CN101063684A - Chip and detecting reagent kit for detecting gastricism relevant indication marks object - Google Patents
Chip and detecting reagent kit for detecting gastricism relevant indication marks object Download PDFInfo
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- CN101063684A CN101063684A CN 200610025973 CN200610025973A CN101063684A CN 101063684 A CN101063684 A CN 101063684A CN 200610025973 CN200610025973 CN 200610025973 CN 200610025973 A CN200610025973 A CN 200610025973A CN 101063684 A CN101063684 A CN 101063684A
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Abstract
This invention discloses one protein chip, which comprises more than two kinds of micro ball fixed with different anti-gastritis label first antibody. This invention also discloses one method for test of the label. This invention also discloses one test agent case with protein chip. This invention protein chip has first antibody volume match design for accurate test.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of protein-chip and relevant detection kit that is used to detect the relevant mark of gastritis particularly.
Background technology
Chronic gastritis is common disease and frequently-occurring disease, and two types are generally arranged: the inflammatory disorders comparison sheet is shallow, is confined to gastric mucosa surface one deck (being no more than 1/3rd) person, is called chronic superficial gastritis; And inflammatory disorders involves the holostrome of gastric mucosa, and with gastric gland body atrophy person, then is atrophic gastritis.Gastroscope generaI investigation confirms, in the population of China incidence of disease of chronic gastritis up to more than 60%, atrophic gastritis account for wherein 20%.Atrophic gastritis by the chronic superficial gastritis development, is a kind of chronic progressive external pathology often.If atrophic gastritis can cause dementia, neurogenic disease, heart disease etc. not after diagnosing and treatment, increase the incidence probability of cancer of the stomach and gastric ulcer.Domestic document announcement, the cancer of the stomach incidence of atrophic gastritis is about 2%-7%.
The early stage means that atrophic gastritis is made a definite diagnosis are that gastrocopy and pathology are made a definite diagnosis.By gastroscope and pathologic finding, degree that can clear and definite atrophic gastritis and whether be in gastric precancerous lesion.Method by these three kinds of blood serum designated object diagnosis atrophic gastritiss of traditional E LISA methods analyst gastrin 17 (Gastrin-17), pepsinogen I (Pepsinogen I) and pepsinogen I I (Pepsinogen II) is also arranged at present, and testing result helps to diagnose whether patient is the position (stomach hole, body of stomach) that gastritis, atrophic gastritis and gastritis take place.This serological detection be a kind of alternative gastrocopy and pathological diagnosis be used for the method for the Noninvasive of initial diagnosis is carried out in stomachache and dyspeptic patient.Yet owing at present the detection of these three factors is finished by 3 ELISA kits, and three kinds of ELISA kits are all inequality to the pre-service of the requirement of sample collection and processing (whether fasting or give the high protein agent because of Gastrin-17 content in the serum is too low stimulate), specimen (56 ℃ water-baths or not), diluted sample degree (from diluting 2 times to 200 times), ELISA reaction time (from 2 hours to 4.5 hours) or the like, and background interference is very serious when detecting, and makes troubles to clinical diagnosis.
To sum up, still there are not at present both at home and abroad quick, the sensitive detection articles for use that can address the above problem and can detect simultaneously a plurality of marks relevant with stomach trouble.Therefore, this area presses for quick, the sensitive product that can detect a plurality of marks relevant with stomach trouble simultaneously of exploitation, so that clinical practice.
Summary of the invention
The purpose of this product just provides a kind of liquid phase protein matter chip that can detect a plurality of gastritis marks rapidly and sensitively simultaneously.
Another object of the present invention just provides the kit that contains described liquid phase protein matter chip.
In a first aspect of the present invention, a kind of protein-chip is provided, described protein-chip comprises (as the 2-100 kind) different microballoon more than 2 kinds, is fixed with different first antibodies on the described microballoon,
Wherein, described first antibody is the first antibody of gastritis mark, and described first antibody is selected from down group: the former I antibody of antipepsin, the former II antibody of antipepsin or anti-gastrin-17 antibody.
In another preference of the present invention, in the described chip, the former I antibody of antipepsin: the former II antibody of antipepsin: the mol ratio of anti-gastrin-17 antibody is (4-10): (4-12): (6-15), and when having described antibody.
In another preference of the present invention, described different microballoon is the microballoon with different microballoon identification signals.
In another preference of the present invention, described microballoon identification signal is a fluorescence signal.
In another preference of the present invention, in the described chip, the quantity of each microballoon is 10
3-10
7Individual; Preferable is 10
3-10
6Individual, better 10
3-10
5Individual.
In another preference of the present invention, the mean diameter of described microballoon is the 1-100 micron.
In another preference of the present invention, described chip contains three kinds of different microballoons, and is fixed with the former I antibody of antipepsin, the former II antibody of antipepsin and anti-gastrin-17 antibody on three kinds of microballoons respectively,
Wherein, the quantity of every kind of microballoon is 10
3-10
7Individual, and the quantity of each first antibody is as follows: the former I antibody of 0.04-0.10 microgram antipepsin, the former II antibody of 0.04-0.12 microgram antipepsin, 0.06-0.15 microgram anti-gastrin-17 antibody.
In another preference of the present invention, described first antibody also comprises: anti-CEA antibody, anti-CA19-9 antibody, anti-CA242 antibody, anti-CA724 antibody or anti-CA50 antibody.
In a second aspect of the present invention, the method for a kind of many gastritis mark parallel detection is provided, said method comprising the steps of:
(a) the described protein-chip of testing sample and claim 1 is mixed, thereby make gastritis mark in the testing sample and first antibody and microballoon form " gastritis mark-first antibody-microballoon " ternary complex;
(b) ternary complex that step (a) is formed mixes with second antibody solution, wherein said second antibody solution contains the second antibody that has detectable signal not of the same race, the anti-respectively a kind of gastritis mark of each second antibody and corresponding to corresponding first antibody in the protein-chip, and corresponding second antibody is 1 with the mol ratio that first antibody can be incorporated into this gastritis mark and first antibody and second antibody simultaneously: 0.1-1: 2, thus formation " second antibody-gastritis mark-first antibody-microballoon " tetraplex;
(c) detect the microballoon identification signal of different microballoons in the tetraplex, thereby whether and the amount that exists the existence of determining each gastritis mark in the detected sample;
Subsidiary condition are testing samples and the mixing of protein-chip and second antibody solution, and can carry out successively, also can carry out simultaneously.
In another preference of the present invention, described detectable signal is a phycoerythrin.
In another preference of the present invention, also comprise step: (d) detectable signal of measuring is compared with contrast or typical curve, thereby the existence of each gastritis mark is whether in definite detected sample, and/or quantity.
In a third aspect of the present invention, a kind of kit that is used to detect many gastritis mark is provided, it comprises following component:
(1) first container, and be loaded on first antibody solution in this container, contain the described protein-chip of claim 1 in the described first antibody solution.
In another preference of the present invention, also comprise in the described kit:
(2) second containers, and be loaded on second antibody solution in this container, wherein said second antibody solution contains the second antibody that has detectable signal not of the same race, the anti-respectively a kind of gastritis mark of described each second antibody and corresponding to corresponding first antibody in the first antibody solution, and the mol ratio that corresponding second antibody and first antibody can be incorporated into this gastritis mark and first antibody and second antibody simultaneously is 1: 0.1-1: 2.
In another preference of the present invention, also comprise in the described kit:
(3) standard items or reference substance.
In another preference of the present invention, comprise in the described kit:
(1 ') container a, and be loaded on the described liquid phase protein matter of claim 1 chip in the described container;
(2 ') container b, and be loaded on the second antibody not of the same race that has detectable signal in the described container, the anti-respectively a kind of gastritis mark of each second antibody and corresponding to corresponding first antibody in the liquid phase protein matter chip;
(3 ') container c, and be loaded on the interior hybridization solution of container (as 10.1mM Na
2HPO
4, 138mM NaCl, 1.76mM KH
2PO
4, 2.7mM KCl, 0.05% (V/V) Tween-20, pH7.4);
(4 ') container d, and after being loaded on hybridization in the container washing lotion (as 10.1mM Na
2HPO
4, 138mMNaCl, 1.76mM KH
2PO
4, 2.7mM KCl, 0.02% (m/V) Timerosal, 1% (m/V) BSA, pH7.4);
(5 ') container e, and be loaded on the interior detection material corresponding to detectable signal (as fluorescent dye) of container;
(6 ') negative control and positive control.
In a fourth aspect of the present invention, the purposes of described protein-chip is provided, be used to prepare the kit of diagnosis or auxiliary diagnosis gastritis; Or be used for preparing the kit whether the vitro detection sample exists the gastritis mark.
In another preference of the present invention, described detection is a detection by quantitative.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the synoptic diagram of the microballoon that is fixed with first antibody, wherein, 25 is first microballoon, 45 is to be connected with microballoon 25 and first kind of fixing first antibody molecule, 27 is second microballoon, 47 is to be connected with microballoon 27 and second kind of fixing first antibody molecule, and first microballoon 25 self has diacritic different fluorescence signal with second microballoon 27.
Fig. 2 has shown the synoptic diagram that adopts protein-chip of the present invention to detect.Wherein, 1 representative has the microballoon of first antibody, and 2 represent the gastritis mark, and 3 represent second antibody, and 4 represent detectable signal.
Fig. 3 is the typical curve of G-17.
Embodiment
The inventor is through extensive studies, to be separately fixed on the different microballoons (Beads) at the first antibody of multiple gastritis mark, make protein-chip, and the inventor designs the amount of gastritis mark antibody in protein-chip according to the concentration difference of various gastritis marks in serum, thereby improved the detected level and the accuracy rate of gastricism relevant indication marks object greatly, reduced the generation of background signal to greatest extent.Finished the present invention based on this.
As used herein, term " gastritis mark " is meant in the generation and progression of gastritis, produced by body, is present in the body fluid (as serum), and reflection gastritis exists and a class material of progress.Representational gastritis mark comprises (but being not limited to): gastrin 17 (G-17), pepsinogen I (PG I), pepsinogen I I (PG II).
As used herein, " capture antibody ", " coated antibody ", " first antibody ", be used interchangeably with " one is anti-", all be meant for fixing to antibody on the microballoon, the anti-a kind of corresponding gastritis mark of specificity, as anti-gastrin 17 antibody, antipepsin I antibody and the former II antibody of antipepsin.
As used herein, " detection antibody ", " second antibody ", be used interchangeably with " two anti-", all be meant the anti-a kind of corresponding gastritis mark of specificity and corresponding to the antibody of corresponding first antibody in the liquid phase protein matter chip.For for a kind of gastritis mark, corresponding first antibody is different with second antibody, and can be incorporated into the different epi-positions of described gastritis mark simultaneously.
As used herein, described " microballoon identification signal " is meant and is positioned on the microballoon, be used to distinguish the identification signal of different first antibodies.For convenience's sake, for the microballoon that is fixed with a kind of first antibody, the microballoon identification signal is preferably identical.Preferably, described microballoon identification signal is a fluorescence signal, and for being fixed with the not microballoon of first antibody of the same race, the color of fluorescence is different, and for the microballoon that is fixed with first antibody of the same race, the color of fluorescence is preferably identical.
As used herein, described " detectable signal " is meant to combine with biotin on the second antibody and is used to the signal that shows that second antibody combines with corresponding gastritis mark.In optimal way of the present invention, described detectable signal is streptavidin-phycoerythrin, and it combines with the biotin of mark on the antibody, produces fluorescence signal by laser excitation.
Ultimate principle
Ultimate principle of the present invention is the double antibodies sandwich method.Conventional way is with an anti-carrier that is fixed in, and an anti-and antigen-reactive resists reaction with two of tape label after the washing more then, and chemiluminescence or enzyme connection chromogenic reaction detection signal is carried out in washing at last.
Carrying out improved to above-mentioned double antibodies sandwich method is with described one anti-being fixed on the microballoon that carries specific detectable signal, preparation liquid phase protein matter chip, by the principle that adopts liquid phase protein matter chip to detect be: make single microballoon by sense channel, and use two kinds of laser simultaneously microballoon identification signal and detectable signal on the microballoon to be detected.A kind of laser excitation be microballoon identification signal on the microballoon, according to the identification signal not of the same race on the microballoon, microballoon can be classified, thereby the association reaction that each is different makes a distinction.Another kind of laser excitation be detectable signal, purpose is to determine the quantity of the detectable signal of combination on the microballoon, thereby determines the quantity of the molecules of interest of combination on the microballoon.Therefore, detect in the time of by two kinds of laser, can determine the kind and the quantity of combined detection thing.
Protein-chip
Described protein-chip comprises (as the 2-50 kind) different microballoon more than 2 kinds, is fixed with different first antibodies on the described microballoon,
Wherein, described first antibody is the first antibody of gastritis mark, and described first antibody is selected from down group: the former I antibody of antipepsin, the former II antibody of antipepsin or anti-gastrin-17 antibody.
Among the present invention, the DOP detailed operating procedure that first antibody is fixed on the microballoon can be used conventional method, for example according to the product description or the website of Luminex company: the method described in the www.luminexcorp.com is carried out coupled, thereby obtains different microballoons and the corresponding one anti-coupled thing that forms.
Described different microballoon is the microballoon with different microballoon identification signals, owing to have different microballoon identification signals, makes between the different microballoons and can be distinguished.In optimal way of the present invention, described microballoon identification signal is a fluorescence signal, and the fluorescence signal on the microballoon is different because of the difference that is fixed in the corresponding first antibody on the microballoon.
In the protein-chip of the present invention, described microballoon is made for adopting materials such as polystyrene, Polyvinylchloride, tygon.And the mean diameter of described microballoon is 1-100 μ m; Preferred, the mean diameter of described microballoon is 2-80 μ m, and is most preferred, and the mean diameter of described microballoon is 2-50 μ m.
In the protein-chip of the present invention, the quantity of the microballoon that adopts is had no particular limits, in a kind of optimal way of the present invention, in a reaction system (100 μ l systems according to appointment), each quantity that is fixed with the microballoon of different first antibodies is 10
3-10
7Individual; Be more preferably 10
3-10
6Individual, most preferred is 10
4-10
5Individual.
In order to detect described gastritis mark better, the inventor is according to gastritis mark (as pepsinogen I, pepsinogen I I, gastrin-17) difference of content in sample (as serum), amount to the first antibody of the different gastritis marks that adopt is carried out unique design, to guarantee from sample, accurately the detecting gastritis mark, improve recall rate.
In optimal way of the present invention, in the described chip, the former I antibody of antipepsin: the former II antibody of antipepsin: the mol ratio of anti-gastrin-17 antibody is (4-10): (4-12): (6-15), and when having described antibody.Also promptly:
When the first antibody of described gastritis mark is: during the former II antibody of former I antibody of antipepsin and antipepsin, their molar ratios in protein-chip are: (4-10): (4-12).Such as, when detecting, in each hole of 96 orifice plates (about 100 μ l systems), their content is respectively 0.04-0.1 μ g/0.5-2.5 * 10
4Individual microballoon and 0.04-0.12 μ g/0.5-2.5 * 10
4Individual microballoon.
When the first antibody of described gastritis mark is: when former I antibody of antipepsin and anti-gastrin-17 antibody, their molar ratios in liquid phase protein matter chip are: (4-10): (6-15).Such as, when detecting, in each hole of 96 orifice plates, their content is respectively 0.04-0.1 μ g/0.5-2.5 * 10
4Individual microballoon and 0.06-0.15 μ g/0.5-2.5 * 10
4Individual microballoon.
When the first antibody of described gastritis mark is: when former II antibody of antipepsin and anti-gastrin-17 antibody, their molar ratios in liquid phase protein matter chip are: (4-12): (6-15).Such as, when detecting, in each hole of 96 orifice plates, their content is respectively 0.04-0.12 μ g/1-2.5 * 10
4Individual microballoon and 0.06-0.15 μ g/0.5-2.5 * 10
4Individual microballoon.
When the first antibody of described gastritis mark is: when the former I antibody of antipepsin, the former II antibody of antipepsin and anti-gastrin-17 antibody, their content ratio in liquid phase protein matter chip are: (4-10): (4-12): (6-15).Such as, when detecting, in each hole of 96 orifice plates, their content divides in addition not Wei 0.04-0.1 μ g/100 μ l, 0.04-0.12 μ g/100 μ l and 0.06-0.15 μ g/100 μ l.
Certainly, in the protein-chip of the present invention, also can fix the antibody of various anti-stomach trouble marks on the described microballoon, include but not limited to: anti-CEA antibody, anti-CA19-9 antibody, anti-CA242 antibody, anti-CA724 antibody, anti-CA50 antibody (described antibody all can available from Sigma, Biodesign, Medix or Abcam).
In the protein-chip of the present invention, the structure of microballoon-first antibody can be referring to Fig. 1.Wherein 25 is first microballoon, 45 is to be connected with microballoon 25 and first kind of fixing first antibody molecule, 27 is second microballoon, and 47 is to be connected with microballoon 27 and second kind of fixing first antibody molecule, and first microballoon 25 self has diacritic different fluorescence signal with second microballoon 27.
In another preference, the used microballoon of chip of the present invention is 5.5 microns of a kind of diameters, with the fluorescently-labeled microballoon of difference.At every kind of microballoon (solid phase carrier) even-coupling have an antibody molecule of different binding specificities.
In a kind of optimal way of the present invention, a kind of protein-chip is provided, and described chip contains three kinds of different microballoons, and is fixed with the former I antibody of antipepsin, the former II antibody of antipepsin and anti-gastrin-17 antibody on three kinds of microballoons respectively, wherein, the quantity of every kind of microballoon is 10
3-10
7Individual, and the quantity of each first antibody is as follows: the former I antibody of 0.04-0.10 microgram antipepsin, the former II antibody of 0.04-0.12 microgram antipepsin, 0.06-0.15 microgram anti-gastrin-17 antibody.
Two anti-and two anti-marks
Among the present invention, the anti-respectively a kind of corresponding gastritis mark of each second antibody, and corresponding to corresponding first antibody.In optimal way of the present invention, because each gastritis mark antigen contains a plurality of epi-positions, so its second antibody is not unique, but the epitope of the epitope of second antibody combination and first antibody combination accordingly is different.
In optimal way of the present invention, when utilizing second antibody to detect, be directed to each hole (about 100 μ l systems) of 96 orifice plates, the content of second antibody (detection antibody) is as follows:
Anti-PGI antibody: 750-1500ng;
Anti-PGII antibody: 750-1500ng;
Anti-G-17 antibody: 750-1500ng.
Among the present invention, can select for use multiple detectable signal to come the described second antibody of mark.Such as, described detectable signal is selected from: FITC, CY3, CY5, phycocyanin, Alexa Fluor Dyes, BODIPY Dyes, Oregon green dye (Oregon Green Dyes), Texas's red (Texas Red Dye), or rhodamine (Rhodamine).
In a kind of optimal way of the present invention, the two biotin labeling methods that resist are as follows: get respectively at adding biotin dimethyl sulfoxide (DMSO) (DMSO) solution behind two antivenom purifications of different gastritis mark antigens, unreacted biotin is removed in the lucifuge reaction, preserves standby.
Detection material corresponding to detectable signal also can be reported the molecule (reporter molecules) of described combination for combining with detectable signal.When adopting biology usually during the mark second antibody, can adopt streptavidin-phycoerythrin as reporter molecules.
Sample
Among the present invention, the sample that available described protein-chip detects comprises humoral sample or the blood sample of any extraction in mammalian body.In optimal way of the present invention, described sample is a blood sample; Preferred, described sample is a blood serum sample.
In optimal way of the present invention, the sample serum that to be the blood obtained of aseptic venipuncture obtain through fresh sedimentation and refrigeration, consistent with routine clinical blood sampling operation.Sample is through 1: 5 a times of dilution, do not need special processing just can use inventor's process microballoon bag by the optimization of condition, the optimization of analytic process and when detecting the chip detection of optimization of antibody guarantees that 3 factors use same analysis condition to finish detection and quantitative test simultaneously in a chip.
Contrast or standard
In order to eliminate false positive and false negative, contrast should be set in testing process.In addition, in order to obtain quantitative result, the standard items of a plurality of gastritis marks that contain concentration known can be set in testing process.The method to set up of contrast or standard items adopts conventional method.
In optimal way of the present invention, being provided with of standard items is as follows:
Standard items I:(G-17/PG I/PG II:8.5pmol/L, 100ug/L, 12ug/L)
Standard items II:(G-17/PG I/PG II:6.375pmol/L, 75ug/L, 9ug/L)
Standard items III:(G-17/PG I/PG II:4.25pmol/L, 50ug/L, 6ug/L)
Standard items IV:(G-17/PG I/PG II:2.125pmol/L, 25ug/L, 3ug/L)
Standard items V:(G-17/PG I/PG II:0.708pmol/L, 8.33ug/L, 1ug/L)
Standard items VI:(G-17/PG I/PG II:0pmol/L, 0ug/L, 0ug/L)
The detected fluorescent value of corresponding standard items is a Y-axis among the result, and the concentration of corresponding standard items is X-axis, tries to achieve typical curve by instrument software.The fluorescent value that system detects is per sample calculated the concentration that detects mark in the sample by typical curve.
The method of many gastritis mark parallel detection
In the inventive method, anti-characteristics that are fixed on the different microballoons that flow have been made full use of, will fix anti-microspheres solution and sample, standard items or a reference substance, two anti-solution of detectable signal mark successively or add in the reaction vessel in the lump simultaneously, thereby following the reaction taken place:
1. one on the microballoon is anti-combines with corresponding antigen (being gastritis mark antigen) in sample, standard items or the reference substance, forms " gastritis mark-first antibody-microballoon " ternary complex,
2. two resist and samples, standard items, or corresponding antigen (being gastritis mark antigen) combination in the reference substance, final " second antibody-gastritis mark-first antibody-microballoon " tetraplex (the two anti-compounds that comprise the antigen-detectable signal mark of the two anti-compounds of micro-sphere crosslinked anti--gastritis mark antigen-detectable signal mark or micro-sphere crosslinked anti--standard items or reference substance) that forms, step that must be not unnecessary in the course of reaction, in liquid phase, can detect the fluorescence of compound by the liquid-phase chip detector, reach from being reacted to qualitative and quantitative analysis one and go on foot the effect of finishing, that is: single stage method.
On the liquid-phase chip detector, these microballoons are lined up single-row by the micro liquid transfer system, and by two bundle laser, thereby the microballoon identification signal of a branch of judgement microballoon determines the kind of tested gastritis mark; Another bundle is measured the detectable signal of combination on the microballoon, draws the content of tested gastritis mark through data processing.
The detection synoptic diagram of protein-chip of the present invention is seen Fig. 2.Wherein, 1 representative has the microballoon of first antibody, and 2 represent the gastritis mark, and 3 representatives have the second antibody of the mark that can combine with the fluoroscopic examination signal, the fluoroscopic examination material that 4 representatives can combine with the second antibody that has mark.
Testing sample mixes with first antibody solution and second antibody solution, can carry out successively, also can carry out simultaneously.
In optimal way of the present invention, the method for described many gastritis mark parallel detection comprises the steps:
1. get personnel to be tested's peripheral blood, separation of serum;
2. the liquid phase protein matter chip that uses different binding specificities integrated is identified serum to be checked, and is specific as follows:
(a) the liquid phase protein matter chip for preparing is added in the 96 hole suction filtration plates, serum to be checked is added in the liquid phase microglobulin matter chip, and add an amount of damping fluid, incubated at room 30-60 minute;
(b) suction filtration is abandoned supernatant, adds the unconjugated detection thing of hybridization back washing lotion flush away;
(c) the second antibody potpourri of the anti-different gastritis marks of specificity of adding tape label in liquid-phase chip, incubated at room 15-30 minute;
(d) add corresponding fluorescent dye in liquid-phase chip, the reaction back is detected with the liquid-phase chip detector.
3. kit
The major technique of the liquid phase protein matter chip inspecting reagent unit of detection gastritis mark of the present invention is characterised in that liquid phase protein matter chip technology is combined with immunological method, the multiple mark relevant with gastritis (as gastrin 17, pepsinogen I and pepsinogen I I) is united the quantitative test of mark.
The kit that is used to detect many gastritis thing of the present invention comprises following component: first container, and be loaded on the described liquid phase protein matter of claim 1 chip in this container.
In optimal way of the present invention, described kit also comprises: second container, and be loaded on second antibody solution in this container, wherein said second antibody solution contains the second antibody that has detectable signal not of the same race, the anti-respectively a kind of gastritis mark of described each second antibody and corresponding to corresponding first antibody in the first antibody solution, and the mol ratio that corresponding second antibody and first antibody can be incorporated into this gastritis mark and first antibody and second antibody simultaneously is 1: 0.1-1: 2.
In another preference of the present invention, also comprise in the described kit: standard items or reference substance.
In a kind of optimal way of the present invention, described kit comprises:
A container a, and be loaded on the liquid phase protein matter chip that above-mentioned being used in the described container detects the gastritis serum marker relative;
Preferred, described kit also comprises a pipe hybridization solution at least, and this hybridization solution is used for the hybridization of liquid phase protein matter chip and analyte;
Preferred, described kit also comprises a pipe hybridization back washing lotion at least, and this hybridization back washing lotion is used for the washing after liquid phase protein matter chip and the analyte hybridization;
Preferred, described kit also comprise at least a pipe can with the fluorescent dye of the antibodies of specific markers;
Preferred, described kit also comprises a pipe positive control at least, and this positive control is used for when detecting 3 goal analysis things testing process being controlled;
Preferred, described kit also comprises a pipe negative control at least, and this negative control is used for when detecting 3 goal analysis things testing process being controlled.
Major advantage of the present invention is:
The first, can carry out quantitative test to a plurality of blood serum designated objects relevant in a certain individual sample simultaneously with gastritis; And, also can carry out quantitative test to a plurality of blood serum designated objects relevant in a plurality of samples simultaneously with gastritis.
The second, owing to the amount of the first antibody that microballoon is had is carried out proportion design, thereby make that the gastritis mark (as G-17) that is not easy to detect also can be detected delicately.And, reduced nonspecific combination that different analytes exist to greatest extent when hybridization, thereby greatly reduced the generation of background signal.Plenty of time and fund have also been saved simultaneously.
The 3rd, whether the present invention is that the position (stomach hole, body of stomach) that gastritis, atrophic gastritis and gastritis take place is diagnosed to patient not only, and for better preventing and treating atrophic gastritis, and the incidence probability of reduction cancer of the stomach and gastric ulcer has been created condition.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise number percent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Antibody sources
The antibody sources of using in following examples sees Table 1.
Table 1
| Antibody | The source |
| Anti-PGI first antibody | Sigma、Biodesign、Medix、Abcam |
| Anti-PGII first antibody | Sigma、Biodesign、Medix、Abcam |
| Anti-G-17 first antibody | Sigma、Biodesign、Medix、Abcam |
| Anti-PGI second antibody | Sigma、Biodesign、Medix、Abcam |
| Anti-PGII second antibody | Sigma、Biodesign、Medix、Abcam |
| Anti-G-17 second antibody | Sigma、Biodesign、Medix、Abcam |
The reagent preparation
The reagent of using in following examples sees Table 2.
Table 2
| Reagent | Prescription |
| The microballoon cleansing solution | 10.1mM Na 2HPO 4，138mMNaCl，1.76mMKH 2PO 4， 2.7mMKCl，0.05％(V/V)Tween-20，pH7.4 |
| Activating solution | 10.1mM mM NaH 2PO 4，5M NaOH，pH6.2 |
| Confining liquid | 10.1mM Na 2HPO 4，138mMNaCl，1.76mMKH 2PO 4， 2.7mMKCl，1％(m/V)BSA，0.02％(m/V)Thimerosal，pH7.4 |
| Storage liquid | 10.1mM Na 2HPO 4，138mMNaCl，1.76mMKH 2PO 4， 2.7mMKCl，1％(m/V)BSA，0.02％(m/V)Thimerosal， 0.02％(V/V)Tween-20，pH7.4 |
| Buffer A | 10.1mM Na 2HPO 4，138mMNaCl，1.76mMKH 2PO 4， 2.7mMKCl，0.05％(V/V)Tween-20，pH7.4 |
| Buffer B | 10.1mM Na 2HPO 4，138mMNaCl，1.76mMKH 2PO 4， 2.7mMKCl，1％(m/V)BSA，0.02％(m/V)Thimerosal，pH7.4 |
The method (microballoon method for coating) of chip preparation
(1) gets about 1.25 * 10
6Individual microballoon is (available from Bio-rad company, as 171-506027,5.5 microns of diameters), concussion, the centrifugal 4min of ultrasonic 30 seconds → 14000 * g, abandon supernatant, the microballoon cleansing solution of 100 μ l is resuspended → the 1400rpm concussion, the centrifugal 4min of ultrasonic 30s → 14000 * g, the activating solution of 80 μ l is resuspended → add EDC and the S-NHS of the 50mg/ml of each 10 μ l, lucifuge room temperature 1400rpm shakes 20min → adding 150 μ l PBS damping fluids, 1800rpm shakes 15s, the centrifugal 4min of 14000 * g → adding 250 μ lPBS damping fluids, 1800rpm shakes 15s, and the centrifugal 4min of 14000 * g → 100 μ l PBS damping fluids are resuspended → and the capture antibody (anti-PGI first antibody) that adds 4 μ g correspondences complements to 500 μ l with the PBS damping fluid with it respectively; Lucifuge, 4 ℃, 1400rpm concussion are spent the night → the centrifugal 4min of 14000 * g the centrifugal 4min of PBS damping fluid washing microballoon → 14000 * g of 500 μ l, 250 μ l confining liquids; The centrifugal 4min of lucifuge room temperature 1400rpm sealing 30min → 14000 * g, the centrifugal 6min of storage liquid washing microballoon → 16000 * g of 500 μ l is with the resuspended microballoon of the storage liquid of 125 μ l; Lucifuge and 4 ℃ of preservations.
With above-mentioned step, preparation contains the microballoon of anti-PGII first antibody, anti-G-17 first antibody respectively, but the addition of two kinds of antibody is respectively 4.5ug and 9ug.
(2) microballoon coated antibody efficient detects
In the biotin labeling sheep anti-mouse igg (concentration is 2ug/ml) of every pipe 50 μ l, add microballoon → lucifuge room temperature 1400rpm that 1 μ l shook, the centrifugal 4min of 30min → 14000g is hatched in vibration, with 50 μ l1: the resuspended microballoon of streptavidin-phycoerythrin after 25 dilutions, the centrifugal 4min of lucifuge incubated at room 10min → 14000g, suck 96 orifice plates with the resuspended microballoon of the cleansing solution of 100 μ l/ pipes → commentaries on classics, sending into instrument detecting → detect fluorescent value at last should surpass 10000.
Embodiment 2
Detect the biotinylation of antibody
(1) with 30 μ l DMSO dissolving BCA-SulfoNHS (Sigma), and be 0.5ml with 0.1mol/l sodium phosphate buffer constant volume, final concentration is 10mg/ml → in the BCA-SulfoNHS solution that adds 38 μ l in antibody (at different gastritis marks, being respectively the second antibody of PGI, PGII, the G-17) solution of the 10mg/ml of 1ml → lucifuge room temperature concussion 30 minutes → the cross antibody protein amount after the biotinylated antibody of Sephadex G25 column separating purification → usefulness BAC method is measured recovery.
(2) antibody biotinylation efficient detects
Get the sample (PGI that mark is good of the good biotin of 50 μ l marks, PGII, the G-17 second antibody), add the 5ul pronase, 37 ℃ of water-baths 90 minutes → with HABA[2-(the 4-hydroxy benzenes azo group) benzoic acid of 3.2ml avidin (Avidin) with 100ul 10mM, 2-(4-Hydroxyphenylazo) benzoic acid] mix, sample or concentration after obtaining Avidin-HABA liquid storage → with the enzyme of the Avidin-HABA liquid storage of 90ul and 10ul and handling are respectively 30nmol/L, 25nmol/L, 20nmol/L, 15nmol/L, 10nmol/L, 5nmol/L, 2.5nmol/L, (light path 10mm) returns to zero with the PBS baseline under the d-biotin solution mixing → A500 of 0nmol/L, the d-biotin of test sample product and each concentration and the OD value of Avidin-HABA liquid storage, do typical curve, thereby try to achieve the concentration of the biotin in the sample → biotin concentration/protein sample concentration=biotin labeling efficient → biotin labeling efficient should be greater than 5.
Embodiment 3
The preparation of detection kit
In the present embodiment, prepare a kind of kit that is used to detect the gastritis mark, described kit comprises following assembly:
(a) container a, and the microballoon mixed liquor (preparation among the embodiment 1) of PGI, PGII, G-17 capture antibody that has been loaded in the described container coupling respectively.
(b) container b, and be loaded on the mixed liquor (preparation among the embodiment 2) that biotin labeled PGI, PGII in the described container, G-17 detect antibody.
(c) container c, and be loaded on 2 * buffer A (10.1mM Na among the container c
2HPO
4, 138mMNaCl, 1.76mMKH
2PO
4, 2.7mMKCl, 0.05% (V/V) Tween-20, pH7.4).
(d) container d, and be loaded on buffer B (10.1mM Na among the container d
2HPO
4, 138mMNaCl, 1.76mMKH
2PO
4, 2.7mMKCl, 1% (m/V) BSA, 0.02% (m/V) Thimerosal, pH7.4).
(e) container e, and be loaded among the container e can with the streptavidin-phycoerythrin of the detection antibodies of mark biotin.
(f) 6 containers of a cover, and PGI, the PGII, G-17 standard items (concentration is at the 7.5-15ug/ml) mixed liquor that are loaded on a series of variable concentrations in the container.
The shrouding film that shrouding was used when (g) one 96 hole suction filtration plate and 4 were used to hatch.
The detection of embodiment 4 serum
(1) detection method
1, antibody coupling microballoon 1400rpm on oscillator was shaken 30 seconds;
2, microballoon was diluted by 1: 100;
3, the amount of antibody coupling microballoon by 50 μ l/ holes added in 96 orifice plates, vacuum is drained;
4, add the buffer A washing microballoon in 100 μ l/ holes, repeat this step;
5, add 100 μ l/ hole samples (having used the dilution in 1: 5 of 1 * buffer A), standard items (need not dilute) respectively, stick the shrouding film;
6, plate is placed on the dull and stereotyped shaking table vibration of 1100rpm lucifuge 1min, 600rpm lucifuge incubated at room 60min then;
7, vacuum filtration adds the 1 * buffer A in 100 μ l/ holes, washs 3 times;
8, the biotin labeling two in adding 100 μ l/ holes is anti-, plate is placed on the dull and stereotyped shaking table 1100rpm lucifuge vibration 1min, 600rpm lucifuge incubated at room 30min.
9, vacuum filtration adds the 1 * buffer A in 100 μ l/ holes, washs 3 times;
10, add 50 μ l/ pore chain Avidin-phycoerythrin (having used buffer B dilution in 1: 12.5), plate is placed on the dull and stereotyped shaking table, the 1100rpm 1min that vibrates, 400rpm lucifuge incubated at room 10min;
11, vacuum filtration adds the 1 * buffer A in 100 μ l/ holes, washs 3 times;
12, add the resuspended microballoon of buffer B in 125 μ l/ holes, plate is placed on the dull and stereotyped shaking table, 1100rpm lucifuge vibration 1min.
13, plate is sent into instrument detecting.
(2) testing result:
Adopt this method that person-portion serum surplus 70 is detected, the result is shown in table 1, table 2.
The detection data that table 3 reads for detector.
The relative concentration of each mark that table 4 obtains for the detection data reduction of reading according to detector.
The result of table 3 and table 4 shows: the mensuration concentration of three kinds of gastritis marks is accurate.
And, the gastroscope survey report that the inventor will adopt whole test result of samples that method of the present invention records and experimenter relatively, the result is in full accord.
Table 3
| Well | Type | Description | Gastrin-17(25) | Pepsinogen I(27) | Pepsinogen II(32) |
| E5 | X1 | N1 | 731.0(113) | 1619.0(100) | 525.0(105) |
| F5 | X2 | N2 | 807.0(138) | 415.0(100) | 225.0(111) |
| G5 | X3 | N3 | 5578.0(115) | 4309.0(120) | 4350.0(100) |
| H5 | X4 | N4 | 1367.0(104) | 1221.0(109) | 1244.0(100) |
| A6 | X5 | N5 | 578.0(133) | 687.0(100) | 784.0(107) |
| B6 | X6 | P1 | 113.0(134) | 555.0(100) | 352.0(141) |
| C6 | X7 | P2 | 615.0(142) | 782.0(100) | 1405.0(103) |
| D6 | X8 | P3 | 1751.0(134) | 1023.0(100) | 689.0(126) |
| E6 | X9 | P4 | 2166.0(115) | 659.0(100) | 1970.0(105) |
| F6 | X10 | P5 | 810.0(126) | 1051.0(100) | 759.0(106) |
| G6 | X11 | 1 | 157.0(151) | 747.0(100) | 682.0(106) |
| H6 | X12 | 2 | 245.0(120) | 817.0(105) | 530.0(100) |
| A7 | X13 | 3 | 565.0(164) | 905.0(115) | 1311.0(100) |
| B7 | X14 | 4 | 243.0(129) | 1098.0(100) | 1038.0(114) |
| C7 | X15 | 5 | 317.0(122) | 556.0(102) | 728.0(100) |
| D7 | X16 | 6 | 335.0(136) | 1710.0(100) | 501.0(110) |
| E7 | X17 | 7 | 216.0(126) | 1608.0(116) | 697.0(100) |
| F7 | X18 | 8 | 9.0(136) | 1918.0(114) | 545.0(100) |
| G7 | X19 | 9 | 1034.0(153) | 359.0(129) | 154.0(100) |
| H7 | X20 | 10 | 99.0(132) | 2475.0(106) | 852.0(100) |
| A8 | X21 | 11 | 628.0(110) | 1129.0(114) | 635.0(100) |
| B8 | X22 | 12 | 47.0(100) | 1081.0(111) | 411.0(114) |
| C8 | X23 | 13 | 95.0(137) | 542.0(100) | 205.0(116) |
| D8 | X24 | 14 | 71.0(153) | 1750.0(100) | 413.0(143) |
| E8 | X25 | 15 | 189.0(104) | 598.0(102) | 357.0(100) |
| F8 | X26 | 16 | 103.0(142) | 812.0(100) | 250.0(100) |
| G8 | X27 | 17 | 339.0(131) | 776.0(100) | 525.0(108) |
| H8 | X28 | 18 | 332.0(128) | 289.0(100) | 1387.0(108) |
| A9 | X29 | 19 | 417.0(119) | 721.0(124) | 303.0(100) |
| B9 | X30 | 20 | 39.0(127) | 257.0(104) | 129.0(100) |
| C9 | X31 | 21 | 401.0(158) | 3582.0(114) | 1430.0(100) |
| D9 | X32 | 22 | 77.0(147) | 753.0(100) | 285.0(102) |
| E9 | X33 | 23 | 183.0(133) | 1416.0(114) | 362.0(100) |
| F9 | X34 | 24 | 22.0(133) | 1618.0(109) | 867.0(100) |
| G9 | X35 | 25 | 27.0(111) | 2050.0(131) | 1585.0(100) |
| H9 | X36 | 26 | 14.0(100) | 623.0(102) | 169.0(101) |
| A10 | X37 | 27 | 50.0(116) | 783.0(106) | 195.0(100) |
| B10 | X38 | 28 | 128.0(144) | 1128.0(125) | 516.0(100) |
| C10 | X39 | 29 | 71.0(129) | 229.0(105) | 188.0(100) |
| D10 | X40 | 30 | 806.0(125) | 155.0(100) | 430.0(102) |
| E10 | X41 | 31 | 463.0(172) | 217.0(100) | 353.0(126) |
| F10 | X42 | 32 | 163.0(146) | 222.0(101) | 138.0(100) |
| G10 | X43 | 33 | 87.0(144) | 471.0(100) | 264.0(117) |
| H10 | X44 | 34 | 239.0(129) | 833.0(100) | 668.0(109) |
| A11 | X45 | 35 | 1500.0(109) | 299.0(100) | 2853.0(105) |
| B11 | X46 | 36 | 408.0(125) | 512.0(100) | 568.0(120) |
| C11 | X47 | 37 | 62.0(135) | 1371.0(114) | 242.0(100) |
| D11 | X48 | 38 | 1454.0(126) | 252.0(100) | 543.0(119) |
| E11 | X49 | 39 | 27.0(191) | 854.0(100) | 638.0(159) |
| F11 | X50 | 40 | 65.0(138) | 448.0(100) | 213.0(106) |
| G11 | X51 | 41 | 528.0(129) | 13660(100) | 880.0(114) |
| H11 | X52 | 42 | 1053.0(187) | 440.0(100) | 1678.0(134) |
| A12 | X53 | 43 | 358.0(121) | 690.0(100) | 397.0(112) |
| B12 | X54 | 44 | 66.0(114) | 240.0(100) | 91.0(118) |
| C12 | X55 | 45 | 214.0(122) | 313.0(100) | 151.0(102) |
| D12 | X56 | 46 | 106.0(116) | 154.0(126) | 125.0(100) |
| E12 | X57 | 47 | 590.0(176) | 2413.0(100) | 803.0(134) |
| F12 | X58 | 48 | 45.0(141) | 740.0(100) | 262.0(120) |
| G12 | X59 | 49 | 65.0(124) | 389.0(101) | 540.0(100) |
| H12 | X60 | 50 | 10.0(126) | 939.0(101) | 235.0(100) |
* Well is listed as each code name and represents various samples corresponding hole number on 96 orifice plates; In the Description row, N1-N5 represents negative control, and P1-P5 represents positive control, and 1-50 represents experimenter's sample; In the testing result, such as, 731.0 (113), the expression detector has read 113 microballoons, and the fluorescent value of reading is 731.0.
Table 4
| Type | Well | Description | G-17Obs Conc | PGI Obs Conc | PGII Obs Conc |
| eS1 | A1，B1，C1 | 0 | 0 | 0 | 0 |
| eS2 | D1，E1，F1 | 1 | 0.71 | 8.32 | 1 |
| eS3 | A2，G1，H1 | 3 | 2.14 | 25.2 | 3.05 |
| eS4 | B2，C2，D2 | 6 | 4.07 | 48.13 | 5.81 |
| eS5 | E2，F2，G2 | 9 | 6.85 | 80.87 | 9.44 |
| eS6 | A3，B3，H2 | 12 | 8.23 | 96.19 | 11.72 |
| X1 | E5 | N1 | 1.71 | 31 | 5.47 |
| X2 | F5 | N2 | 1.87 | 6.47 | 2.51 |
| X3 | G5 | N3 | 32.1 | 118.05 | 45.62 |
| X4 | H5 | N4 | 3.23 | 22.15 | 12 |
| X5 | A6 | N5 | 1.38 | 11.43 | 7.87 |
| X6 | B6 | P1 | 0.4 | 8.98 | 3.8 |
| X7 | C6 | P2 | 1.46 | 13.24 | 13.43 |
| X8 | D6 | P3 | 4.33 | 18.03 | 7 |
| X9 | E6 | P4 | 5.7 | 10.9 | 18.49 |
| X10 | F6 | P5 | 1.88 | 18.6 | 7.64 |
| X11 | G6 | 1 | 0.5 | 12.57 | 6.93 |
| X12 | H6 | 2 | 0.69 | 13.92 | 5.52 |
| X13 | A7 | 3 | 1.35 | 15.65 | 12.59 |
| X14 | B7 | 4 | 0.69 | 19.57 | 10.16 |
| X15 | C7 | 5 | 0.84 | 9 | 7.36 |
| X16 | D7 | 6 | 0.88 | 33.13 | 5.24 |
| X17 | E7 | 7 | 0.63 | 30.74 | 7.07 |
| X18 | F7 | 8 | OOR＜ | 38.16 | 5.66 |
| X19 | G7 | 9 | 2.39 | 5.49 | 1.76 |
| X20 | H7 | 10 | 0.36 | 52.84 | 8.49 |
| X21 | A8 | 11 | 1.49 | 20.21 | 6.5 |
| X22 | B8 | 12 | 0.21 | 19.22 | 4.38 |
| X23 | C8 | 13 | 0.35 | 8.74 | 2.3 |
| X24 | D8 | 14 | 0.29 | 34.08 | 4.39 |
| X25 | E8 | 15 | 0.57 | 9.77 | 3.85 |
| X26 | F8 | 16 | 0.37 | 13.82 | 2.77 |
| X27 | G8 | 17 | 0.89 | 13.13 | 5.47 |
| X28 | H8 | 18 | 0.87 | 4.3 | 13.27 |
| X29 | A9 | 19 | 1.05 | 12.07 | 3.31 |
| X30 | B9 | 20 | 0.18 | 3.76 | 1.49 |
| X31 | C9 | 21 | 1.02 | 88.42 | 13.65 |
| X32 | D9 | 22 | 0.3 | 12.69 | 3.13 |
| X33 | E9 | 23 | 0.56 | 26.39 | 3.9 |
| X34 | F9 | 24 | 0.09 | 30.97 | 8.62 |
| X35 | G9 | 25 | 0.12 | 41.48 | 15.03 |
| X36 | H9 | 26 | OOR＜ | 10.23 | 1.92 |
| X37 | A10 | 27 | 0.22 | 13.26 | 2.2 |
| X38 | B10 | 28 | 0.43 | 20.19 | 5.38 |
| X39 | C10 | 29 | 0.29 | 3.3 | 2.13 |
| X40 | D10 | 30 | 1.87 | 2.1 | 4.56 |
| X41 | E10 | 31 | 1.14 | 3.1 | 3.81 |
| X42 | F10 | 32 | 0.52 | 3.18 | 1.59 |
| X43 | G10 | 33 | 0.33 | 7.46 | 2.92 |
| X44 | H10 | 34 | 0.68 | 14.23 | 6.8 |
| X45 | A11 | 35 | 3.59 | 4.47 | 26.91 |
| X46 | B11 | 36 | 1.03 | 8.2 | 5.87 |
| X47 | C11 | 37 | 0.26 | 25.4 | 2.69 |
| X48 | D11 | 38 | 3.46 | 3.68 | 5.64 |
| X49 | E11 | 39 | 0.12 | 14.64 | 6.53 |
| X50 | F11 | 40 | 0.27 | 7.05 | 2.39 |
| X51 | G11 | 41 | 1.28 | 25.29 | 8.74 |
| X52 | H11 | 42 | 2.44 | 6.91 | 15.86 |
| X53 | A12 | 43 | 0.93 | 11.49 | 4.24 |
| X54 | B12 | 44 | 0.27 | 3.48 | 1.06 |
| X55 | C12 | 45 | 0.63 | 4.71 | 1.73 |
| X56 | D12 | 46 | 0.38 | 2.09 | 1.44 |
| X57 | E12 | 47 | 1.41 | 51.11 | 8.04 |
| X58 | F12 | 48 | 0.2 | 12.44 | 2.89 |
| X59 | G12 | 49 | 0.27 | 6.01 | 5.61 |
* eS1-eS6 represents six standard items in the Type row, and X1-59 represents unknown sample; Well is listed as each code name and represents various samples corresponding hole number on 96 orifice plates; In the Description row, N1-N5 represents negative control, and P1-P5 represents positive control, and 1-49 represents experimenter's sample; Next 3 Obs conc represent concentration (G-17 concentration unit: pmol/L, PG-I concentration unit ug/L, the PG-II concentration unit: μ g/L) that this gastritis mark is tried to achieve by typical curve.The concentration that OOR＜expression converses after detecting is standard curve range head and shoulders above.Table 4 is the concentration that fluorescent value is converted in typical curve and tries to achieve, and does Excel table output integration and forms.
Fig. 3 is the typical curve of G-17, and the concentration of G-17 standard items is 0,0.126,0.252,0.503,1.062,2.125,4.25,8.5pmol/L the detected fluorescent value of corresponding standard items is a Y-axis among the result, the concentration of corresponding standard items is X-axis, tries to achieve typical curve by instrument software.The fluorescent value that system detects is per sample calculated the concentration that detects mark in the sample by typical curve.
The result that embodiment 5 adopts protein-chip of the present invention and hospital to detect compares
Method according to embodiment 1 and embodiment 2 prepares protein-chip and biotin labeled second antibody etc., and according to embodiment 4 described methods, the inventor has detected a plurality of samples again.These samples are provided by Shandong Qilu Hospital clinical basic chamber, are detected by gastroscope and are confirmed to be the gastritis positive sample.
The part testing result of gastrin 17, pepsinogen I and pepsinogen I I is shown in table 5, table 6, table 7.
When pepsinogen I was lower than 5 μ g/L, showing had moderate or serious body of stomach mucous membrane place's gastritis or body of stomach atrophy, is the sign that need make further stomach hole gastroscope, bigger because its low content shows that these patients get the risk of cancer of the stomach.
When the ratio of pepsinogen I/II among the result was lower than 2.5, the possibility that this sufferer is suffered from the body of stomach atrophy promoted greatly.
In the patients serum of the helicobacter pylori antibody positive, the value of gastrin-17 illustrates it has moderate or severe at stomach Dou Chu gastritis and lipogastry less than 5pmol/L.
The concentration of the gastrin that records-17 sees Table 5; The concentration of the pepsinogen I that records sees Table 6; The concentration of the pepsinogen I I that records sees Table 7.
Table 5 table 6
| Type | Well | Obs Conc | |
| 1 | eS1 | A9，B9，C9 | 0.00 |
| 2 | eS2 | D9，E9，F9 | 0.71 |
| 3 | eS3 | A10，G9，H9 | 2.14 |
| 4 | eS4 | B10，D10 | 4.22 |
| 5 | eS5 | F10，G10 | 6.31 |
| 6 | eS6 | A11，B11，H10 | 8.60 |
| 7 | X1 | A1 | 3.28 |
| 8 | X2 | B1 | 0.14 |
| 9 | X3 | C1 | 0.42 |
| 10 | X4 | D1 | 2538.95 |
| 11 | X5 | E1 | 0.81 |
| 12 | X6 | F1 | 1.68 |
| 13 | X7 | G1 | 0.29 |
| 14 | X8 | H1 | 0.64 |
| 15 | X9 | A2 | 1.06 |
| 16 | X10 | B2 | 1.02 |
| 17 | X11 | C2 | 2.16 |
| 18 | X12 | D2 | 1.50 |
| 19 | X13 | E2 | 1.21 |
| 20 | X14 | F2 | 12.91 |
| 21 | X15 | G2 | 1.84 |
| 22 | X16 | H2 | 1.07 |
| 23 | X17 | A3 | 0.48 |
| 24 | X18 | B3 | 2.42 |
| 25 | X19 | C3 | 0.56 |
| 26 | X20 | D3 | 2.51 |
| 27 | X21 | E3 | 1.57 |
| 28 | X22 | F3 | 0.85 |
| 29 | X23 | G3 | 0.42 |
| 30 | X24 | H3 | 0.68 |
| 31 | X25 | A4 | 1.09 |
| 32 | X26 | B4 | OOR＜ |
| 33 | X27 | C4 | 5.80 |
| 34 | X28 | D4 | 0.41 |
| 35 | X29 | E4 | 0.57 |
| Type | Well | Obs Conc | |
| 1 | eS1 | A9，B9，C9 | 0.06 |
| 2 | eS2 | D9，E9，F9 | 8.32 |
| 3 | eS3 | A10，G9，H9 | 25.29 |
| 4 | eS4 | B10，D10 | 47.75 |
| 5 | eS5 | F10，G10 | 80.10 |
| 6 | eS6 | A11，B11，H10 | 97.54 |
| 7 | X1 | A1 | 5.42 |
| 8 | X2 | B1 | 8.33 |
| 9 | X3 | C1 | 6.72 |
| 10 | X4 | D1 | 222.89 |
| 11 | X5 | E1 | 11.21 |
| 12 | X6 | F1 | 9.96 |
| 13 | X7 | G1 | 11.72 |
| 14 | X8 | H1 | 17.33 |
| 15 | X9 | A2 | 13.23 |
| 16 | X10 | B2 | 3.01 |
| 17 | X11 | C2 | 13.10 |
| 18 | X12 | D2 | 11.21 |
| 19 | X13 | E2 | 4.39 |
| 20 | X14 | F2 | 26.82 |
| 21 | X15 | G2 | 11.77 |
| 22 | X16 | H2 | 10.76 |
| 23 | X17 | A3 | 15.42 |
| 24 | X18 | B3 | 17.55 |
| 25 | X19 | C3 | 17.08 |
| 26 | X20 | D3 | 11.64 |
| 27 | X21 | E3 | 16.98 |
| 28 | X22 | F3 | 9.75 |
| 29 | X23 | G3 | 9.22 |
| 30 | X24 | H3 | 7.39 |
| 31 | X25 | A4 | 6.96 |
| 32 | X26 | B4 | 6.29 |
| 33 | X27 | C4 | 17.50 |
| 34 | X28 | D4 | 12.35 |
| 35 | X29 | E4 | 11.24 |
Table 7
| Type | | Obs Conc | ||
| 1 | eS1 | A9，B9，C9 | 0.01 | |
| 2 | eS2 | D9，E9，F9 | 1.00 | |
| 3 | eS3 | A10，G9，H9 | 3.14 | |
| 4 | eS4 | B10，D10 | 5.59 | |
| 5 | eS5 | F10，G10 | 8.81 | |
| 6 | eS6 | A11，B11，H10 | 12.85 | |
| 7 | X1 | A1 | 2.02 | |
| 8 | X2 | B1 | 0.82 | |
| 9 | X3 | C1 | 1.49 | |
| 10 | X4 | D1 | 9.99 | |
| 11 | X5 | E1 | 2.39 | |
| 12 | X6 | F1 | 1.78 | |
| 13 | X7 | G1 | 0.85 | |
| 14 | X8 | H1 | 1.28 | |
| 15 | X9 | A2 | 3.85 | |
| 16 | X10 | B2 | 6.24 | |
| 17 | X11 | C2 | 6.06 | |
| 18 | X12 | D2 | 2.32 | |
| 19 | X13 | E2 | 2.00 | |
| 20 | X14 | F2 | 6.15 | |
| 21 | X15 | G2 | 3.59 | |
| 22 | X16 | H2 | 3.85 | |
| 23 | X17 | A3 | 1.12 | |
| 24 | X18 | B3 | 2.75 | |
| 25 | X19 | C3 | 1.74 | |
| 26 | X20 | D3 | 3.02 | |
| 27 | X21 | E3 | 2.57 | |
| 28 | X22 | F3 | 2.85 | |
| 29 | X23 | G3 | 0.83 | |
| 30 | X24 | H3 | 2.02 | |
| 31 | X25 | A4 | 1.83 | |
| 32 | X26 | B4 | 0.76 | |
| 33 | X27 | C4 | 5.05 | |
| 34 | X28 | D4 | 1.42 | |
| 35 | X29 | E4 | 1.40 |
* in table 5, table 6, the table 7, eS1-eS6 represents six standard items in the Type row, and X1-29 represents unknown sample; Well is listed as each code name and represents various samples corresponding hole number on 96 orifice plates; Obs conc represents concentration (G-17 concentration unit: pmol/L, PG-I concentration unit ug/L, the PG-II concentration unit: μ g/L) that this gastritis mark is tried to achieve by typical curve.The concentration that OOR＜expression converses after detecting is standard curve range head and shoulders above.
These results that the inventor will adopt protein-chip of the present invention to measure compare with the clinical detection result of hospital, find that the result who obtains conforms to fully.
In time, adopt protein-chip of the present invention only to need 1.5 hour time as a result the time measuring these, and one-time detection has just obtained result accurately; And hospital is when adopting the ELISA method to detect, because three factors detect respectively, the accumulative total elapsed time is 10.5 hours, and hospital is when detecting, and the sample that has need be verified repeatedly because of having powerful connections interferences.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can make various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a protein-chip is characterized in that, described protein-chip comprises microballoons different more than 2 kinds, is fixed with different first antibodies on the described microballoon,
Wherein, described first antibody is the first antibody of gastritis mark, and described first antibody is selected from down group: the former I antibody of antipepsin, the former II antibody of antipepsin or anti-gastrin-17 antibody.
2. chip as claimed in claim 1 is characterized in that, in the described chip, the former I antibody of antipepsin: the former II antibody of antipepsin: the mol ratio of anti-gastrin-17 antibody is (4-10): (4-12): (6-15), and when having described antibody.
3. chip as claimed in claim 1 is characterized in that, described different microballoon is the microballoon with different microballoon identification signals.
4. chip as claimed in claim 1 is characterized in that, in the described chip, the quantity of each microballoon is 10
3-10
7Individual.
5. chip as claimed in claim 1 is characterized in that, described chip contains three kinds of different microballoons, and is fixed with the former I antibody of antipepsin, the former II antibody of antipepsin and anti-gastrin-17 antibody on three kinds of microballoons respectively,
Wherein, the quantity of every kind of microballoon is 10
3-10
7Individual, and the quantity of each first antibody is as follows: the former I antibody of 0.04-0.10 microgram antipepsin, the former II antibody of 0.04-0.12 microgram antipepsin, 0.06-0.15 microgram anti-gastrin-17 antibody.
6. chip as claimed in claim 1 is characterized in that, described first antibody also comprises: anti-CEA antibody, anti-CA19-9 antibody, anti-CA242 antibody, anti-CA724 antibody or anti-CA50 antibody.
7. the method for the parallel detection of gastritis mark more than a kind is characterized in that, may further comprise the steps:
(a) the described protein-chip of testing sample and claim 1 is mixed, thereby make gastritis mark in the testing sample and first antibody and microballoon form " gastritis mark-first antibody-microballoon " ternary complex;
(b) ternary complex that step (a) is formed mixes with second antibody solution, wherein said second antibody solution contains the second antibody that has detectable signal not of the same race, the anti-respectively a kind of gastritis mark of each second antibody and corresponding to corresponding first antibody in the protein-chip, and corresponding second antibody is 1 with the mol ratio that first antibody can be incorporated into this gastritis mark and first antibody and second antibody simultaneously: 0.1-1: 2, thus formation " second antibody-gastritis mark-first antibody-microballoon " tetraplex;
(c) detect the microballoon identification signal of different microballoons in the tetraplex, thereby whether and the amount that exists the existence of determining each gastritis mark in the detected sample;
Subsidiary condition are testing samples and the mixing of protein-chip and second antibody solution, and can carry out successively, also can carry out simultaneously.
8. a kit that is used to detect many gastritis mark is characterized in that, it comprises following component:
(1) first container, and be loaded on first antibody solution in this container, contain the described protein-chip of claim 1 in the described first antibody solution.
9. kit as claimed in claim 7 is characterized in that, also comprises:
(2) second containers, and be loaded on second antibody solution in this container, wherein said second antibody solution contains the second antibody that has detectable signal not of the same race, the anti-respectively a kind of gastritis mark of described each second antibody and corresponding to corresponding first antibody in the first antibody solution, and the mol ratio that corresponding second antibody and first antibody can be incorporated into this gastritis mark and first antibody and second antibody simultaneously is 1: 0.1-1: 2.
10. the purposes of the described protein-chip of claim 1 is characterized in that, is used to prepare the kit of diagnosis or auxiliary diagnosis gastritis; Or be used for preparing the kit whether the vitro detection sample exists the gastritis mark.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610025973 CN101063684A (en) | 2006-04-24 | 2006-04-24 | Chip and detecting reagent kit for detecting gastricism relevant indication marks object |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610025973 CN101063684A (en) | 2006-04-24 | 2006-04-24 | Chip and detecting reagent kit for detecting gastricism relevant indication marks object |
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| CN101063684A true CN101063684A (en) | 2007-10-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200610025973 Pending CN101063684A (en) | 2006-04-24 | 2006-04-24 | Chip and detecting reagent kit for detecting gastricism relevant indication marks object |
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| CN (1) | CN101063684A (en) |
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| WO2016127320A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Reagent kit used for detecting gastrin-17, and preparation method and application for reagent kit |
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| CN112898430A (en) * | 2019-12-04 | 2021-06-04 | 东莞市朋志生物科技有限公司 | Binding protein of CA242, application thereof, detection method and kit |
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| WO2016127320A1 (en) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | Reagent kit used for detecting gastrin-17, and preparation method and application for reagent kit |
| CN104820096A (en) * | 2015-04-30 | 2015-08-05 | 必欧瀚生物技术（合肥）有限公司 | Combined standard quantity for determining quantity of marker used in early gastric cancer risk detection and application thereof in early gastric cancer screening |
| CN104820096B (en) * | 2015-04-30 | 2017-04-19 | 必欧瀚生物技术（合肥）有限公司 | Combined standard quantity for determining quantity of marker used in early gastric cancer risk detection and application thereof in early gastric cancer screening |
| CN112898430A (en) * | 2019-12-04 | 2021-06-04 | 东莞市朋志生物科技有限公司 | Binding protein of CA242, application thereof, detection method and kit |
| CN112898430B (en) * | 2019-12-04 | 2022-11-04 | 东莞市朋志生物科技有限公司 | Binding protein of CA242, application thereof, detection method and kit |
| CN111024964A (en) * | 2019-12-20 | 2020-04-17 | 深圳市蔚景生物科技有限公司 | Composite quality control product for detecting stomach function and detection kit |
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