CN104418939B - People PGI epitope peptides, antigen, antibody, purposes and kit - Google Patents

Abstract

The present invention relates to people PGI epitope peptides, antigen, antibody, purposes and kit.The amino acid sequence of people's PGI epitope peptides of the present invention, is one of sequence shown in sequence table SEQ ID NO.1 and SEQ ID NO.2.The PGI antigens of the present invention are made by people PGI epitope peptides and carrier protein couplet.The PGI monoclonal antibodies or polyclonal antibody of the present invention is made by the PGI antigens of the present invention.The PGI monoclonal antibodies or polyclonal antibody of the present invention is used to prepare PGI external diagnosis reagent cases.People's PGI epitope peptides of the present invention have good antigenicity, the antigen prepared with it（Immunogene）Immune animal can produce the monoclonal antibody and polyclonal antibody of high degree of specificity, so as to the vitro detection applied to people PGI.

Description

People PGI epitope peptides, antigen, antibody, purposes and kit

Technical field

The invention belongs to chemiluminescent polypeptide and field of immunology, and in particular to human pepsinogen I（PGI）Epitope peptide, The PGI specific antigens prepared with the epitope peptide and corresponding monoclonal antibody or polyclonal antibody, the antibody are in system Purposes, people's PGI external diagnosis reagent cases on standby people's PGI external diagnosis reagent cases, and it is a kind of for quantitatively detecting determinand Fluorescence immune chromatography test paper of middle people PGI albumen and preparation method thereof.

Background technology

Propepsin（PG）It is the inactive precursor of pepsin in gastric juice.Human pepsinogen presses its electrophoretic mobility By that to slow, can be divided into seven kinds of isozymogens of PG1 to PG7 soon, and according to its biochemical property, immunogenicity, cell derived and Distribution can be divided into two subgroups of PGI and PGII in tissue.PG1 in seven kinds of isozymogens is approximate to PG5 immunogenicities, referred to as stomach Pepsinogen I（PGI）, it is mainly secreted by the chief cell and mucus neck cell of gastric gland；PG6 to PG7 is referred to as propepsin II（PGII）, except by body of stomach and stomach bottom mucous membrane secrete the cell secretes of acid gland in addition to, secrete mucus neck cell, the cardiac gland of acid gland PGII can be also produced with the mucilage cell of the pyloric gland of antrum and the Brunner glands of duodenum epimere.Stomach is almost PG's Exclusive source, and secretion the stage secretory volume can change.Serum PG I and PGII reflect gastric mucosa different parts Secreting function.PG after synthesis largely enters gastral cavity, activates into pepsin under acidic gastric juice effect, only a small amount of PG （About 1%）Through stomach lining hair.Therefore, serum PG concentration can reflect its secretion level.PGI in normal human serum is PGII 6 times, when stomach lining occur pathological change when, serum PG content also changes therewith.

Serum PG content and helicobacter pylori（HP）The correlations such as infection, chronic gastritis, stomach cancer.In different disease of stomach, Respective change occurs for serum PG I, PGII and PGI/PGII value, and it is raised, reduction or constant not consistent, as diagnosis or The sensitivity for the treatment of results index and specificity are also different.Therefore, for different disease of stomach, serum PG I, PGII and PGI/PGII value also has different criterions.

PGI is detection oxyntic gland（Fundus gland）The pointer of cell function, gastric acid secretion increases, then PGI is raised；Hydrochloric acid in gastric juice point Reduction or gastric mucosa body of gland atrophy are secreted, then PGI is reduced.For example, superficial gastritis and helicobacter pylori in gastroxia （HP）In the gastritis of infection, PGI secretion can increase；And in chronic severe atrophy gastritis, when chief cell is reduced, PGI contains Amount declines.The PGI of high concentration is also as duodenal ulcer and the subclinical indication of complication risk, and be also used as Observe HP（Helicobacter pylori）Infect an index of eradication therapy curative effect.

Stomach cancer is one of common malignant tumour of China, and its case fatality rate is occupied first of various malignant tumours, and it is early diagnosed, early Phase treatment, which turns into, to improve life in patients, reduces the unique channel of case fatality rate.Domestic and foreign scholars become to Serum Obtained From Advance Gastric Cancer PG It has been turned into substantial amounts of research to find：When getting a cancer of the stomach, serum PG I levels reduction, PGII levels keep normal or rise substantially, enter And, determining serum PG I level and PGI/PGII ratio contributes to the antidiastole of stomach cancer, too low PGI and PGI/PGII Early carcinoma of stomach should be vigilant.Because the content of serum PG directly reflects the function of stomach lining, therefore Serum Obtained From Advance Gastric Cancer PGI levels are bright It is aobvious to decline, show that patients with gastric cancer stomach lining secretion capacity declines.More than 80% stomach cancer is with atrophic gastritis, and atrophic stomach Inflammation can cause stomach lining chief cell to be lost, so as to influence its secreting function；Serum Obtained From Advance Gastric Cancer PGI levels are decreased obviously and stomach cancer Patient's Mucosal atrophy, intestinesization are relevant so as to secrete reduction.Therefore serum PG I and PGI/PGII are decreased obviously to monitoring early stage stomach Cancer has important clinical meaning, can be efficiently applied to the preliminary examination of high incidence area of gastric cancer crowd.PG detections are used as Noninvasive Method, it is possible to decrease the pain that patients with gastric disease or stomach cancer people at highest risk check, and it is easy, economical, it is significant.

The optimal method for detecting the PGI levels in serum is immunoassays.Therefore, finding suitably has immunogene The PGI epitope peptides of property, prepare specific PGI antigens and antibody for emphasis.

The content of the invention

For solve it is above-mentioned the problems of in the prior art, the invention provides a kind of people PGI epitope peptides, with this PGI specific antigens and corresponding monoclonal antibody or polyclonal antibody prepared by epitope peptide, it is external in preparation people PGI Purposes on diagnostic kit, and people's PGI external diagnosis reagent cases.

Specifically, the invention provides：

A kind of people PGI epitope peptides, wherein the amino acid sequence of the PGI epitope peptides is one of both：

(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。

Present invention also offers a kind of PGI antigens, it is by making described people PGI epitope peptides (1) and carrier egg What white coupling was prepared from.

Present invention also offers a kind of PGI antigens, it is by making described people PGI epitope peptides (2) and carrier egg What white coupling was prepared from.

Present invention also offers a kind of people PGI antibody, its be the monoclonal antibody that is prepared from by described PGI antigens or Polyclonal antibody, wherein the PGI antigens are by making described people PGI epitope peptides (1) be prepared with carrier protein couplet .

Present invention also offers a kind of people PGI antibody, its be the monoclonal antibody that is prepared from by described PGI antigens or Polyclonal antibody, wherein the PGI antigens are by making described people PGI epitope peptides (2) be prepared with carrier protein couplet .

Present invention also offers the purposes according to described people PGI antibody on people's PGI external diagnosis reagent cases are prepared.

Present invention also offers a kind of people PGI external diagnosis reagent cases, it is anti-as coating comprising described people PGI antibody Body, wherein the coated antibody is preferably monoclonal antibody.

Preferably, the kit also includes binding antibody, and the binding antibody is described people's PGI antibody, and should Binding antibody is preferably polyclonal antibody, and when the binding antibody from described people PGI epitope peptides (1) and One of (2) when, the coated antibody is from the other of described people PGI epitope peptides (1) and (2).

Preferably, the secondary antibody that the kit is also marked comprising enzyme.

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

1. people's PGI epitope peptides of the present invention have good antigenicity, the antigen prepared with it（Immunogene）It is immune Animal can produce the monoclonal antibody and polyclonal antibody of high degree of specificity.

2. with the PGI monoclonal antibodies that prepare of the present invention and polyclonal antibody can high special with blood sample PGI is combined.

3. the people PGI external diagnosis reagent cases of the present invention can effectively monitor the level of the PGI in serum, and prepare Method is simple, and cost is low, suitable for large-scale production.

4. fluorescence analysis are combined by the present invention with flash chromatography immunological technique to be used to quantitatively detect there is provided one kind The fluorescence immune chromatography test paper of people PGI albumen in determinand, with the test paper detect determinand in people's PGI albumen, it is easy to operate, Quickly, 10 minutes are only needed with regard to sample detection can be completed, and detection range is wide, specific high, sensitivity is good, can be timely rapidly The ground assisted diagnosis state of an illness, monitors prognosis.

5. the present invention passes through substantial amounts of experiment during the fluorescence immune chromatography test paper of the people PGI albumen is prepared Grope, optimize the preparation condition of each side so that when being detected with the fluorescence immune chromatography test paper of the present invention, the fluorescence letter back of the body Than greatly improving, so as to improve detection sensitivity and credible result degree；In addition, the present invention also passes through the detection zone and matter of test paper The change for controlling the fluorescence intensity ratio in area carrys out the content of PGI in response sample, and this only examines or check detection zone with traditional chromatographic technique Absolute fluorescence intensity compare, the influence of external condition and background etc. is reduced to the full extent, further increase detection knot Fruit confidence level.

Brief description of the drawings

Fig. 1 is testing result of the testing result with known agent box for showing people's PGI external diagnosis reagent cases of the present invention Correlation figure, wherein abscissa is the value of PGI concentration in the sample measured with known agent box, and unit is ng/ml, indulges and sits The value of PGI concentration in the sample that is measured with kit of the present invention is designated as, unit is ng/ml.

Embodiment

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

First, people PGI epitope peptides

People's PGI albumen specifically described herein is known in the art, and its amino acid sequence is known in the art, Ke Yi Found in the specialized databases such as NCBI.

The invention provides a kind of people PGI epitope peptides (1) and (2), its amino acid sequence is respectively such as sequence table SEQ Shown in ID No.1 and SEQ ID No.2, it is：

(1)Y-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 by substantial amounts of theoretical research and experiment, and screening, which obtains two, has good antigen The epitope peptide of property.

PGI epitope peptides (1) are with people's PGI albumen（PG3）One section of N-terminal the 19th to 30 is containing 12 amino acid residues Peptide fragment is added with Y as antigenic determinant, and in its N-terminal.It is in N-terminal plus Y in order that the epitope peptide passes through BDB （Bis-diazotizedbenzidine dichloride）It is linked to carrier protein（Such as hemocyanin（KLH））On, so that Antibody is prepared as antigen.The characteristics of PGI epitope peptides (1) have hydrophily high, antigenic strong and are readily synthesized.

PGI epitope peptides (2) are with people's PGI albumen（PG3）One section of C-terminal the 372nd to 381 is containing 10 amino acid residues Peptide fragment as antigenic determinant, and in its N-terminal added with 3 amino acid residues：Y、K、N.The PGI antigen tables so constituted The characteristics of position peptide (2) also has hydrophily high, antigenic strong and is readily synthesized.

At present, present invention research finds that PGI epitope peptides of the invention have following function：

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

The preparation method of the PGI epitope peptides of the present invention can use chemical synthesis：Using American AB I431A types polypeptide certainly Dynamic synthesizer, passes through Solid phase synthesis epitope peptide.The epitope peptide (1) of the present invention and the molecular weight of (2) are respectively 1657.24 and 1616.93, it can be determined with mass spectrum, and the epitope peptide sequence synthesized by identification is determined by peptide sequence Row.The purity of peptide fragment is evaluated with thin-layer chromatography and high performance liquid chromatography, and determines the concentration of epitope peptide.

2nd, PGI antigens

Present invention also offers a kind of PGI antigens, it is by making in people PGI epitope peptides (1) and (2) of the invention One is prepared from carrier protein couplet.Specifically, the invention provides PGI antigens (1) and (2), the PGI antigens (1) by making the people PGI epitope peptides (1) of the present invention be prepared from carrier protein couplet；The PGI antigens (2) pass through The people PGI epitope peptides (2) of the present invention are made to be prepared from carrier protein couplet.The PGI antigens of the present invention have immunogene Property and specificity, be a kind of immunogene, can be used to immune animal to prepare specific PGI antibody.In the present invention, it can use Carrier protein example include 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 more, and immune effect is preferable, and with immune animal affiliation farther out, It is difficult to cause cross reaction as carrier protein with it, therefore is preferred.

3rd, PGI monoclonal antibodies, PGI polyclonal antibodies and people's PGI external diagnosis reagent cases

Present invention also offers people PGI monoclonal antibodies and people's PGI polyclonal antibodies, the antibody can be utilized respectively this The PGI antigens (1) of invention and (2)（Immunogene）Immune animal is prepared.Preparation method can use the ordinary skill in the art, It for details, reference can be made to embodiment 2.

The PGI monoclonal antibodies and polyclonal antibody of the present invention can be used for preparing people's PGI external diagnosis reagent cases, the examination Agent box can be detected based on immunization method to the PGI in blood preparation.

Therefore, the invention provides a kind of people PGI external diagnosis reagent cases, its people PGI monoclonal comprising the present invention resists Body or polyclonal antibody.

The immunization experiment method available for clinical examination is currently known mainly including following several：ELISA method, chemiluminescence Method, fluorescent chromatographic method, colloid gold immune determination method etc..

And ELISA method includes following several types：Double antibody sandwich method detection antigen, dual-antigen sandwich method detection antibody, Indirect method surveys antibody, competition law and surveys antibody, competition law survey antigen, capture coating method survey antibody etc..

People's PGI external diagnosis reagent cases of the present invention are preferred to use ELISA double antibody sandwich methods to detect PGI albumen.Should Secondary antibody and/or necessary instrument and reagent that kit can be marked comprising coated antibody, binding antibody, enzyme etc..

Preferably, the people PGI external diagnosis reagent cases are anti-as coating using the people PGI monoclonal antibodies of the present invention Body.Here, term " coated antibody " refers to be coated in the antibody on the ELISA Plate of solid phase.In addition, the people PGI in-vitro diagnosis examination Agent box further preferably comprising people PGI polyclonal antibodies using as binding antibody, wherein, when the binding antibody is from the present invention's When one of people PGI epitope peptides (1) and (2), the coated antibody is derived from the epitope peptide (1) and (2) Another one.Here, term " binding antibody " refers to the spy that can be combined in kit with determined antigen and enzyme-labeled secondary antibody Heterogenetic antibody.The secondary antibody that the kit can also be marked comprising enzyme, the secondary antibody can be goat anti-rabbit igg antibody, The enzyme mark can be horseradish peroxidase, alkaline phosphatase etc..

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

4th, for quantitatively detecting the fluorescence immune chromatography test paper of people's PGI albumen

It is used for the fluorescence immune chromatography test paper of people's PGI albumen in quantitatively detection determinand present invention also offers a kind of, should Test paper detects the people PGI albumen by double antibody sandwich method, wherein：

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

The double antibody sandwich method is also used as detection antibody, the 2nd PGI monoclonals using the 2nd PGI monoclonal antibodies Antibody sources are in the other of people PGI epitope peptides (1) and (2)；

The people PGI epitope peptides (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。

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

In the present invention, the first PGI monoclonal antibodies can be prepared from by the first PGI antigens, and the first PGI resists Original can be by making one of the people PGI epitope peptides (1) and (2) are prepared from carrier protein couplet；And institute Stating the 2nd PGI monoclonal antibodies can be prepared from by the 2nd PGI antigens, and the 2nd PGI antigens can be by making the people PGI The other of epitope peptide (1) and (2) are prepared from carrier protein couplet.

In the present invention, the example of available carrier protein includes 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 more, and immune effect is preferable, And with immune animal affiliation farther out, it is difficult to cause cross reaction as carrier protein with it, therefore is preferred.

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

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

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

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

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

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

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

It is of the present invention can be with being marked with the antiantibody that the first PGI monoclonal antibody specificities of fluorescent microsphere are combined Can be sheep anti-mouse igg monoclonal antibody or rabbit anti-mouse igg monoclonal antibody, wherein preferably sheep anti-mouse igg monoclonal antibody, Compared with polyclonal antibody, monoclonal antibody specificity is higher.

In a specific embodiment, fluorescence immune chromatography test paper of the invention when in use, drips in sample pad Plus sample liquid（Such as blood sample containing PGI）, under capillarity, sample liquid is moved to absorbent filter one end, at pad The first PGI monoclonal antibodies formation immune complex having with described mark, the immune complex is further moved It is dynamic, the immune complex of double-antibody sandwich is combined to form with the 2nd PGI monoclonal antibodies in detection line, and do not formed and exempted from The first PGI monoclonal antibodies that the mark of epidemic disease compound has then are combined with the antiantibody on nature controlling line.This process is needed 10 minutes to 15 minutes are wanted, afterwards, is detected with fluorescence detector, if not occurring band at nature controlling line, illustrates test paper Failure；If occurring band at nature controlling line, and do not occur band at detection line, then illustrate to be free of people's PGI albumen in sample；If Occur band on nature controlling line and detection line, then illustrate the albumen of PGI containing someone in sample.

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

1）The first PGI monoclonal antibodies for being marked with fluorescent microsphere are provided；

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

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

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

It will be appreciated by persons skilled in the art that order that can be according to actual needs to above-mentioned steps is adjusted, Such as step 3）Can be in step 1）Before, or in step 1）With 2）Between.

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

The present inventor by it is substantial amounts of experiment grope, optimize prepare the present invention be used for detect people's PGI albumen Fluorescence immune chromatography test paper method each step condition so that the present invention fluorescence immune chromatography test paper can The result for meeting clinical detection requirement is obtained for people PGI albumen, i.e. detection range is wide, specific high, sensitivity is good.

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

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

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

c）Use step b）The PGI monoclonal antibodies of fluorescent microsphere mark the first obtained, it is preferable that by step b）Obtained Fluorescent microsphere mixed with the first PGI monoclonal antibodies, with BSA- monoethanolamine buffer blinds, centrifugation is water-soluble with BSA- tweens Liquid disperses, through ultrasonication, so as to obtain being marked with the first PGI monoclonal antibodies of fluorescent microsphere.

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

a）Carbodiimide activation fluorescent microsphere is used, wherein, 1 (w/v) % fluorescent microsphere aqueous dispersions are taken, 10000rpm is extremely 15000rpm low temperature（Such as 10 DEG C）Centrifugation 5 to 10 minutes, removes supernatant, sediment is distributed to 500 μ l distilled water or previous cleaning Buffer solution（The 0.1M MES aqueous solution）In, ultrasonic wave（240W）Processing 1 to 2 minute, repeats above procedure three times, adds carbon two Imines 10mg to 50mg, is stirred 10~15 minutes, so as to activate the fluorescent microsphere；

b）Washing step a）The fluorescent microsphere of the activation obtained, wherein, by step a）The fluorescence of the activation obtained is micro- Ball is centrifuged 5 to 10 minutes under 1000rpm to 15000rpm, and sediment is distributed into 1ml coupling buffers（20~100mM's N- hydroxy thiosuccinimides-citrate buffer solution）In, ultrasonic wave（240W）Processing 1 to 2 minute, repeats above procedure three It is secondary；

c）Use step b）The PGI monoclonal antibodies of fluorescent microsphere mark the first obtained, wherein, according to 1 μ l to 3 μ l antibody （10mg/ml）The ratio of the fluorescent microsphere activated described in/100 μ l, by step b）The fluorescent microsphere and the first PGI Dan Ke obtained Grand antibody mixing, room temperature（25℃）Lower stirring 1.5~3 hours（It is preferred that 2 hours）, add 1ml Block buffers（1(w/v)% BSA-0.05M monoethanolamines）, continue to stir 1 hour, centrifuged 5 to 10 minutes under 10000rpm to 15000rpm, repeated centrifugation 3 It is secondary, sediment is distributed to the whole wash buffers of 500 μ l（0.5 (w/v) %BSA-0.11 (v/v) % Tween solutions）In, ultrasonic wave （240W）Processing 1 to 2 minute, 500 μ l are settled to the whole wash buffer.

Preferably, in the method for the invention, the step 2）Including：The first PGI Dan Ke of fluorescent microsphere will be marked with Grand antibody antibody diluent（1%(w/v)BSA-0.01M PBS（pH7.2）Buffer solution）It is diluted, to be diluted to 0.5~ 2mg/ml, preferably 1mg/ml, then with micropipettor even application on pad, drying afterwards or vacuum refrigeration are dry It is dry.In the step 2 of the method for the present invention）In, the coating concentration for the first PGI monoclonal antibodies that described mark has For 0.5~2mg/ml, preferably 1mg/ml.

Preferably, the step 3）Including：The 2nd PGI monoclonal antibodies and antiantibody are drawn with metal spraying machine and arrive nitric acid Cellulose membrane（Solid phase carrier）On using as detection zone and quality control region, make detection zone and quality control region at intervals of 3mm to 8mm, institute The concentration for stating the 2nd PGI monoclonal antibodies and the antiantibody is respectively 0.5~2mg/ml, preferably 1mg/ml.

Preferably, as a result detection utilizes special fluorescence detector（It is purchased from Anqun Bioengineering Co., Ltd., Shenzhen, Model AQ-3000）Quality control region and detection zone are detected, in the ratio and testing sample of detection zone and quality control region fluorescence intensity PGI content be directly proportional.Using detection zone with the ratio of quality control region fluorescence intensity rather than directly using the absolute of detection zone Fluorescent value can be reduced as far as the influence of reaction condition, matrix etc., and avoid ambient interferences as far as possible.

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

Embodiment

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

Embodiment 1：PGI epitope peptides (1) and the preparation of (2).

Preparation method chemical synthesis：Using many automatic peptide synthesizers of American AB I431A types, closed respectively by solid phase method Into PGI epitope peptides (1) and (2).The purity of epitope peptide is evaluated with high performance liquid chromatography, and determines the dense of peptide fragment Degree.The epitope peptide (1) and the molecular weight of (2) of the present invention is respectively 1657.24,1616.93, is determined using mass spectrum, Peptide sequence synthesized by identification is determined by peptide sequence.

First, the synthesis of PGI epitope peptides (1) and (2)

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

The PGI epitope peptides (1) of the present invention and the respective specific preparation process of (2) are as follows：

1. it is raw materials used：

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

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

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

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

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

Piperidine（Piperidines, is purchased from sigma companies）

DMAP（Dimethyl aminopyridine, is purchased from sigma companies）

HOBT（Hydroxybenzotriazole, is purchased from sigma companies）

DCC（Dicyclohexylcarbodiimide, is purchased from sigma companies）

TFA（Trifluoroacetic acid, is purchased from sigma companies）

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

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

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

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

2. use instrument：

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

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

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

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

3. synthetic method and process：

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

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

The amino acid of Fmoc protections

（2）Amino acid is connected to resin

（3）Slough the Fmoc protection groups of amino acid

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

（5）Coupling

Peptide-resin of new coupling

（6）Repeat step（3）Extremely（5）Until end of synthesis.

（7）Peptide resin：

Resin is transferred in beaker, TFA is used（Trifluoroacetic acid）Peptide chain is cut, EDT is used（2.5 volume %）, thioanisole （2.5 volume %）Make scavenger, react 3.0 hours at room temperature, remove cutting reagent, then extracted with ether, respectively obtain PGI The crude product 119.8mg of crude product 124.3mg and the PGI peptide fragment (2) of peptide fragment (1).

2nd, the purifying of PGI epitope peptides (1) and (2) crude product：

Purified using high performance liquid chromatography separation：

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

Chromatograph：Waters600, Waters, US

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

B：0.1%TFA（Trifluoroacetic acid）In 60% acetonitrile

Detection wavelength：214nm

Flow velocity：4ml/ minutes

Gradient：20-60%B, 30 minutes

HPLC（High performance liquid chromatography）Analysis

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

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

B：0.1%TFA（Trifluoroacetic acid）In acetonitrile

Detection wavelength：214nm

Flow velocity：1ml/ minutes

Gradient：0-60%B, 30 minutes

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

3rd, the identification of PGI epitope peptides (1) and (2)

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

（1）Reagent raw material

TFA（Trifluoroacetic acid, is purchased from sigma companies）

HCCA（Alpha-cyano -4- hydroxycinnamic acids, are purchased from sigma companies）

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

（2）Instrument

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

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

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

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

As a result, the molecular weight for measuring gained PGI epitope peptides (1) is the molecule of 1658.5, PGI epitope peptides (2) Measure as 1618.4, it is consistent with theoretical molecular 1657.24,1616.93, it was demonstrated that synthesis polypeptide is purpose product.

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

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

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

（3）Reagent raw material

Phenyl isothiocyanate PITC, is purchased from sigma companies

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

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

TFA（Trifluoroacetic acid, is purchased from sigma companies）

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

Chlorobutane, is purchased from sigma companies

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

（4）Determine

Carried out by instrument specification.

As a result：Identified, the sequence of gained PGI epitope peptides (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 result is consistent with target section of synthesized peptide.

Embodiment 2：The PGI epitope peptides (1) of the gained of embodiment 1 and (2) are connected with carrier protein to prepare respectively PGI antigens (1) and (2), are immunized animal, so as to be prepared using antigen (1) specific respectively using gained antigen (1) and (2) Monoclonal antibody and polyclonal antibody, and prepare specific monoclonal antibody and polyclonal antibody using antigen (2).

1. the preparation of antigen：Use BDB（Bis-diazotizedbenzidine dichloride）Method is by PGI peptide fragments (1) (2) respectively with carrier protein KLH（Keyhole limpet hemocyanin）Connection is prepared into PGI antigens (1) and (2).

PGI peptide fragments (1) or (2) 10.0mg are taken, 1ml0.1M PBSs are used（pH7.4）Dissolving；KLH10mg, uses 0.2M Borate buffer solution（pH9.0）20ml dissolves；Then both are mixed, is cooled to 0 DEG C, takes BDBCl₂110 μ L, react at room temperature Dispensed after 1.5h, dialysed overnight, -20 DEG C of preservations.

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

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

2. immune animal prepares monoclonal antibody：

2.1. PGI antigens (1) and (2) of above-mentioned preparation are taken（Immunogene）Respectively with isometric Freund's complete adjuvant（Purchase From Shanghai Yuan Ju biotech firms）After being sufficiently mixed, Balb/c mouse are individually immunized, 50 μ g antigens/only, subcutaneous multiple spot note Penetrate.Serum titer is surveyed after 4 weeks, the good mouse of immunoreactivity booster immunization again is selected：Take antigen and isometric Freund endless After full adjuvant is sufficiently mixed, 25 μ of antigen dose g/, subcutaneous multi-point injection, the number of times of booster immunization is 6 times, continuous before fusion Twice, extracting spleen cell uses 50%PEG to booster immunization according to a conventional method with Sp2/0 myeloma cell afterwards（MW4000）（Purchased from Central Plains Chemical company）Mediation is merged, and uses HAT conditioned mediums（Purchased from sigma companies）Selection culture.CO is put into after fusion₂ In incubator after 37 DEG C of cultures 9~11 days, appearance larger cell clone hole in.Start to be screened with indirect ELISA within 11 days. 4 time cloning cultures are carried out using limiting dilution assay to the positive hole of primary dcreening operation（Even if a large amount of schizogamies of cell after screening）, Afterwards amplifying cells, freeze, prepare ascites.

2.2. by Balb/c mouse norphytanes（Purchased from sigma companies）0.5ml/ is only handled, and pneumoretroperitoneum inoculation in one week is miscellaneous Hand over oncocyte 2 × 10⁶Individual/only, collect ascites after 10 days.

2.3. antibody titer is determined：The monoclonal antibody (1) prepared using PGI antigens (1) is determined with indirect ELISA method Potency, as a result show that the potency of monoclonal antibody reaches 1:More than 32000.

The potency of the monoclonal antibody (2) prepared using PGI antigens (2) is also measured using identical method, and it is imitated Valency also reaches 1:More than 32000.

3. immune animal prepares polyclonal antibody：

3.1. the NZw for being about 2kg or so from three monthly ages, body weight is used as immune animal.In fundamental immunity, By the PGI antigens (1) of the above-mentioned preparations of 1-2mg and (2)（Immunogene）Mixed respectively with isometric Freund's complete adjuvant-fully newborn Individually multiple spot hypodermic injection is carried out after change at rabbit back.Every 4 weeks booster immunizations once, antigen and incomplete Freund After agent is fully emulsified, only it is subcutaneously injected with 100 μ g/ in back multiple spot.Arteria carotis bloodletting in 10th day after final boost, separation Serum.

3.2. antibody titer is determined：The polyclonal antibody (1) prepared with indirect elisa method measure using PGI antigens (1) Potency, as a result shows that antibody titer reaches 1:More than 16000.

The potency of the polyclonal antibody (2) prepared using PGI antigens (2) is also measured using identical method, and it is imitated Valency also reaches 1:More than 16000.

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

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

5. freezed after antibody packing, Cord blood.

Embodiment 3：People PGI monoclonal antibodies (1) and the specificity identification of (2)

Detected with ELISA.Respectively with people PGI albumen and PGII albumen（It is purchased from Shanghai osmanthus Kanggong department）It is anti-for detection Primordial covering elisa plate, the spy of prepared PGI monoclonal antibodies (1) and (2) and people's PGI albumen is detected by ELISA respectively Opposite sex reaction, makees negative control, PBS liquid makees blank control with normal BALB/c mouse serum.

As a result：It is the positive (P/N that PGI monoclonal antibodies (1) and (2) are only reacted with PGI respectively>2.1), reacted with PGII For feminine gender, the PGI monoclonal antibodies (1) and (2) for illustrating the present invention have specificity respectively.

Embodiment 4：People PGI polyclonal antibodies (1) and the specificity identification of (2)

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

As a result show：PGI polyclonal antibodies (1) and (2) are respectively the positive (P/N with PGI reactions>2.1) it is, and anti-with PGII It should be negative, the PGI polyclonal antibodies (1) and (2) for illustrating the present invention have specificity respectively.

Embodiment 5：PGI external diagnosis reagent cases are prepared using PGI monoclonal antibodies and PGI polyclonal antibodies.

In the present embodiment, this will be used as by the use of monoclonal antibody (1) prepared by PGI epitope peptides (1) in embodiment 2 Coated antibody in kit；Combination will be used as by the use of polyclonal antibody (2) prepared by PGI epitope peptides (2) in embodiment 2 Antibody.

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

1. the preparation of various buffer solutions and reagent：

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

Na₂CO₃：16.0 grams

NaHCO₃：29.0 grams

Distillation is water-soluble to 1000ml

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

Na₂HPO₄·12H₂O：58 grams

KH₂PO₄：4 grams

NaCl：100 grams

KCl：4 grams

Distillation is water-soluble to 1000ml

Plus Tween20：20ml

C, enzyme marker dilution：

10×PBS-Tween20：10ml

FCS（Calf serum）：20ml

Distillation is water-soluble to 1000ml

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

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

D, developer A：

Citric acid：35.5 grams

Urea peroxide：10 grams

Distillation is water-soluble to 1000ml

Tween20：10ml

E, developer B：

Citric acid：120 grams

EDTA-2Na：1 gram

TMB·2HCl：2 grams

Distillation is water-soluble to 1000ml

F, terminate liquid：2M H₂SO₄

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

Distilled water：177.3ml

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

2. the preparation of pre-coated plate：

PGI monoclonal antibodies (1) are dissolved in the 0.05M of pH=9.6 carbonate buffer solution, pre-coated liquid are made, in enzyme Target（It is purchased from Shenzhen Jin Canhua companies）It is upper to add 100 μ l by 0.1 μ g/ holes per hole, put 4 DEG C and place 18-24 hours, take out, Coating buffer is got rid of, is washed, is fitted into after BSA closings 16 hours, dried overnight in aluminide-coating bag and vacuumizes sealing, 4 DEG C of guarantors are placed in Deposit.

3. binding antibody（PGI polyclonal antibodies (2)）With enzyme-linked thing（The goat anti-rabbit igg of horseradish peroxidase-labeled resists Body（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 objects（Raw material is purchased from Shanghai osmanthus Kanggong department）：6：6×1.0ml（Concentration is respectively 0ng/ml, 20ng/ ml、40ng/ml、80ng/ml、160ng/ml、320ng/ml）

PGI binding antibodies：1×10ml（Through 1:5000 dilutions）

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

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

Developer A：1×6.0ml

Developer B：1×6.0ml

Terminate liquid：1×6.0ml

5. the operating procedure of kit：

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

6. result judgement：

1. carry out serum PG I detections using the kit of the present invention

Table 1：Standard concentration and corresponding mean light absorbency（OD）Value

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

Standard curve, the R of standard curve are drawn with standard concentration and correspondence absorbance²=0.997。

The PGI concentration results in detected sample are calculated according to standard curve.

To 28 superficial gastritis patients, 30 atrophic gastritis patients, 34 Patients with Gastric Cancer and 70 healthy persons by upper The mode of stating carries out serum PG I detections, and testing result is shown in Table 2.

Table 2：Four groups of sample PGI concentration compare

The result of table 2 shows that the difference between each group is statistically significant（P<0.01）, can be corresponding with clinical detection result.

2. the testing result of kit of the present invention is related to the testing result of known detection PGI ELISA kit Property analysis

Utilize DRG kitsHuman Pepsinogen I ELISA, it is public purchased from ALPCO Diagnostics Department）50 parts of same blood samples are detected respectively with the kit of the present invention（Including superficial gastritis blood sample of patient, atrophic stomach Scorching blood sample of patient and patients with gastric cancer blood sample）.Using the measured value of DRG kits as abscissa, with the measured value of kit of the present invention For ordinate, set up linear equation and calculate coefficient correlation.

Result to two kinds of kits carries out paired t-test, judges whether there were significant differences for both quantitative results.

As shown in figure 1, kit of the present invention and the coefficient R of the measured value of DRG kits²=0.997, dependent equation The measured value of kit of the present invention is represented for y=0.9968x+0.0144, wherein y, x represents the measured value of DRG kits.By Fig. 1 Understand, the correlation of the measured value and the measured value of DRG kits of kit of the present invention is good.

Statistical procedures are carried out with paired t-test to the measured values of two kinds of kits, obtained | t |=0.996, v=n-1=49, Look into t dividing value tables and obtain P>0.05.The result shows that the testing result of two kinds of kits, without significant difference, illustrates kit of the present invention The correlation of measured value and the measured value of DRG kits is good, so that the kit of the present invention is with a high credibility, and can be further For PGI/PGII joint-detections.

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

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

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

1.1st, the activation of fluorescent microsphere：

Take 500 μ l, content 1 (w/v) % fluorescent microsphere（Purchased from Guangzhou Growth hormone secretagogue company）Aqueous dispersions, at 10 DEG C, with 12000rpm is centrifuged 10 minutes, removes supernatant, sediment is distributed to 500 μ l distilled water or first wash buffer（0.1M MES water Solution）In, ultrasonic wave（240W）Processing 2 minutes, repeats above procedure three times, adds carbodiimide（Purchased from Shanghai Jing Chun companies） 50mg, is stirred 15 minutes, so as to activate the fluorescent microsphere.

1.2nd, with the fluorescent microsphere labelled antibody activated：

The fluorescent microsphere activated is centrifuged 10 minutes under 12000rpm, supernatant is removed, sediment is distributed to 1ml couplings Buffer solution（The citrate buffer solution of 50mM N- hydroxy thiosuccinimides）In, ultrasonic wave（240W）Processing 2 minutes, is repeated Above procedure three times, obtains the buffer solution 1ml for being dispersed with fluorescent microsphere.According to 3 μ l antibody（10mg/ml）What/100 μ l had been activated The ratio of fluorescent microsphere, adds the PGI monoclonal antibodies (1) prepared by embodiment 2, stirs 2 hours at normal temperatures thereto, plus Enter 1ml Block buffers（1 (w/v) %BSA-0.05M monoethanolamines）, continue to stir 1 hour, afterwards, 10 centrifuged under 12000rpm Minute, sediment is distributed to the whole wash buffers of 500 μ l by repeated centrifugation 3 times（0.5 (w/v) %BSA-0.11 (v/v) % tween water Solution）In, ultrasonic wave（240W）Processing 2 minutes, 500 μ l are settled to above-mentioned whole wash buffer.

2nd, it is coated with pad

The PGI monoclonal antibodies (1) that the mark of above-mentioned preparation has are used into antibody diluent（1%(w/v)BSA- 0.01M PBS（pH7.2）Buffer solution）1mg/ml is diluted to, working solution is obtained, then uses micropipettor（It is purchased from Labsystems companies）By 4 μ l/cm amount even application on pad, afterwards with 37 DEG C of oven for drying, under 45% humidity Save backup.

2nd, the preparation of reaction film

The PGI monoclonal antibodies (2) and sheep anti-mouse igg monoclonal antibody prepared according to embodiment 2（Purchased from China fir gold in Beijing Bridge company）1mg/ml is diluted to respectively with 50mM pH7.2 PBS, by metal spraying machine（Purchased from Hangzhou Feng Hang companies）Inspection Survey line and nature controlling line spacing parameter are set to 6mm, package amount are respectively set into 1.0 μ l/cm, with metal spraying machine in nitrocellulose Upper PGI monoclonal antibodies (2) and sheep anti-mouse igg monoclonal antibody are drawn on film, normal temperature dries standby.

3rd, the assembling and cutting of test paper

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

4th, the preparation of PGI fluorescence immunoassays detection card：

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

5th, the detection of sample

PGI standard items（Purchased from Shanghai osmanthus Kanggong department）Use sample diluting liquid（1%(w/v)BSA-0.01M PBS（pH7.2）It is slow Fliud flushing）It is configured to the calibration object of following series concentration：400ng/ml、200ng/ml、100ng/ml、50ng/ml、20ng/ml、 10ng/ml, 0ng/ml, 50 μ l above calibration objects are added drop-wise on well respectively, fluorescence detector is used after 10 minutes（Purchased from depth Zhen Shi peace all living creatures's things Engineering Co., Ltd, model AQ-3000）Detection, can collect fluorescence in detection line and Quality Control line position. Using sample concentration as abscissa, the ratio of detection line and the fluorescence intensity at nature controlling line is ordinate drafting calibration curve, R²For 0.996.Nature controlling line is used for test paper Effective judgement and makees corresponding correction to detection line signal, and such as nature controlling line does not occur band, Then illustrate that test paper fails.

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

6th, PGI fluorescence immunoassays test paper performance evaluation

1. evaluate the index of test paper performance

1）The range of linearity：Each concentration calibration product repeat detection 3 times, draw calibration curve, through data fitting and statistical analysis, Test paper linear detection range of the present invention is 5ng/ml-400ng/ml.

2）Minimum detectability：By PGI null value blood samples（Without PGI compositions）（Purchased from Shenzhen Central Plains company）It is divided into 20 parts to be examined Survey, calculate mean concentration and 2 times of standard deviation sums, obtain test paper lowest detection of the present invention and be limited to 3.1ng/ml.

3）Precision：With the present invention PGI fluorescence immunoassays test paper detect respectively PGI concentration be respectively 300ng/ml, 100ng/ml, 30ng/ml blood sample, repeat detection 10 times, carry out withinrun precision measure.Daily to the sample of above-mentioned 3 concentration Originally it is measured, 1 day 1 time, it is continuous to survey 20 days, betweenrun precision measure is carried out, it is as a result as shown in table 3 below：

Table 3

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

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

Claims (11)

1. a kind of people PGI epitope peptides, wherein the amino acid sequence of the PGI epitope peptides is one of both：

(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。

2. a kind of PGI antigens, it is by making the people PGI epitope peptides (1) described in claim 1 and carrier protein couplet system Standby.

3. a kind of PGI antigens, it is by making the people PGI epitope peptides (2) described in claim 1 and carrier protein couplet system Standby.

4. a kind of people PGI antibody, it is the monoclonal antibody or polyclonal being prepared from by the PGI antigens described in claim 2 Antibody.

5. a kind of people PGI antibody, it is the monoclonal antibody or polyclonal being prepared from by the PGI antigens described in claim 3 Antibody.

6. purposes of the people PGI antibody on people's PGI external diagnosis reagent cases are prepared according to claim 4 or 5.

7. a kind of people PGI external diagnosis reagent cases, it is used as coated antibody comprising people's PGI antibody described in claim 4 or 5.

8. people PGI external diagnosis reagent cases according to claim 7, wherein the kit also includes binding antibody, institute It is people's PGI antibody described in claim 4 or 5 to state binding antibody, and when the binding antibody is anti-from described people PGI When one of former epitope peptide (1) and (2), the coated antibody is derived from described people PGI epitope peptides (1) and (2) Another one.

9. people PGI external diagnosis reagent cases according to claim 8, wherein the kit also includes the second of enzyme mark Antibody.

10. people PGI external diagnosis reagent cases according to claim 7, wherein the coated antibody is monoclonal antibody.

11. people PGI external diagnosis reagent cases according to claim 8, wherein the binding antibody is polyclonal antibody.