrophic gastritis patients, 34 routine Patients with Gastric Cancer and 70 routine healthy persons are pressed to above-mentioned sideFormula is carried out serum PG II detection, and testing result is in table 4.

Table 4: respectively organize sample PGII concentration ratio

Table 4 result shows, the detected value difference between each group is not obvious, can be corresponding with Clinical detection result.

2. the testing result of the testing result of kit of the present invention and the ELISA kit of known detection PGII is relevantProperty is analyzed

Utilize DRG kit (HumanPepsinogenIIELISA, purchased from ALPCODiagnosticsCompany) and kit of the present invention detect respectively 50 parts of same blood samples (comprising patients w ith peptic ulcer disease blood sample, atrophic gastritisBlood sample of patient and patients with gastric cancer blood sample). Taking the measured value of DRG kit as abscissa, taking the measured value of kit of the present invention asOrdinate, sets up linear equation and calculates coefficient correlation.

As shown in Figure 3, the coefficient R of the measured value of kit of the present invention and DRG kit²=0.997, dependent equationFor y=1.0159x-0.488, wherein y represents the measured value of kit of the present invention, and x represents the measured value of DRG kit. By Fig. 3Known, the correlation of the measured value of the measured value of kit of the present invention and DRG kit is good.

The measured value of two kinds of kits is carried out to statistical procedures with paired t-test, 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 kits is without significant difference, and kit of the present invention is describedThe correlation of the measured value of measured value and DRG kit is good, thereby kit of the present invention is with a high credibility, and can be furtherFor PGI/PGII joint-detection.

Embodiment 7: the preparation of the fluorescence immune chromatography test paper of joint-detection people PGI albumen and people PGII albumen.

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

1, be marked with the preparation of the monoclonal antibody of fluorescent microsphere

1.1, the activation of fluorescent microsphere:

Get fluorescent microsphere (purchased from the Guangzhou Growth hormone secretagogue company) aqueous dispersions of 500 μ l, content 1 (w/v) %, at 10 DEG C, withCentrifugal 10 minutes of 12000rpm, removes supernatant, sediment is distributed to distilled water or first wash buffer (the MES water of 0.1M of 500 μ lSolution) in, ultrasonic wave (240W) is processed 2 minutes, repeats above process three times, adds carbodiimide (purchased from Shanghai Jing Chun company)50mg, stirs 15 minutes, thereby activates described fluorescent microsphere.

1.2, with the fluorescent microsphere labelled antibody having activated:

By the fluorescent microsphere having activated under 12000rpm centrifugal 10 minutes, remove supernatant, sediment is distributed to 1ml couplingIn buffer solution (citrate buffer solution of the N-hydroxy thiosuccinimide of 50mM), ultrasonic wave (240W) is processed 2 minutes, repeatsAbove process three times, obtains the buffer solution 1ml that is dispersed with fluorescent microsphere. Activate according to 3 μ l antibody (10mg/ml)/100 μ lThe ratio of fluorescent microsphere, adds the PGI monoclonal antibody (1) of preparing by embodiment 2 wherein, stirs at normal temperatures 2 hours, addsEnter 1ml sealing buffer solution (1 (w/v) %BSA-0.05M monoethanolamine), continue to stir 1 hour, afterwards, under 12000rpm centrifugal 10Minute, repeated centrifugation 3 times, is distributed to whole wash buffer (0.5 (w/v) %BSA-0.11 (v/v) the % tween water of 500 μ l by sedimentSolution) in, ultrasonic wave (240W) is processed 2 minutes, is settled to 500 μ l with above-mentioned whole wash buffer.

The PGII monoclonal antibody that the fluorescent microsphere mark activating according to identical method utilization is prepared by embodiment 2(3)。

2, coated pad

By the PGI monoclonal antibody (1) that is marked with fluorescent microsphere of above-mentioned preparation with to be marked with the PGII of fluorescent microsphere mono-Clonal antibody (3) is used respectively antibody diluent (1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) be diluted to 1mg/ml, obtainObtain working solution, then use micropipettor (purchased from labsystems company) to be evenly sprayed on respectively pad by the amount of 4 μ l/cmUpper, use afterwards 37 DEG C of oven for drying, under 45% humidity, save backup.

Two, the preparation of reaction film

The PGI monoclonal antibody (2) of preparing according to embodiment 2 and PGII monoclonal antibody (4) and sheep anti-mouse igg listClonal antibody (purchased from Beijing company of Zhong Shan Golden Bridge) is diluted to respectively 1mg/ml with the PBS buffer solution of 50mMpH7.2, by metal sprayingThe first detection line and the second detection line spacing parameter of machine (purchased from Hangzhou Feng Hang company) are set to 3mm, and by the second detectionLine and nature controlling line spacing parameter are set to 5mm, and package amount is set to respectively to 1.0 μ l/cm, use metal spraying machine at nitrocellulose filterUpper above PGI monoclonal antibody (2), PGII monoclonal antibody (4) and the sheep anti-mouse igg monoclonal antibody of drawing, normal temperature dries for subsequent use.

Three, the assembling of test paper and cutting

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

Four, the preparation of PGI fluorescence immunoassay test card:

The test paper of above-mentioned well cutting is fixed on plastic bottom card, test paper surface compresses with face card, and face is stuck in test stripsOn the position of sample pad and reaction film, have well and observation window. After test card assembles, pack in aluminium foil bag, add dryAgent sealing is preserved, and under drying at room temperature condition, can preserve more than 1 year.

Five, the detection of sample

Draw the calibration curve of PGI: PGI standard items (purchased from osmanthus, Shanghai Kanggong department) sample diluting liquid (1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) be mixed with the calibration object of following series concentration: 400ng/ml, 200ng/ml, 100ng/ml,50ng/ml, 20ng/ml, 10ng/ml, 0ng/ml, be added drop-wise to the above calibration object of 50 μ l respectively on well, after 10 minutes, usesFluorescence detector (purchased from Anqun Bioengineering Co., Ltd., Shenzhen, model AQ-3000) detects, can be in the first detection line and matterOn control line position, collect fluorescence. Taking sample concentration as abscissa, the ratio of the fluorescence intensity at the first detection line and nature controlling line placeFor ordinate is drawn calibration curve, R²Be 0.996. Nature controlling line is for the judgement of test paper validity and do corresponding to detection line signalProofread and correct, as band does not appear in nature controlling line, illustrate that test paper lost efficacy.

Draw the calibration curve of PGII: draw the calibration curve of PGII according to method same as described above, wherein, PGII markSample diluting liquid for accurate product (purchased from osmanthus, Shanghai Kanggong department) (1% (w/v) BSA-0.01MPBS(pH7.2) buffer solution) be mixed with asThe calibration object of lower series concentration: 50ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2ng/ml, 1ng/ml, 0ng/ml, by 50 μ lAbove calibration object is added drop-wise to respectively on well. Taking sample concentration as abscissa, the fluorescence at the second detection line and nature controlling line place is strongThe ratio of degree is that ordinate is drawn calibration curve, R²Be 0.994.

The blood sample to be checked of getting 50 μ l, is added drop-wise on well, detects, if first and the after 10 minutes with fluorescence detectorThere is band in two detection lines, in interpret sample, containing PGI and PGII, its concentration can obtain according to calibration curve.

Six, joint-detection PGI and PGII fluorescence immunoassay test paper performance evaluation

1. evaluate the index of test paper performance

1) range of linearity: respectively to the each concentration calibration object of above-mentioned PGI and the each concentration calibration object of above-mentioned PGII duplicate detection 3Inferior, draw respectively calibration curve, through data fitting and statistical analysis, the PGI linear detection range of test paper of the present invention is 5ng/ml-400ng/ml; PGII linear detection range is 0.1ng/ml-50ng/ml.

2) minimum detectability: respectively by PGI null value blood sample (without PGI composition) (purchased from Central Plains, Shenzhen company) and PGII null valueBlood sample (without PGII composition) (purchased from Central Plains, Shenzhen company) is divided into 20 parts and detects respectively, respectively calculating concentration mean value and 2Times standard deviation sum, obtains test paper PGI lowest detection of the present invention and is limited to 3.1ng/ml; PGII lowest detection is limited to 0.05ng/ml.

3) precision:

I) with PGI fluorescence immunoassay test paper of the present invention detect respectively PGI concentration be respectively 300ng/ml, 100ng/ml,The blood sample of 30ng/ml, duplicate detection 10 times, carries out withinrun precision mensuration. Survey every day to the sample of above-mentioned 3 concentrationFixed, 1 day 1 time, survey continuously 20 days, carry out betweenrun precision mensuration, result is as shown in table 5 below:

Table 5

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

In addition, as seen from the above table, the range of linearity of this detection paper PGI is wide, sensitivity good.

II) with PGII fluorescence immunoassay test paper of the present invention detect respectively PGII concentration be respectively 40ng/ml, 15ng/ml,The blood sample of 2ng/ml, duplicate detection 10 times, carries out withinrun precision mensuration. Measure every day to the sample of above-mentioned 3 concentration,1 day 1 time, survey continuously 20 days, carry out betweenrun precision mensuration, result is as shown in table 6 below:

Table 6

In addition, as seen from the above table, the range of linearity of this detection paper PGII is wide, sensitivity good.

2. test paper Evaluation on specificity

Get the PGII(50ng/ml of above-mentioned high concentration) calibration object is added drop-wise on well, after 10 minutes, uses fluorescence detectorDetect. There is band in result only the second detection line.

Separately get the PGI(400ng/ml of above-mentioned high concentration) calibration object is added drop-wise on well, after 10 minutes, uses fluoroscopic examinationInstrument detects. There is band in result only the first detection line.

The above results illustrates that this test paper specificity is good, and PGI and PGII do not interfere with each other.

3. detect blood sample

Draw PGI calibration curve and PGII calibration curve according to sample detection method mentioned above, then get gastric cancerPeople's blood sample to be checked 50 μ l are added drop-wise on well, after 10 minutes, detect with fluorescence detector. The first and second detection lines as a resultOccur band, in interpret sample, containing PGI and PGII, its concentration can obtain according to calibration curve.

Claims

1. for combining a fluorescence immune chromatography test paper for quantitative detection determinand people PGI albumen and people PGII albumen, be somebody's turn to doTest paper detects respectively described people PGI albumen and described people PGII albumen by double antibody sandwich method, wherein:

(a), for described people PGI albumen, described double antibody sandwich method adopts a PGI monoclonal that is marked with fluorescent microsphere to resistBody is as capture antibody, and a described PGI monoclonal antibody derives from the one in people PGI epitope peptide (1) and (2); AndAnd

Described double antibody sandwich method adopts the 2nd PGI monoclonal antibody as detecting antibody, and described the 2nd PGI monoclonal antibody is comeCome from the another one in people PGI epitope peptide (1) and (2);

Described people PGI epitope peptide (1) and (2) are respectively:

(1)Tyr-Lys-Val-Pro-Leu-Ile-Arg-Lys-Lys-Ser-Leu-Arg-Arg；

(2) Tyr-Lys-Asn-Phe-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Gln; And

(b), for described people PGII albumen, described double antibody sandwich method adopts a PGII monoclonal that is marked with fluorescent microsphereAntibody is as capture antibody, and a described PGII monoclonal antibody derives from one in people PGII epitope peptide (3) and (4)Person; And

Described double antibody sandwich method adopts the 2nd PGII monoclonal antibody as detecting antibody, described the 2nd PGII monoclonal antibodyDerive from the another one in people PGII epitope peptide (3) and (4);

Described people PGII epitope peptide (3) and (4) are respectively:

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

(4)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 PGI monoclonal antibody is by firstPGI antigen is prepared from, a PGI antigen be by make one in described people PGI epitope peptide (1) and (2) withCarrier protein couplet is prepared from; And

Described the 2nd PGI monoclonal antibody is prepared from by the 2nd PGI antigen, and the 2nd PGI antigen is by described in makingAnother one in people PGI epitope peptide (1) and (2) and carrier protein couplet are prepared from.

3. fluorescence immune chromatography test paper according to claim 1, a wherein said PGII monoclonal antibody is by firstPGII antigen is prepared from, and a PGII antigen is by making the one in described people PGII epitope peptide (3) and (4)Be prepared from carrier protein couplet; And

Described the 2nd PGII monoclonal antibody is prepared from by the 2nd PGII antigen, and the 2nd PGII antigen is by makingState that another one in people PGII epitope peptide (3) and (4) and carrier protein couplet be prepared from.

4. fluorescence immune chromatography test paper according to claim 1, the particle diameter of wherein said fluorescent microsphere be 320nm extremely400nm。

5. fluorescence immune chromatography test paper according to claim 1, the fluorescent material on wherein said fluorescent microsphere is different sulphurCyanic acid fluorescein, RB 200, TRITC or X-rhodamine; The micro-sphere material of described fluorescent microsphereFor the copolymer of polystyrene, polymethyl methacrylate or methyl methacrylate.

6. fluorescence immune chromatography test paper according to claim 5, the fluorescent material on wherein said fluorescent microsphere is sieve X-Red bright.

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

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

9. fluorescence immune chromatography test paper according to claim 1, wherein said test paper has base plate, and on this base plateChromatography direction during along use is provided with the way of contact successively: sample pad, pad, reaction film, absorbent filter, described combinationPad is coated with a described PGI monoclonal antibody that is marked with fluorescent microsphere and described first of the fluorescent microsphere that is marked withPGII monoclonal antibody, described reaction film comprises the first detection zone, the second detection zone and Quality Control district, described the first detection zone is coatedHave the one in described the 2nd PGI monoclonal antibody and described the 2nd PGII monoclonal antibody, described the second detection zone is coated withAnother one in described the 2nd PGI monoclonal antibody and described the 2nd PGII monoclonal antibody, described Quality Control district is coated with can be withA described PGI monoclonal antibody that is marked with fluorescent microsphere and a described PGII monoclonal that is marked with fluorescent microsphereThe antiantibody of the equal specific binding of antibody.

10. fluorescence immune chromatography test paper according to claim 9, wherein said reaction film is to be greater than the wavelength of 550nmUnder not fluorescent nitrocellulose filter substantially.

11. fluorescence immune chromatography test papers according to claim 9, wherein said base plate does not have photoluminescent property substantially.

12. fluorescence immune chromatography test papers according to claim 9, wherein said antiantibody is that sheep anti-mouse igg monoclonal is anti-Body or rabbit anti-mouse igg monoclonal antibody.

13. fluorescence immune chromatography test papers according to claim 12, wherein said antiantibody is that sheep anti-mouse igg monoclonal is anti-Body.

Prepare according to the method for the fluorescence immune chromatography test paper described in any one in claim 1 to 13 for 14. 1 kinds, it comprisesFollowing steps:

1) provide a PGII monoclonal that is marked with a PGI monoclonal antibody of fluorescent microsphere and is marked with fluorescent microsphere anti-Body;

2) provide pad, wherein on described pad, a coated described PGI monoclonal that is marked with fluorescent microsphere is anti-Body and a described PGII monoclonal antibody that is marked with fluorescent microsphere;

3) provide reaction film, chromatography direction interval when wherein edge is used on described reaction film arranges the first detection zone, secondDetection zone and Quality Control district, described the first detection zone is fixed with in the 2nd PGI monoclonal antibody and the 2nd PGII monoclonal antibodyOne, described the second detection zone is fixed with the another one in the 2nd PGI monoclonal antibody and the 2nd PGII monoclonal antibody, described inQuality Control district is fixed with antiantibody; With

4) chromatography direction when edge is used on base plate arranges sample pad, described pad, described reaction with the way of contact successivelyFilm, absorbent filter, thus described fluorescence immune chromatography test paper made.

15. methods according to claim 14, wherein said step 1) comprising:

A) activate fluorescent microsphere with carbodiimide;

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

C) fluorescent microsphere mark the one PGI monoclonal antibody and a PGII monoclonal antibody respectively of using step b) to obtain.

16. methods according to claim 15, wherein wherein said step 1) comprising:

A) aqueous dispersions of fluorescent microsphere or MES buffer solution dispersion liquid are processed and mixed with carbodiimide through ultrasonic wave, therebyActivate described fluorescent microsphere;

B) by steps A) N-hydroxy thiosuccinimide for fluorescent microsphere-citrate buffer solution washing of the activation that obtains,Disperse, and through ultrasonic wave processing;

C) by step B) fluorescent microsphere that obtains is mixed with a PGI monoclonal antibody and a PGII monoclonal antibody respectivelyClose, with the sealing of BSA-monoethanolamine buffer solution, centrifugal, with the dispersion of BSA-Tween solution, through ultrasonic wave processing, thereby obtain respectivelyBe marked with a PGI monoclonal antibody and a PGII monoclonal antibody that is marked with fluorescent microsphere of fluorescent microsphere.

17. methods according to claim 14, wherein in described step 2) in, described first of the fluorescent microsphere that is marked withThe coated concentration of PGI monoclonal antibody and a described PGII monoclonal antibody that is marked with fluorescent microsphere is respectively 0.5～2mg/ml。

18. methods according to claim 14, wherein in described step 3) in, described the first detection zone and described the second inspectionSurvey the 3mm that is spaced apart between district, between described the second detection zone and described Quality Control district, be spaced apart 5mm, described the 2nd PGI Dan KeThe coated concentration of grand antibody, described the 2nd PGII monoclonal antibody and described antiantibody is respectively 0.5～2mg/ml.