CN107271669A - Propepsin, helicobacter pylori antibody and G17 detection method and its kit - Google Patents
Propepsin, helicobacter pylori antibody and G17 detection method and its kit Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The present invention proposes the purposes of kit, kit in detection pepsinogen Cgene, PGⅡ, helicobacter pylori antibody and G17 and the method using kit detection pepsinogen Cgene, PGⅡ, helicobacter pylori antibody and G17.Kit includes：First coated film；With the second coated film；At least one piece region in first coated film is coated with the G17 antibody of the pepsinogen Cgene antibody of fluorescent microsphere mark, the PGⅡ antibody of fluorescent microsphere mark, the first Heliobacter pylori antigen of fluorescent microsphere mark and fluorescent microsphere mark, second coated film includes first area, second area, the 3rd region, the 4th region and the 5th region of separation, forms the material of the fluorescent microsphere and includes：Polystyrylmethyl methyl acrylate copolymer.There is sensitivity height, high specificity using the kit and method of the present invention, quick, simplicity, the measure that objectifies can be achieved.
Description
Technical field
The present invention relates to biomedicine field.In particular it relates to propepsin, helicobacter pylori antibody and
G17 detection method and its kit.More particularly it relates to kit, kit detection pepsinogen Cgene,
Purposes in PGⅡ, helicobacter pylori antibody and G17 and using kit detection pepsinogen Cgene,
The method of PGⅡ, helicobacter pylori antibody and G17.
Background technology
Propepsin (PG) is the precursor of pepsin, and two Asias are divided into according to its biochemical property and immunogenicity
Group.The identical title PG I of immunogenicity of 1-5 components, is mainly secreted, component 6-7 claim PG by the chief cell and mucus neck cell of gastric gland
II, by the cell secretes for secreting acid gland of the stomach bottom mucous membrane of body of stomach, secrete the pylorus of mucus neck cell, cardiac gland and the antrum of acid gland
The mucilage cell of gland and the Brunner glands of duodenum epimere can also produce PG II, and prostate and pancreas also produce a small amount of PG
Ⅱ.About 1% PG enters blood circulation under normal circumstances, and the amount of entrance is sufficiently stable, therefore the reflection stomach lining gland of serum PG I, II
The secreting function of the quantity of body and cell, also indirect reaction stomach lining different parts.When pathological change occurs for stomach lining, serum
PG contents also change therewith, and the ratio of simultaneous determination Serum Pepsinogen I and II is referred to as " the serology biopsy " of stomach lining.
Helicobacter pylori (H.pylori) is generally acknowledged stomach cancer pathogenic bacteria, and H.pylori infection makes stomach lining undergo inflammation, atrophy, on intestines
Skin metaplasia, dysplasia, and then develop into cancer.Can be effectively to suffering from according to serum PG detection and H.pylori antibody test results
The stomach cancer risk of person is estimated.The main G cells by antrum of gastrin (Gastrin) are secreted, and are directly entered people
Blood circulation, gastrin in circulation mainly include composition, gastrin 14, G17,34 4 kinds of bioactivity of gastrin into
Point.Serum gastrin 17 and 34 accounts for more than the 90% of blood circulation gastrin.Serum gastrin 17 can react gastric mucosal cell function
State, the level of serum gastrin 17 can as atrophic antrum gastritis blood serum designated object.
The detection method of current common Serum Pepsinogen, G17 and helicobacter pylori antibody mainly has：Breast
Glue enhancing immunoturbidimetry, ELISA, Timed resolved fluoroimmunoassay, colloidal gold immunity chromatography etc..Latex increases
Strong immunoturbidimetry, ELISA, Timed resolved fluoroimmunoassay, operating process is complicated, it is necessary to which big measuring appratus is with setting
Standby and professional operation, detection time is longer, and detection sensitivity is high.Colloidal gold immunity chromatography is simple to operate, but sensitivity
It is not high, it is impossible to accurate quantification.
The content of the invention
It is contemplated that at least solving one of above-mentioned technical problem to a certain extent or providing at a kind of useful business
Industry is selected.
Therefore, in one aspect of the invention, the present invention proposes a kind of kit.Embodiments in accordance with the present invention, it is described
Kit includes：First coated film；With the second coated film, one end of first coated film and one end of second coated film
It is connected；It is micro- that at least one piece region in first coated film is coated with the pepsinogen Cgene antibody of fluorescent microsphere mark, fluorescence
The stomach that PGⅡ antibody, the first Heliobacter pylori antigen of fluorescent microsphere mark and the fluorescent microsphere of ball mark are marked
The antibody of secretin 17, first area of second coated film comprising separation, second area, the 3rd region, the 4th region and the 5th
Region, the first area, the second area, the 3rd region and the 5th region described in the 4th region ratio are close to the described first bag
Envelope, the first area is coated with former I antibody of antipepsin, and the second area is coated with antipepsin original II and resisted
Body, the 3rd region is coated with the second Heliobacter pylori antigen, and the 4th region is coated with the antibody of anti-gastrin 17, described
5th region is coated with antiantibody, and the antiantibody specifically binds the pepsinogen Cgene antibody, the PGⅡ
Antibody, the first Heliobacter pylori antigen and G17 antibody, forming the material of the fluorescent microsphere includes：Polystyrene-first
Base methyl acrylate copolymer.
There is the more difficult control of synthesis condition, coating in traditional fluorescent microsphere material, mostly single styrene polymer
The shortcomings of fluorescent yield is low, microspherulite diameter heterogeneity, hydrophily are poor.And use styrene-methylmethacrylate copolymer
Prepared fluorescent microsphere can obtain higher fluorescent yield, and particle diameter is more homogeneous, good hydrophilic property, for biomarker effect more
It is good.
It should be noted that the present invention is for the pepsinogen Cgene for being coated with fluorescent microsphere mark in the first coated film
Antibody, the PGⅡ antibody of fluorescent microsphere mark, the first Heliobacter pylori antigen of fluorescent microsphere mark and fluorescence are micro-
The size in the region of the G17 antibody of ball mark is not restricted, and can be whole first coated film or the first bag
A part for envelope, in one embodiment of the invention, the first coated film are referred to as sample pad.Likewise, being coated with to second
First area, second area, the 3rd region, the 4th region and the 5th respective size in region of separation in film are not made especially yet
Limitation, if this five regions any two or it is wantonly three between do not have connect or overlapping region.In addition, first area,
Second area, the 3rd region and four-range distribution are not restricted, can be with cross direction profiles, can also genesis analysis.5th area
Domain, which can be further divided into multiple regions, each region, can be coated with antiantibody, as long as ensureing along the first coated film to second
Coated film direction, be coated with the region of antiantibody separately with first area, second area, the 3rd region and the 4th region
It is parallel.
In one embodiment of the invention, as shown in figure 1, the first coated film 100 and the second coated film 200 of kit
All it is rectangular, a broadside (" broadside " is the shorter one side of the length of side) for the first coated film and one of the second coated film
Broadside (" broadside " is the shorter one side of the length of side) is mutually glued, and fluorescent microsphere mark propepsin is coated with the first coated film
The region of I antibody, be coated with fluorescent microsphere mark PGⅡ antibody region, be coated with fluorescent microsphere mark first imprison
Helicobacter pylori antigen and be coated with fluorescent microsphere mark G17 antibody region be about 2cm, width about 3mm it is rectangular
First area 210, second area 220, the 3rd region 230, the 4th region 240 and the 5th region 250 in shape, the second coated film
It is wire, the region that first area endoperidium has former I antibody of antipepsin is referred to as detection line 1 or T1 lines, second area
The region that endoperidium has former II antibody of antipepsin is referred to as detection line 2 or T2 lines, and the 3rd region is coated with the second pylorus spiral shell
The region of bacteroides antigen is referred to as detection line 3 or T3 lines, and the region that the 4th region is coated with G17 antibody is referred to as detection
Line 4 or T4 lines, the region that the 5th region endoperidium has antiantibody are referred to as nature controlling line or C lines, detection line 1, detection line 2, Quality Control
Wire plane where line 1, detection line 3, detection line 4, nature controlling line 2 is parallel to the bonding side of two coated films, described wire
Width be about 0.5~1mm, the width of a length of place film.According to another embodiment of the invention, kit of the invention
There can be the structure shown in Fig. 2.
This lateral chromatography detection reagent of the present invention is coated with respectively using the first coated film and the second coated film can be special
The opposite sex combines the coated antibody/antigen and fluorescent labeled antibody/antigen of same antigen or antibody, adds after testing sample and utilizes
Film layer analyse, on the second coated film formed double antibody/antigen sandwich compound, based on detection compound in fluorescence intensity and
Standard concentration curve carrys out the concentration of contained antigen/antibody in determination sample.This lateral chromatography detection reagent of the present invention can
The obvious specificity for improving detection, the time required to shortening detection, can significantly improve detection sensitivity.
According to one embodiment of present invention, the material further comprises at least one following：Quantum dot；And rare earth
Complex compound.Inventor has found by many experiments, and quantum dot and/or rare-earth complex excite lower launched glimmering in ultraviolet source
Light long lifespan, is difficult bleaching, and fluorescence intensity can effectively quantify detection.According to a preferred embodiment of the invention, the quantum
Point is CdSe/ZnS, and the rare-earth complex is Eu (TTA)3Phen。
According to one embodiment of present invention, the fluorescent microsphere has functionalized surface.Thereby, it is possible to cause antibody knot
Close on fluorescent microsphere.
According to one embodiment of present invention, the fluorescent microsphere is obtained in the following manner：By styrene and first
Base methacrylate is according to 1:1 mass ratio is mixed, then adds into resulting mixture 1 volume % rare-earth complexs
Eu(TTA)3Phen or 0.5 volume %CdSe/ZnS quantum dots, ultrasound are mixed, and obtain a liquid；0.05 volume % carboxylated is gathered
Vinyl alcohol and 0.05 volume % sodium acid carbonates are dissolved in water, obtain b liquid；The a liquid is added in the b liquid, ultrasound is after 15 minutes,
Nitrogen is passed through into resulting mixed liquor 30 minutes, be stirred simultaneously, be heated to 80 DEG C, and add into the mixed liquor
Enter 0.01~0.1 volume % potassium peroxydisulfates, react 12 hours, to obtain the fluorescent microsphere.Thereby, it is possible to obtain with work(
The fluorescent microsphere on surface can be changed, and detection sensitivity and accuracy can be effectively improved using the fluorescent microsphere.
According to one embodiment of present invention, the particle diameter of the fluorescent microsphere is 50~500nm, preferably 100~250nm.
Thus, when being detected for lateral chromatography, with higher sensitivity and homogeneity, and detection can effectively be quantified.
According to one embodiment of present invention, the other end of second coated film is connected with adsorptive pads.Adsorptive pads can be
Strong absorbent material, thus can give directed forces when detecting liquid sample, make liquid sample from the first coating membrane orienting
Chromatograph to the second coated film.
According to one embodiment of present invention, first coated film, second coated film and the adsorptive pads are fixed
On same solid-phase matrix.Solid-phase matrix predominantly has individual carrying, convenient operation, the type of solid-phase matrix when using coated film
It is not particularly limited, can is the inert material that will not be reacted with testing sample or not influence antigen-antibody to be combined, than
Such as cardboard, plastic plate.
According to one embodiment of present invention, first coated film is the plain film of glass fibre, and second coated film is
Nitrocellulose filter.Glass fibre membrane is in chemical inertness, without adhesive, is fabricated by using 100% pyrex fiber,
Be coated with thereon fluorescent microsphere mark pepsinogen Cgene antibody, PGⅡ antibody, the first Heliobacter pylori antigen and
G17 antibody, specifically binds beneficial to antibody/antigen with target antigen/antibody in testing sample.And NC film sheets
Body is to have with the addition of surfactant to improve hydrophilic ability, and there are certain buffer system, with capillary
Fibre structure, can adsorb moisture more more than equal cellulosic filter paper, and flow velocity is fast, high temperature resistant, beneficial to coated anti-stomach egg thereon
Former II antibody of the white antibody of proenzyme I, antipepsin, the second Heliobacter pylori antigen and the antibody of anti-gastrin 17 respectively with it is foregoing
Pepsinogen Cgene antibody-antigene, the PGⅡ antibody-antigene marked with fluorescent microsphere, the band marked with fluorescent microsphere
The first Heliobacter pylori antigen-helicobacter pylori antibody of fluorescent microsphere mark and the G17 marked with fluorescent microsphere resist
Specific binding reaction occurs for body-antigen, excites fluorescence.
According to one embodiment of present invention, the pepsinogen Cgene antibody, PGⅡ antibody and the first pylorus
Pylori antigen is combined with the fluorescent microsphere by covalent peptide bonds respectively.
According to one embodiment of present invention, pepsinogen Cgene antibody and former I antibody of antipepsin can specificity
With reference to antigen pepsinogen Cgene, and it is preferred that two kinds of antibody can determine group-specific with the different surfaces of pepsinogen Cgene
With reference to；Former II antibody of PGⅡ antibody and antipepsin can molecule of the antigen binding PGⅡ, and
And it is preferred that two kinds of antibody can be specifically bound with the different surfaces determinant of PGⅡ；First helicobacter pylori and
Second helicobacter pylori can specifically combine helicobacter pylori antibody, and it is preferred that both antigens have not
Same antigenic determinant；G17 antibody and the antibody of anti-gastrin 17 can molecule of the antigen binding G17, and
It is preferred that two kinds of antibody can be specifically bound with the different surfaces determinant of G17.So it is beneficial to pepsinogen Cgene, stomach
The accurate progress of pepsinogen II, helicobacter pylori antibody and G17 detection.The kit of the present invention is based on to glimmering
The research of light microballoon mark, antigen and antibody characteristic, is carried out by the suitable fluorescent microsphere mark of selection with specific antibody
Covalent chemical coupling is oriented, the analysis of fluorescent microsphere labelled antibody is obtained, and by optimizing the various of double-antibody sandwich immune response
Condition, prepares above-mentioned detection pepsinogen Cgene, PGⅡ, helicobacter pylori antibody and the stomach of can be used in and secretes
The kit of element 17.
According to one embodiment of present invention, the antiantibody is sheep anti-mouse igg antibody, and it can be with pepsinogen Cgene
Antibody, PGⅡ antibody, the first Heliobacter pylori antigen and G17 antibody specificity are combined, i.e., with it is unnecessary glimmering
Light microballoon mark pepsinogen Cgene antibody, fluorescent microsphere mark PGⅡ antibody, fluorescent microsphere mark the first pylorus spiral shell
Bacteroides antigen and fluorescent microsphere mark G17 antibody binding form immune complex, excite fluorescence, be able to can it is qualitative and
Quantitatively detect the immune complex.
In another aspect of this invention, the present invention proposes a kind of kit described above in detection propepsin
Ith, the purposes in PGⅡ, helicobacter pylori antibody and G17.Above for the advantage described by kit
And technical characteristic, the purposes of the kit is still applied to, be will not be repeated here.
In still another aspect of the invention, the present invention proposes one kind and detects pepsin using kit described above
Former I, the method for PGⅡ, helicobacter pylori antibody and G17, embodiments in accordance with the present invention, methods described
Including：Sample to be tested is added to the stomach egg of the pepsinogen Cgene antibody for being coated with fluorescent microsphere mark, fluorescent microsphere mark
The region of the G17 of the white antibody of proenzyme II, the first Heliobacter pylori antigen of fluorescent microsphere mark and fluorescent microsphere mark；
The fluorescence intensity in the first area, second area, the 3rd region, the 4th region and the 5th region is detected, first area is obtained
Fluorescence intensity, second area fluorescence intensity, the 3rd region fluorescence intensity, the 4th region fluorescence intensity and the 5th region fluorescence are strong
Degree；Based on the first ratio and its corresponding ratio-concentration standard curve, determine that the pepsinogen Cgene in the sample to be tested is dense
Degree, based on the second ratio and its corresponding ratio-concentration standard curve, determines that the PGⅡ in the sample to be tested is dense
Degree, based on the 3rd ratio and its corresponding ratio-concentration standard curve, determines that the anti-helicobacter pylori in the sample to be tested resists
Bulk concentration, based on the 4th ratio and its corresponding ratio-concentration standard curve, determines that the G17 in the sample to be tested is dense
Degree, wherein, the region fluorescence intensity of first ratio=first area fluorescence intensity/the 5th, the area of second ratio=second
The region fluorescence intensity of domain fluorescence intensity/the 5th, the region fluorescence intensity of the region fluorescence intensity of the 3rd ratio=the 3rd/the 5th,
The region fluorescence intensity of the region fluorescence intensity of 4th ratio=the 4th/the 5th.
Fluorescent microsphere mark pepsinogen Cgene antibody, fluorescent microsphere mark pepsin are coated with first coated film
The region of former II antibody, the Heliobacter pylori antigen of fluorescent microsphere mark first and fluorescent microsphere mark G17 antibody is with treating
The region that target antigen/antibody (if there is antigen/antibody in sample to be tested) in test sample sheet is combined, in the reality of the present invention
Apply in example, the region is referred to as to be loaded end.Based on ratio and concentration standard curve formula, the stomach egg in sample to be tested is quantitative determined
White proenzyme I, PGⅡ, helicobacter pylori antibody and G17 concentration.In one embodiment of the invention, lead to
Hand-held instrument fluorescence intensity is crossed, first area fluorescence intensity, second area fluorescence intensity, the 3rd region fluorescence intensity is obtained
With the ratio of the 4th region fluorescence intensity respectively with the 5th region fluorescence intensity, four ratios are substituted into corresponding detection respectively
In value-concentration standard curve, testing result is obtained so as to objective.
Using the kit and detection method of the present invention, it can realize to pepsinogen Cgene, PGⅡ, resist and imprison
Helicobacter pylori antibody and G17 carry out quick and Sensitive Determination, can with sensitivity height, high specificity, quick, simplicity
Realize the advantage for the measure that objectifies.Utilize this kit and method detection pepsinogen Cgene, the propepsin of the present invention
IIth, helicobacter pylori antibody and G17, the sensitivity to pepsinogen Cgene reach 0.5ng/mL, to PGⅡ
Sensitivity reach 0.5ng/mL, 0.5ng/mL is reached to the sensitivity of helicobacter pylori antibody, the sensitivity to G17 reaches
1ng/L。
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from description of the accompanying drawings below to embodiment is combined
Substantially and be readily appreciated that, wherein：
Fig. 1 shows the structural representation of kit according to an embodiment of the invention；
Fig. 2 shows the structural representation of kit in accordance with another embodiment of the present invention；And
Fig. 3 shows canonical plotting according to an embodiment of the invention.
Embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Pepsinogen Cgene to be marked used, PGⅡ, G17 antibody are available from elder brother in following embodiments
The numbering at the bright big monoclonal antibody center of cloud is P1-12, P2-14, G17-11 antipepsin original I, PGⅡ, G17
Monoclonal antibody, Heliobacter pylori antigen to be marked used is the helicobacter pylori recombinant antigen that numbering is HP01.
Pepsinogen Cgene, PGⅡ in following embodiments, that G17 coated antibody is available from Kunming cloud is big
The numbering at monoclonal antibody center is P1-13, P2-15, G17-12 antipepsin original I, PGⅡ, the monoclonal of G17
Antibody, coating Heliobacter pylori antigen used is the helicobacter pylori recombinant antigen that numbering is HP02..
Be used to make the glass fibre membrane of sample pad in following embodiments be purchased from Millipore companies, cat. no
GF-CP20300。
It is CF- to be used to make the adsorptive pads of adsorptive pads purchased from Millipore companies, cat. no in following embodiments
SP22300。
The nitrocellulose filter for being used to make coated film in following embodiments is purchased from Millipore companies, cat. no
For Hi-Flow Plus HF135.
PH in following embodiments is prepared as follows for 7.4 0.02M PBSs：Weigh 2.3g
Na2HPO4、0.524g NaH2PO4.H2O, 8.77g NaCl are dissolved in pure water, and 1L is settled to pure water, adjust pH to 7.4, obtaining pH is
7.4 0.02M PBSs.
Film process buffer solution in following embodiments is prepared as follows：Tween-20, BSA, sucrose are dissolved in
The weight/mass percentage composition that stating the 0.02M PBSs that pH is 7.4 makes Tween-20 weight/mass percentage composition be 0.2%, BSA is
1%th, the weight/mass percentage composition of sucrose is 2%, adjusts pH to 7.4, obtains film process buffer solution.
50mM pH in following embodiments are prepared as follows for 8.5 borate buffer solution：Weigh 1.9g
Na2B4O7.10H2O is dissolved in 100ml pure water, adjusts pH to 8.5 to obtain the borate buffer solution that 50mM pH are 8.5.
Sample treatment liquid in following embodiments is prepared as follows：It is 7.4 that Tween-20 is dissolved in into above-mentioned pH
0.02M PBSs make Tween-20 weight/mass percentage composition be 0.2%, adjust pH to 7.4, obtain sample treatment liquid.
Detect pepsinogen Cgene, PGⅡ, G17 and the detection examination of helicobacter pylori antibody lateral chromatography
The method that is typically prepared of agent comprises the following steps：
(1) preparation of the fluorescent microsphere label probe of carboxyl modified
Styrene and methymethacrylate are pressed 1:1 ratio mix, add 1% rare-earth complex Eu (TTA) 3Phen or
Person's 0.5%CdSe/ZnS quantum dots, ultrasound is mixed, and obtains a liquid.By 0.05% carboxylated polyethylene alcohol and 0.05% sodium acid carbonate
Water is dissolved in, b liquid is obtained.After a liquid is added into b liquid ultrasound 15 minutes, lead to nitrogen stirring deoxygenation in 30 minutes, be then heated to 80 degree.
Add 0.01-0.1% potassium peroxydisulfates to react 12 hours, obtain polymer fluorescent microspheres, it is clear through filtering, centrifugation and deionized water
Wash, the functional fluorescence microballoon purified.
After the functional fluorescence microballoon that upper step is obtained, the carboxyl for activating its surface, distinguished by the way of covalent coupling
Pepsinogen Cgene, PGⅡ, G17 labelled antibody and helicobacter pylori labelled antigen orientation are connected to the carboxylic
The fluorescent microsphere surface of base modification.
(2) at test section T lines and C lines antibody coating
Using spray film instrument, spray at pepsinogen Cgene coated antibody, T2 lines and spray at the T1 lines of coated film test section
PGⅡ coated antibody, sprays sheep anti-mouse igg antibody at C1 lines, and pylorus is sprayed at the T3 lines of coated film test section
Helicobacter envelope antigen, sprays G17 coated antibody at the T4 lines of coated film test section, and sheep anti mouse is sprayed at C2 lines
IgG antibody.
(3) at sample pad label probe coating
Using spraying apparatus, antipepsin original I, the stomach of fluorescent microsphere mark are sprayed respectively in sample pad specific location
The mixtures of antibodies of pepsinogen II, the helicobacter pylori labelled antigen of fluorescent microsphere mark and the mixing of G17 labelled antibody
Thing.The ad-hoc location is one piece of region in sample pad, and the region is as follow-up " sample-adding end ".
(4) assembled formation of lateral chromatography detection reagent
Structural diagrams according to lateral chromatography detection reagent are intended to Fig. 1, are pasted in the middle of plastic support backboard as test
The coated film in area, sample pad is pasted in T2 the or T4 line ends of coated film, and C1, C2 line end paste adsorptive pads.Using test paper cutting machine,
The paper slip for certain broadband is cut, and loads intermediate plate, is packed with the aluminium foil bag equipped with drier.
(5) formation of antigen-antibody fluorescent immune complex
Testing sample is added at the sample-adding end of the reaction plate of above-mentioned assembled formation, the pepsinogen Cgene in sample with it is glimmering
Chromatography arrives the pepsinogen Cgene coated antibody sprayed at T1 lines after the pepsinogen Cgene labelled antibody of light microballoon mark is combined,
Coated antibody-antigen-fluorescent microsphere labelled antibody immune complex, unnecessary fluorescent microsphere mark pepsin are formed at T1 lines
The fluorescent mark immunity compound of former I antibody then at C1 lines with sheep anti-mouse igg formation；PGⅡ in sample with it is glimmering
Chromatography arrives the PGⅡ coated antibody sprayed at T2 lines after the PGⅡ labelled antibody of light microballoon mark is combined,
Coated antibody-antigen-fluorescent microsphere labelled antibody immune complex, unnecessary fluorescent microsphere mark stomach cardia are formed at T2 lines
The fluorescent mark immunity compound of the antibody of proenzyme II then at C1 lines with sheep anti-mouse igg formation.Anti-helicobacter pylori in sample
Helicobacter pylori coating of the chromatography to spraying at T3 lines after antibody is combined with the helicobacter pylori labelled antigen that fluorescent microsphere is marked
Antigen, forms envelope antigen-antibody-fluorescent microballoon labelled antigen immune complex at T3 lines.G17 in sample with
Chromatography arrives the G17 coated antibody sprayed at T4 lines after the G17 labelled antibody of fluorescent microsphere mark is combined, in T4 lines
Place forms coated antibody-antigen-fluorescent microsphere labelled antibody immune complex, unnecessary fluorescent microsphere mark G17 antibody
The fluorescent mark immunity compound then formed at C2 lines with sheep anti-mouse igg.
(6) fluorescent mark immunity complex fluorescence intensity detection
Determined with fluorescence detector at T1 lines, at T2 lines, at T3 lines and at T4 lines, fluorescence intensity at C1 lines and at C2 lines,
Calculated by fluorescence intensity and standard concentration curve equation and obtain quantitative result, C lines measurement result is then as the assay method
Quality Control internal standard.
The fluorescence intensity of described fluorescent mark immunity compound, refers in T1 lines, T2 lines and C1 lines, T3 lines, T4 lines and
The quantity of combination fluorescent microsphere under being detained respectively at C2 lines numerical value resulting after being determined with fluorescence detector.By dual anti-
The condition of body/antigen sandwich immune response, draws standard concentration curve, and marked through a large amount of various criterion concentration samples that determine
Quasi- concentration curve equation, the concentration of detection sample is calculated with this standard concentration curve equation.C lines measurement result is then used as the survey
Determine the Quality Control internal standard of method.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
The preparation of the stomach Function detection lateral chromatography detection reagent of embodiment 1
(1) fluorescent microsphere mark pepsinogen Cgene, PGⅡ labelled antibody, helicobacter pylori labelled antigen and
The preparation of G17 labelled antibody
Styrene and methymethacrylate are pressed 1:1 ratio mix, add 1% rare-earth complex Eu (TTA) 3Phen or
Person's 0.5%CdSe/ZnS quantum dots, ultrasound is mixed, and obtains a liquid.By 0.05% carboxylated polyethylene alcohol and 0.05% sodium acid carbonate
Water is dissolved in, b liquid is obtained.After a liquid is added into b liquid ultrasound 15 minutes, lead to nitrogen stirring deoxygenation in 30 minutes, be then heated to 80 degree.
Add 0.01-0.1% potassium peroxydisulfates to react 12 hours, obtain polymer fluorescent microspheres, it is clear through filtering, centrifugation and deionized water
Wash, the functional fluorescence microballoon purified.
Take after the fluorescent microsphere of 20mg above-mentioned carboxyl modified washs and centrifuged with MES buffer solutions (0.1M, pH4.7), use
1ml MES buffer solutions (0.1M, pH4.7) are resuspended, and add 1- ethyls-(3- dimethylaminopropyls) carbodiimide (EDC) to end
Concentration is 5mM, adds NHS (N- hydroxysuccinimides) to final concentration of 10mM, and room temperature lucifuge, reaction half an hour is activated
The fluorescent microsphere of carboxyl modified afterwards.
The fluorescent microsphere of carboxyl modified after the activation is washed with 50mM pH8.5 borate buffer solution, is taken respectively on 0.37mg
State pepsinogen Cgene to be marked, PGⅡ antibody, G17 labelled antibody and helicobacter pylori labelled antigen with
The fluorescent microsphere of carboxyl modified, which is mixed into 50mM pH8.5 borate buffer solution, after the above-mentioned activation of 5mg fully mixes.Room temperature is kept away
Reacted 2 hours under light, allow antibody/antigen and fluorescent microsphere formed stable covalent peptide bonds combine obtain fluorescent microsphere and antibody/
The conjugate of antigen.After reaction terminates, the BSA solution of final concentration of 1% (weight/mass percentage composition) is added to fluorescent microsphere and is resisted
Residual activity carboxyl site is closed on the conjugate of body/antigen, and room temperature lucifuge is reacted 0.5 hour.After the completion of, use pH7.4
The washing of 0.02M PBSs, be resuspended and obtain 5mg/ml fluorescent microspheres mark pepsinogen Cgene, PGⅡ, stomach and secrete
Plain 17 labelled antibodies, helicobacter pylori labelled antigen liquid, 4 DEG C of preservations are stand-by.
(2) detection pepsinogen Cgene, PGⅡ, G17, the detection of helicobacter pylori antibody lateral chromatography
The preparation of reagent
With pepsinogen Cgene, PGⅡ, G17 coated antibody, helicobacter pylori envelope antigen, sheep anti mouse
IgG antibody prepares coated film, and specific method is as follows：
Using pH7.4 0.02M PBSs, by sheep anti-mouse igg antibody (Changsha Bo You bio tech ltd,
ABGAM-0500 concentration 1mg/ml solution) is formulated as, (Kunming cloud is big by pepsinogen Cgene and PGⅡ coated antibody
The numbering at monoclonal antibody center is P1-13, P2-15) concentration be formulated as concentration 2mg/ml solution respectively, G17 coating see and
Helicobacter pylori envelope antigen (numbering at the big monoclonal antibody center of Kunming cloud is G17-12, HP01) concentration is formulated as concentration 2mg/ respectively
Sheep anti-mouse igg antibody, the nature controlling line of coated film (nitrocellulose filter) is sprayed onto from BioDot XYZ3050 spray membranous systems by ml
(C1 lines, C2 lines) position, is sprayed onto detection line (T1 lines) position by pepsinogen Cgene coated antibody, PGⅡ is coated with
Antibody is sprayed onto detection line (T2 lines) position, helicobacter pylori envelope antigen is sprayed onto into detection line (T3 lines) position, by G17
Coated antibody is sprayed onto detection line (T4 lines) position, is done after relative humidity carries out dehumidifier 4 hours for less than 10% drying plant
It is dry stand-by, obtain the coated film with detection line and nature controlling line.
Soaked all-glass paper half an hour with above-mentioned film process buffer solution, the temperature of immersion is 37 DEG C, in same dehumidifier
After condition carries out dehumidifier 4 hours, pepsinogen Cgene is marked with fluorescent microsphere obtained by above-mentioned film process buffer solution dilution step (one)
It is with PGⅡ antibody liquid to fluorescent microsphere mark pepsinogen Cgene and PGⅡ antibody content
After 0.05mg/ml mixed liquors, above-mentioned treated glass fibre element is sprayed into using BioDot XYZ3050 spray membranous systems
Prepared on film and form sample pad, be dried in same dehumidifier condition；With above-mentioned film process buffer solution dilution step (one) institute
Fluorescent microsphere mark helicobacter pylori labelled antigen and G17 labelled antibody liquid to helicobacter pylori labelled antigen and
G17 labelled antibody content be 0.05mg/ml mixed liquors after, using BioDot XYZ3050 spray membranous system be sprayed into
Prepared on the above-mentioned plain film of treated glass fibre and form sample pad, be dried in same dehumidifier condition.It is clean at 100,000 grades
Above-mentioned dried coated film, above-mentioned sample pad, adsorptive pads, the back of the body with detection line and nature controlling line in net and dry workshop
Plate is carried out after collocation assembling as shown in Figure 1, uses BioDot CM4000 cutting systems by the Paperboard cutting posted for 4mm/ bars
Width, load detection it is stand-by with intermediate plate.
The assessment of the stomach Function detection lateral chromatography detection reagent of embodiment 2
(1) detection sensitivity
Using pepsinogen Cgene, PGⅡ, G17 antigen and helicobacter pylori antibody as testing sample
Determine detection pepsinogen Cgene, PGⅡ, G17 and the helicobacter pylori antibody lateral chromatography of embodiment 1
The sensitivity of detection reagent.
It is with the pH containing 5% calf serum by pepsinogen Cgene, PGⅡ antigen and helicobacter pylori antibody
7.4 0.02M PBSs be configured to respectively series concentration (0,0.5,1,5,10,50,100,500ng/mL), by gastrin
17 with containing 5% calf serum pH for 7.4 0.02M PBSs be configured to respectively series concentration (0,0.5,1,5,10,
50th, 100,500ng/L), it is separately added into the detection pepsinogen Cgene obtained by embodiment 1, PGⅡ antigen, stomach and secretes
In the sample-adding end of element 17 and helicobacter pylori antibody lateral chromatography detection reagent, and it is strong using fluorescence detector detection fluorescence
Degree.Detecting step：Detected sample is first recovered into room temperature (25 DEG C) before detection, takes the μ l of detected sample 60 to hang down with accurate pipettor
Directly it is slowly dropped into detection pepsinogen Cgene, PGⅡ antigen, G17 and anti-helicobacter pylorus that embodiment 1 is obtained
The sample-adding end of bacteria antibody lateral chromatography detection reagent, is tested after 10 minutes with fluorescence detector.
Its testing result is as shown in table 1 below.It can show that the detection pepsinogen Cgene of embodiment 1 resists from testing result
Former sensitivity is 0.5ng/mL, and the sensitivity of detection PGⅡ antigen is 0.5ng/mL, detects the spirit of G17
Sensitivity is 1ng/L, and the sensitivity of detection helicobacter pylori antibody is 0.5ng/mL.Calibration is made according to detected value and concentration bent
Line, detects the coefficient R 2=0.9981 of pepsinogen Cgene antigen, the range of linearity is 0-500ng/mL；Detect pepsin
The coefficient R 2=0.9996 of former II antigen, the range of linearity is 0-500ng/mL；Detect the coefficient correlation of G17 antigen
R2=0.9991, the range of linearity is 0-500ng/L；Detect the coefficient R of helicobacter pylori antibody2=0.9997, linear model
Enclose for 0-500ng/mL.Pepsinogen Cgene, PGⅡ, G17 and the inspection of helicobacter pylori antibody lateral chromatography
Test agent detected value and concentration curve are as shown in Figure 2 and Figure 3.
The lateral chromatography detection reagent detected value of the different sample concentrations of table 1
(2) accuracy is detected
Pepsinogen Cgene antigen (T1), PGⅡ antigen (T2), the anti-pylorus of 3 parts of various concentrations are chosen respectively
Pylori antibodies (T3), G17 antigen (T4) sample, duplicate measurements 10 times according to the method described in the present invention, according to 10 times
Result calculate batch in average deviation CV% values.The 3 batches of lateral chromatography detection reagents prepared according to the method described in the present invention, point
Not Xuan Qu 3 parts of various concentrations sample, duplicate measurements 10 times respectively, average deviation CV% values between being calculated batch according to result.It is examined
Survey result as shown in table 2 below.As a result show that the accuracy of the inventive method is higher.
Difference between batch is determined in 2 batches, table
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.Moreover, specific features, structure, material or the feature of description can be with office
Combined in an appropriate manner in one or more embodiments or example.In addition, in the case of not conflicting, the skill of this area
Art personnel can be tied the not be the same as Example or the feature of example and non-be the same as Example or example described in this specification
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Claims (10)
1. a kind of kit, it is characterised in that including：
First coated film；With
Second coated film, one end of first coated film is connected with one end of second coated film；
It is micro- that at least one piece region in first coated film is coated with the pepsinogen Cgene antibody of fluorescent microsphere mark, fluorescence
The stomach that PGⅡ antibody, the first Heliobacter pylori antigen of fluorescent microsphere mark and the fluorescent microsphere of ball mark are marked
The antibody of secretin 17,
Second coated film includes first area, second area, the 3rd region, the 4th region and the 5th region of separation, institute
First area, the second area, the 3rd region and the 5th region described in the 4th region ratio are stated close to first coated film,
The first area is coated with former I antibody of antipepsin,
The second area is coated with former II antibody of antipepsin,
3rd region is coated with the second Heliobacter pylori antigen,
4th region is coated with the antibody of anti-gastrin 17,
5th region is coated with antiantibody, and the antiantibody specifically binds the pepsinogen Cgene antibody, pepsin
Former II antibody, the first Heliobacter pylori antigen and G17 antibody,
Forming the material of the fluorescent microsphere includes：Polystyrene-methacrylic acid methyl terpolymer.
2. kit according to claim 1, it is characterised in that the material further comprises at least one following：
Quantum dot；And
Rare-earth complex,
Preferably, the quantum dot is CdSe/ZnS,
The rare-earth complex is Eu (TTA)3Phen。
3. kit according to claim 1, it is characterised in that the fluorescent microsphere has functionalized surface.
4. kit according to claim 1, it is characterised in that the fluorescent microsphere is obtained in the following manner：
By styrene and methymethacrylate according to 1:1 mass ratio is mixed, then adds 1 into resulting mixture
Volume % rare-earth complexs Eu (TTA)3Phen or 0.5 volume %CdSe/ZnS quantum dots, ultrasound are mixed, and obtain a liquid；
0.05 volume % carboxylated polyethylenes alcohol and 0.05 volume % sodium acid carbonates are dissolved in water, b liquid is obtained；
The a liquid is added in the b liquid, ultrasound is passed through nitrogen into resulting mixed liquor 30 minutes, simultaneously after 15 minutes
It is stirred, is heated to 80 DEG C, and 0.01~0.1 volume % potassium peroxydisulfates are added into the mixed liquor, reacts 12 hours,
To obtain the fluorescent microsphere.
5. kit according to claim 1, it is characterised in that the particle diameter of the fluorescent microsphere is 50~500nm, preferably
100~250nm.
6. kit according to claim 1, it is characterised in that the other end of second coated film is connected with water suction
Pad,
Optionally, first coated film, second coated film and the adsorptive pads are fixed on same solid-phase matrix,
Optionally, first coated film is the plain film of glass fibre, and second coated film is nitrocellulose filter.
7. kit according to claim 1, it is characterised in that the pepsinogen Cgene antibody, PGⅡ resist
Body, the first Heliobacter pylori antigen and G17 antibody are combined with the fluorescent microsphere by covalent peptide bonds respectively.
8. kit according to claim 1, it is characterised in that the antiantibody is sheep anti-mouse igg antibody.
9. any one of claim 1~8 kit is in detection pepsinogen Cgene, PGⅡ, anti-helicobacter pylori
Purposes in antibody and G17.
10. a kind of detect pepsinogen Cgene, PGⅡ using any one of claim 1~8 kit, resist and imprison
The method of Helicobacter pylori antibody and G17, it is characterised in that including：
Sample to be tested is added to the stomach egg of the pepsinogen Cgene antibody for being coated with fluorescent microsphere mark, fluorescent microsphere mark
The region of the G17 of the white antibody of proenzyme II, the first Heliobacter pylori antigen of fluorescent microsphere mark and fluorescent microsphere mark；
The fluorescence intensity in the first area, second area, the 3rd region, the 4th region and the 5th region is detected, first is obtained
Region fluorescence intensity, second area fluorescence intensity, the 3rd region fluorescence intensity, the 4th region fluorescence intensity and the 5th region fluorescence
Intensity；
Based on the first ratio and its corresponding ratio-concentration standard curve, determine that the pepsinogen Cgene in the sample to be tested is dense
Degree,
Based on the second ratio and its corresponding ratio-concentration standard curve, the PGⅡ in the sample to be tested is determined
Concentration,
Based on the 3rd ratio and its corresponding ratio-concentration standard curve, the anti-helicobacter pylori in the sample to be tested is determined
Antibody concentration,
Based on the 4th ratio and its corresponding ratio-concentration standard curve, the G17 concentration in the sample to be tested is determined,
Wherein,
The region fluorescence intensity of first ratio=first area fluorescence intensity/the 5th,
The region fluorescence intensity of second ratio=second area fluorescence intensity/the 5th,
The region fluorescence intensity of the region fluorescence intensity of 3rd ratio=the 3rd/the 5th,
The region fluorescence intensity of the region fluorescence intensity of 4th ratio=the 4th/the 5th.
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