CN102662059B - Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof - Google Patents
Latex-enhanced immunoturbidimetry kit for measuring Helicobacter pylori antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a latex-enhanced immunoturbidimetry kit for measuring a Helicobacter pylori antibody as well as a preparation method and application thereof. The kit comprises a diluent, latex reagent and blank liquid of Helicobacter pylori antigen, a standard product and a quality control product, wherein the Helicobacter pylori antigen can be one or more of full-tropina antigen of Helicobacter pylori, urease antigen of Helicobacter pylori, cytotoxin-related protein A antigen of Helicobacter pylori or cell vacuole toxin A antigen of Helicobacter pylori; the latex reagent is a mixture of two latex granules which have different particle sizes and are adsorbed by Helicobacter pylori; and the Helicobacter pylori antibody for preparing the standard product and the quality control product is derived from IgM (immunoglobulin M) and/or IgG (immunoglobulin G) in serum of a patient infected by Helicobacter pylori. The kit is suitable for various full-automatic biochemical analyzers and semiautomatic biochemical analyzers, and has the advantages of rapid detection, high sensitivity, strong specifically, good accuracy and the like.
Description
Technical field
The present invention relates to a kind of detection kit, particularly a kind of latex immune reagent kit for detection of helicobacter pylori antibody in human serum and preparation method thereof.The invention belongs to technical field of biological
Background technology
Helicobacter pylori (Helicobacter pylori is called for short HP) is the bacterium of a kind of one pole, many flagellums, the blunt circle of end, helically bent, is Grain stain feminine gender, is often typical spiral fashion or arc on gastric epithelial cell surface.
Helicobacter pylori infections is that chronic active gastritis, digestibility stomach are burst and die young and the moving lymphadenomatous main pathogenic of film associated lymphoid tissue (MALT) of stomach, and with the generation closely related .1994 World Health Organization (WHO)/international cancer research institution (WHO/IARC) of cancer of the stomach, helicobacter pylori is decided to be to I class procarcinogen.In the Gastric Biopsy of Patients with Chronic Gastritis, helicobacter pylori recall rate can reach 80%～90%. and the burst patient Geng Gao that dies young of digestibility stomach can reach more than 95%, even approaches 100%.Due to local epithelial cell there is alienation in oneself to cancer of the stomach, and therefore its recall rate height report differs.Oneself is a worldwide problem for the infection of helicobacter pylori, and it is carried out to the monitoring that Accurate Diagnosis is conducive to control HP spread and epidemic and eradicates HP treatment of infection.
At present, the HP detection method of having set up is both at home and abroad divided into invasive and the large class of Noninvasive two from detection means.
One, invasive detection method
The feature of these class methods is to detect after using endoscope to obtain gastric biopsy, and test method mainly contains Rapid urease test method, pathology detection method and bacterial cultivation.
1, fast furosemide enzyme detection method: having abundant urease according to HP can produce ammonia by decomposing urea, is developed the color by pH indicator.This method is easy, rapid, sensitive, but also likely because existing and produce false positive containing other bacteriums of urease.In addition, used in the recent period the sample that reduces stomach amount of bacteria or directly suppress the medicine of urease activity can produce false negative.
2, pathology detection method: by mucosa tissue is carried out to section statining inspection, can directly show HP thalline and confirm, wherein argentation verification and measurement ratio is high, but the otherness that different pathological scholar observes has certain difficulty for the diagnosis of lipogastry sample.
3, bacterial cultivation is got gastric mucosa living tissue and is done HP cultivation, because culture of bacteria is less, operating process is not suitable for generally applying compared with complicated, time cycle compare Shi, somewhat expensive.
Two, Noninvasive detection method
This type of method characteristic is not need to use endoscope to take gastric biopsy, thereby avoids patient because the infringement repeatedly doing OGD and bring is painful or the infection of other type occurs.Test method mainly contains urea breath test, PCR detection method and immunoserology method.
1, urea breath test: because HP is rich in urease, after being taken by experimenter with the urea of 13C or 14C mark, detect that according to gas nuclide mass spectrometer decomposing the isotope-labeled carbon dioxide sample of being with producing judges HP gradient of infection.This method is highly sensitive, can be quantitative, but have certain Radio Active Hazard, and be subject to device-restrictive to be difficult to generally promote.
2, PCR detection method: mainly detect HP urease (UReA) gene and toxin associated protein A (CagA) gene in gastric juice, mucous membrane, but complicated operation, easily pollute, having relatively high expectations of professional knowledge to reviewer and operative technique, and greatly increase patient's economy spending, therefore application is clinically also comparatively limited.
3, immunoserology method: existing method comprises euzymelinked immunosorbent assay (ELISA), Western blot and colloidal gold method at present, for the epidemiology survey of HP provides favourable convenient means, but detection speed is slow, except euzymelinked immunosorbent assay (ELISA) can be used full-automatic enzyme non-analysis meter (and need 2 ~ 3 hours, step many), all the other all need manual operations, it is qualitative analysis that patient obtains testing result, is unsuitable for treatment monitoring and curative effect evaluation.
At present, although it is various to detect the method for Helicobacter pylori infection, also there is no a kind of suitable Biochemical Analyzer, can be easy, quick, in enormous quantities and quantitative detect.If set up this kind of method, can make greatly easy of testing process, operating personnel only need set set factors, reagent and sample are placed in behind relevant position, Biochemical Analyzer can automatically complete detection, and can obtain rapidly result, and be easy to apply in medical institutions at different levels, there is great clinical meaning and universal value for the control of helicobacter pylori.
Summary of the invention
The object of the invention is to overcome the defect of existing method and technology, a kind of latex immune reagent kit of measuring helicobacter pylori antibody and preparation method thereof is provided and uses this reagent to carry out the method for clinical sample detection.This reagent is applicable to all types of Biochemical Analyzers, has that sample is high without dilution, simple to operate, quick, quantitatively accurate, reproducible, good stability, susceptibility, the feature of high specificity.
For realizing object of the present invention, adopt following technical scheme:
Of the present invention a kind of for measuring the latex enhancing immune of helicobacter pylori antibody than turbid kit, it is characterized in that comprising: the emulsion reagent of dilution, Heliobacter pylori antigen and blank solution;
Wherein, the emulsion reagent of described Heliobacter pylori antigen is the potpourri that has adsorbed the latex particle of two kinds of different-grain diameters of Heliobacter pylori antigen, and described latex particle is polystyrene microsphere.
In the present invention, preferably, described dilution is the damping fluid that contains surfactant and increased response agent, described damping fluid is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid, is preferably phosphate buffer; Described increased response agent is any one or several in PEG-300, PEG2000 or PEG6000, is preferably PEG6000; In preferred described dilution, also contain the sodium chloride of 2mol/L.
Wherein, inorganic salts sodium chloride can regulate ionic strength, and increased response agent can be accelerated the immune response speed of antigen-antibody, shortens detection time, and surfactant (Tween 20) can be removed nonspecific association reaction.
In the present invention, preferred, described emulsion reagent is that the particle diameter that adsorbed Heliobacter pylori antigen is the potpourri of 60-90nm and the particle diameter latex particle that is 160-200nm.
Preferably, described latex particle is Properties of Polystyrene Nano Particles, and this combination can either improve the sensitivity of reaction, and can improve the sensing range of reagent.
In the present invention, preferred, described emulsion reagent can prepare in accordance with the following methods:
(1) latex pre-treatment
Getting the unlabelled latex particle of 1ml adds in 10ml 0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml 0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 DEG C of preservations are stand-by;
(2) latex particle mark
After step (1) is processed, latex is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, then add 1mg EDC, mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02MPH7.4 phosphate buffer, add 1mg Heliobacter pylori antigen in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle uses confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up, 4 DEG C of preservations,
(3) use above-mentioned steps respectively the latex of particle diameter 60-90nm and 160-200nm to be distinguished to mark, after mark finishes, described 60-90nm latex and described 160-200nm latex are mixed, by quality percent by volume, the concentration that makes 60-90nm latex is 0.673-0.732%, the concentration that makes 160-200nm latex is 0.135-0.323%, after mixing, preserve, the above-mentioned flow process of preferred use is distinguished mark to the latex of particle diameter 70-90nm and 160-180nm respectively, after mark finishes, described 70-90nm latex and described 160-180nm latex are mixed, by quality percent by volume, making 70-90nm latex concentration is 0.708-0.732%, making 160-180nm latex concentration is 0.135-0.235%, after mixing, preserve,
Described confining liquid is the phosphate buffer of the 0.02M PH7.4 that contains 5g/L BSA;
Described dispersion liquid is by BSA, Tween-20, PVP-K30 and biological preservative are formulated by the phosphate buffer of 0.02M PH7.4, and wherein BSA concentration is 1g/L, and Tween-20 concentration is 0.05%(v/v), PVP-K30 concentration is 2g/L, and biological antiseptic agent concentration is 0.05%(v/v).
In the present invention, preferably, described antigen comprises helicobacter pylori whole cell albumen, helicobacter Pylori urease, Cytotoxin-associated gene-A (Cytotoxin associated gene A, Cag A) or helicobacter pylori vacuolate cytotoxin A (Vacuolating cytotoxinA, VacA) in one or more.
Helicobacter pylori can produce a large amount of ureases, and this urease is to bacterium field planting and pathogenic playing an important role in vivo.Urease is mainly made up of A (UreA) and B (UreB) Liang Ge subunit, and molecular weight is about respectively 30kD and 63kD, has already confirmed that urease B subunit (ureB) is an important protective antigens for helicobacter pylori.
Existing research further shows, the helicobacter pylori that accounts for greatly 50-60% can produce cytotoxic activity in vitro, wherein cytotoxic expression is closely related with the cytotoxin-associated gene A that is exposed to helicobacter pylori surface, the albumen of this gene expression exists only in and produces in cytotoxic helicobacter pylori, and there is good immunogenicity, think now, this albumen can provide foundation for the diagnosis of clinical gastric illness as antigen.
Vacuolate cytotoxin A (VacA) is also the specific expressed albumen of helicobacter pylori, has important reference value for the detection of clinical helicobacter pylori.
The preparation process of Heliobacter pylori antigen of the present invention comprises:
Step 1 microbe growth, comprises solid-state cultivation, liquid cultivation in a small amount and liquid a large amount of cultivation;
Step 2: Bacteria Identification, fragmentation, centrifugal, extraction supernatant, purifying and determination of protein concentration.
In the specific embodiment of the invention, a kind of method of preparing helicobacter pylori whole cell antigen is disclosed as a reference.
In kit of the present invention, preferred, also contain standard dilution, calibration object and quality-control product.
Preferably, described standard dilution is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid, is preferably phosphate buffer.
In the present invention, described calibration object, quality-control product are that one is used for and sample comparison, carry out the helicobacter pylori antibody solution of result calculating and quality control, preferably, described calibration object, quality-control product is a kind of helicobacter pylori antibody solution, comprising PBS damping fluid, stabilizing agent, antiseptic and helicobacter pylori specific antibody;
Described stabilizing agent is selected from one or more in protein, amino acid, inorganic salts, surfactant, is preferably the phosphate buffer containing 0.05%BSA,
Described antiseptic is selected from one or more in 0.1% Sodium azide, Procl in-300, gentamicin.
The concentration of calibration object can be high concentration single-point reference calibrations product, become in use the reference calibrations product of multiple variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of multiple variable concentrations, can also be the single-point reference calibrations product of fixed concentration, jointly draw calibration curve with physiological saline.The single-point reference calibrations product of the preferred fixed concentration of the present invention, the concentration of calibration object, quality-control product is respectively 50AU/mL, 31AU/mL.In specific embodiment, a kind of method of preparing calibration object, quality-control product is disclosed.
The invention also discloses the application of described kit in formation determination helicobacter pylori antibody reagent.
Further, the present invention also provides a kind of three joint inspection kits for diagnosis of gastrosis or cancer of the stomach, it is characterized in that comprising of the present invention for measuring the latex enhancing immune of helicobacter pylori antibody than turbid kit, also comprise pepsinogen I detection kit and pepsinogen I I detection kit.
As a reference, provided the preparation method of pepsinogen I detection kit and pepsinogen I I detection kit in specific embodiments of the invention part.
The principle that the present invention measures sample is latex enhancing immune turbidimetry, selected sample is human serum, sample and reagent Rl preincubate (fully exposed the antibody combining site in sample) after 3-5 minute, add reagent R2 (Heliobacter pylori antigen emulsion reagent), continue to hatch 3-5 minute, the Heliobacter pylori antigen of helicobacter pylori specific antibody in human serum in reagent R2 is combined, form insoluble antigen one antibody complex, produce certain turbidity, its pool, degree height are directly proportional to the specific antibody concentration detecting in sample.Under provision wavelengths, measure the absorbance of this insoluble antigen antibody complex, know that with oneself the helicobacter pylori specific antibody calibration object of concentration compares, can calculate the concentration of helicobacter pylori antibody in sample.
Reagent provided by the invention is liquid double reagent, is applicable to all types of automatic clinical chemistry analyzers and semi-automatic biochemical analyzer, compared with prior art, has following characteristics
1, quantitatively detect, highly sensitive, can reach 2.OAU/mL, the range of linearity is wide, can reach 245AU/mL;
2, accuracy in detection and precision are good;
3, high specificity, and be difficult for being disturbed:
4, good stability, can lucifuge store at least 12 months, after each reagent, at least can preserve 14 days for 2-8 DEG C:
5, sample need not dilute in advance, simple to operate, quick, from detecting out that result only needs 10 minutes, be specially adapted to examination in enormous quantities, detection speed can improve with the raising of Biochemical Analyzer detection speed.
Brief description of the drawings
Fig. 1 is kit range of linearity correlativity schematic diagram of the present invention;
Fig. 2 is the schematic flow sheet that uses kit of the present invention to detect.
Fig. 3 is at the bottom of pepsinogen I and stomach stomach and the gastric mucosa situational relationship figure of body of stomach;
Fig. 4 is the gastric mucosa situational relationship figure of pepsinogen I I and stomach stomach hole and stomach angular position.
Embodiment
The invention discloses a kind of latex immunoreagent and preparation and application thereof of measuring helicobacter pylori antibody, those skilled in the art can use for reference content herein, realize by suitable improvement technological parameter.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, within they are all deemed to be included in the present invention's technical scheme required for protection.Product of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention, these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The purchase source of the related main material of the embodiment of the present invention and reagent:
Q-2 anion-exchange chromatography post, DEAE-Sephadex A52(DEAE-52) chromatographic column buys from GE company of the U.S.;
Freund's complete adjuvant, biological preservative Proclin-300,2-(N-morpholine) ethyl sulfonic acid (MES) is bought from SIGMA;
10%(w/w) Properties of Polystyrene Nano Particles solution is bought from U.S. Bangs;
Propepsin I antibody PGI-8003 and PGI-8015 buy from Medix lot number: 0021636,
PGⅡ antibody PGII-8103 and PGII-8101 buy from Medix lot number: 0023880
PVP-K30(Polyvinylpyrrolidone), sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride PFG-6000, EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, Tween-20 etc. are purchased from traditional Chinese medicines group;
Bovine serum albumin(BSA) (BSA) is purchased from the prosperous Golden Horse of unit;
HP bacterial classification, purchased from Shanghai Tiancheng Technology Co., Ltd., numbers 43504;
Helicobacter pylori antibody, purchased from Shanghai Ling Chao biotech firm, is numbered: L1H01202;
The preparation (taking whole cell proteantigen as example) of embodiment 1 Heliobacter pylori antigen
(1) microbe growth
(a) solid-state cultivation: HP bacterial classification is inoculated in chocolate culture-medium, cultivate 3-4 days for 37 DEG C, produce Flat belt point transparent and like gluey bacterium colony, this is helicobacter pylori, provoke several bacterium colonies with oese, after bacterium is scratched with three zoning collimation methods on chocolate culture-medium, cultivate 3-4 days for 37 DEG C, went down to posterity every four days once to keep the activity of bacterium.
(b) liquid cultivation in a small amount: the helicobacter pylori bacterium colony that time state is turned out, hang lower several bacterium colonies with oese, and shake off in 125ml conical flask, in bottle, fill BHI nutrient solution 50ml, wherein containing 1ml newborn calf serum and O.1ml helicobacter pylori screen liquid, conical flask bottleneck is covered tightly with tampon, cultivate 3 ~ 4 days for 37 DEG C.
(c) a large amount of liquid cultivations: draw 0.5ml and cultivate bacterium liquid in a small amount, carry out preliminary urea qualification and outward appearance and observe, confirm not to be subject to other living contaminantses.Draw 15ml bacterium liquid and add in 500ml conical flask, in bottle, fill BHI nutrient solution 250ml, wherein containing 5ml newborn calf serum and helicobacter pylori screening liquid O.5ml, conical flask bottleneck is covered tight with tampon, be placed in 37 DEG C of cultivations 3 ~ 4 days.
(2) Bacteria Identification, fragmentation, centrifugal and extraction supernatant
(a) Bacteria Identification: outward appearance state observation: observing the growth kenel of bacterium colony on chocolate culture-medium, is the transparent seemingly gluey bacterium colony of Flat belt point.
Urea method of inspection: the urease of helicobacter pylori is very abundant, 15% urease catalyzes hydrolysis of urea of about mycetome albumen can produce ammonia.Get 0.5ml urea test solution and be placed in test tube, add 0.5ml bacterium liquid, react about half an hour in 37 DEG C.If contain helicobacter pylori, test solution can change pink into by yellow.
(b) bacteria breaking, centrifugal and extraction supernatant: get 250mL helicobacter pylori bacterium liquid, in 8000rpm centrifugal 20 minutes, abandoning please, precipitation adds 10m10.05M glycine buffer (pH=7.0) to clean, and in 8000rpm centrifugal 20 minutes, abandons supernatant, precipitation adds lOml 0.05M glycine buffer (pH=7.0) to mix, carrying out ultrasonic bacteria breaking, in l8.000rpm centrifugal 20 minutes, take out supernatant and placed 80 DEG C for subsequent use.
(c) antigen protein concentration determination: with the quantitative Heliobacter pylori antigen protein concentration of Bio-Rad protein determination kit, first hyclone albumen (BSA) is dissolved in to the standard items of preparing following each concentration in PBS: Omg/mL, O.lmg/ml, O.2mg/ml, O.3mg/ml, 0.4mg/ml, O.5mg/ml time, then with 5 times of dilution analysis damping fluids of intermediate water (containing Coomassie brilliant blue R-250). then in 96 micropore dishes, add the analysis buffer after 100 μ l dilutions, finally add respectively the Heliobacter pylori antigen protein solution that obtains in the each concentration standard product of 10 μ l and (2), in room temperature reaction 5 minutes, under 595nm wavelength, read OD value with visible spectrophotometer or microplate reader, drawing standard curve, and calculate Heliobacter pylori antigen protein concentration.
The assembling of embodiment 2 kits of the present invention
1 damping fluid configuration
The preparation of 1.1 dilutions (reagent R1)
Reagent in following table 1 is weighed and puts into clean container, add purified water and mix, measuring pH value is 7.4, constant volume 1000ml.
The preparation of table 1 dilution (reagent R1)
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 2.88g |
| Sodium dihydrogen phosphate | 2.4g |
| Sodium chloride | 116.88g |
| PEG-6000 | 2g |
| PVP－k30 | 20g |
| BSA | 10.0g |
| Tween-20 | 0.5ml |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
1.2 the preparation of standard dilution
Reagent in following table 2 is weighed and puts into clean container, add purified water dissolving and mix, measuring pH value is 7.4, is settled to 1000ml.
The preparation of table 2 standard dilution
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 1.44g |
| Potassium dihydrogen phosphate | 1.36g |
| Sodium chloride | 8g |
| Potassium chloride | 0.2g |
| BSA | 10g |
| Glycerine | 5ml |
| Tween-20 | 0.5mL |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
2 preparations of the emulsion reagent (reagent 2) containing Heliobacter pylori antigen
The preparation of 2.1 damping fluids
Reagent in following table 3 has been weighed to the each damping fluid of preparation
The preparation of table 3 damping fluid
2.2 latex pre-treatments
2.2.1 get the unmarked latex particle of 1ml, add in 10ml0.02M pH7.4 phosphate buffer by 1ml 10% Properties of Polystyrene Nano Particles solution, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 DEG C of preservations are stand-by.
2.3 latex particle marks
According to the quality of latex particle, latex after 2.2 processing is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, add 1mg EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M PH7.4 phosphate buffer, add 1mg Heliobacter pylori antigen (embodiment 1 prepares) in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, is separated to other containers for future use by supernatant; Particle uses confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and precipitation is redissolved in dispersion liquid; The latex particle that mark is completed is distributed into 1ml/ and props up 4 DEG C of preservations.
Use above-mentioned flow process respectively the latex of particle diameter 75nm and 167nm to be distinguished to mark, after mark finishes, described 75nm latex and described 167nm latex are mixed, making 75nm latex concentration is 0.724%(w/v), making 167nm latex concentration is 0.147%(w/v).
5, determine extension rate
Get antigenic mark residue supernatant liquid and use respectively OD
260and OD
280measure its absorbance, utilize measurement result and calculate the concentration and the labeling effciency that remain antigen in supernatant according to following formula; Finally determine extension rate according to labelled protein amount.
Antigen concentration=1.45*A
280-0.74*A
260
Protein content in labelled antigen amount=mark total protein concentration-supernatant
Labeling effciency=(protein content in mark total protein concentration-supernatant)/mark total protein concentration * 100%
6, latex particle dilution
According to antigen excess principle, the antigen of mark is diluted with dispersion liquid according to fixed required labelled antigen concentration.
Dilution process: the emulsion reagent that mark is completed dilutes, dilution ratio is 1:8; 1:9; 1:10, uses respectively the emulsion reagent after dilution to carry out calibration test, and choosing linear optimal is best dilution ratio.
7, packing
By detect qualified present latex particulate working fluid carry out packing, labelled after, be placed in 2～8 DEG C of preservations.
8, calibration object and quality-control product preparation
Get 3, sizeable container, use that standard diluent preparing concentration point is 0, the calibration object of 50AU/ml, and the quality-control product of 31AU/ML, each concentration point will mix with the raw material strong solution adding successively.
9, packing
Through after the assay was approved, the calibration object preparing and quality-control product are divided and installed in 1ml plastic bottle, 1.0ml/ bottle.
The object of quality-control product: after calibration completes, first test quality-control product, its concentration should be within the scope indicating, thinks that as exceeded the unsuccessful or kit of kit calibration lost efficacy.In the present embodiment, quality-control product concentration is 31AU/ML, if detectable concentration is between 26-36AU/ML, thinks that kit is effective, exceeds this scope and assert that measurement result is invalid.
The assay method of embodiment 2 helicobacter pylori antibody latex immunoreagents
The ultimate principle that this kit adopts is latex enhancing immune turbidimetry.Pylori antigen is adsorbed on emulsion particle particle, is combined with antigen by the specific antibody of hatching in the human serum that makes dilution, after hatching, the absorbance variable quantity of generation is directly proportional to the specific antibody concentration detecting in sample.
The chief component composition of table 5 kit of the present invention
| Composition | Loading amount | Principal ingredient |
| Dilution (reagent R1) | 25mL/ bottle | PBS damping fluid |
| Emulsion reagent (reagent R2) | 5mL/ bottle | The latex solution of anti-human PG I monoclonal antibody |
| Blank solution | 1mL/ bottle | PBS damping fluid |
| Calibration object | 1mL/ bottle | Helicobacter pylori antibody raw material |
| Quality-control product | 1mL/ bottle | Helicobacter pylori antibody raw material |
| Instructions | 1 part |
Condition of storage and the term of validity: kit should be at 2-8 DEG C kept dry, when storage, avoid weight and should protection against the tide, lucifuge, be Protected from Heat; The term of validity: 12 months.
This kit is applicable to Beckman, Hitachi, Olympus, Toshiba, Roche, Abbott Laboratories, Siemens, step the full-automatic or semi-automatic biochemical analyzer of the brand such as auspicious, and assay method is as follows:
1. reagent configuration: reagent i.e. use, and damping fluid and emulsion reagent will fully mix before mensuration;
2. test condition:
First add R1 reagent 200ul, then add serum sample 5ul, 37 DEG C of reactions added R2 reagent 30ul to read OD after 5 minutes, reacted after 5 minutes reading out data under the wavelength of 578nm, used schematic flow sheet that kit of the present invention detects as shown in Figure 2.
3. result is calculated: do typical curve with standard items concentration, the antigen concentration in sample is read by typical curve.
Embodiment 3 utilizes the mensuration of kit of the present invention to helicobacter pylori antibody
1, adopt kit of the present invention according to the methods analyst of embodiment 5 without 280 of stomach trouble crowds (after health check-up confirm without disease of digestive tract, liver, kidney disease, without stomachache history crowd) and stomach trouble group 329 examples (all through gastrocopy, pathology is made a definite diagnosis.Be divided into 5 groups: duodenal bulbar ulcer group 75 examples; Gastric ulcer group 55 examples; Atrophic gastritis group 61 examples; Cancer of the stomach group 113 examples; Cardia cancer group 5 examples.The results are shown in Table 4:
Table 6 utilizes the mensuration of kit of the present invention to helicobacter pylori antibody
| Group | Number of cases | HP（AU/ML） |
| Control group | 280 | 5.2±4.4 |
| Duodenal bulbar ulcer group | 75 | 10.5±3.6 |
| Gastric ulcer group | 55 | 22.7±8.7 |
| Atrophic gastritis group | 61 | 23.8±9.8 |
| Cancer of the stomach group | 113 | 84.3±27.2 |
| Cardia cancer group | 5 | 66.5±21.3 |
According to clinical test results, in conjunction with literature research, kit of the present invention adopts following diagnosis index, and this evaluation is using 1-10X as evaluating cancer of the stomach degree of risk, and wherein 1X is minimum, and 10X is the highest:
hP≤12AU/ml, without disease of stomach, cancer of the stomach risk index low (1X);
12AU/ml≤HP≤29AU/ml, slight helicobacter pylori infections, cancer of the stomach risk index low (1X)
30AU/ml≤HP≤60AU/ml, height helicobacter pylori infections, cancer of the stomach risk index higher (7-9X), suggestion endoscopy.
hP>=60AU/ml, severe helicobacter pylori infections, cancer of the stomach risk index (9-10X), suggestion endoscopy is to make a definite diagnosis.
2, the result that adopts kit of the present invention to diagnose model case
Case 1
Case 2
Case 3
4 one kinds of assemblings for three joint inspection kits of diagnosis of gastrosis or cancer of the stomach of embodiment
This kit comprises that embodiment 2 prepares helicobacter pylori antibody detection kit and pepsinogen I, pepsinogen I I detection kit.
One, the preparation of pepsinogen I I detection kit:
1 damping fluid configuration
The preparation of 1.1 dilutions (R1)
Reagent in following table 7 is weighed and puts into clean container, add purified water and mix, measuring pH value is 7.4, constant volume 1000ml.
The preparation of table 7 dilution (R1)
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 2.88g |
| Sodium dihydrogen phosphate | 2.4g |
| Sodium chloride | 116.88g |
| PEG-6000 | 2g |
| BSA | 10.0g |
| Tween-20 | 0.5ml |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
1.2 the preparation of standard dilution
Reagent in following table 8 is weighed and puts into clean container, add purified water dissolving and mix, measuring pH value is 7.4, is settled to 1000ml.
The preparation of table 8 standard dilution
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 1.44g |
| Potassium dihydrogen phosphate | 1.36g |
| Sodium chloride | 8g |
| Potassium chloride | 0.2g |
| BSA | 10g |
| Tween-20 | 0.5mL |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
2 preparations of the emulsion reagent (reagent 2) containing pepsinogen I I antibody
2.1 the configuration of damping fluid is as shown in table 9
The configuration of table 9 damping fluid
2.2 latex pre-treatments
2.2.1 get the unmarked latex particle of 1ml, add in 10ml0.02M pH7.4 phosphate buffer by 1ml 10% Properties of Polystyrene Nano Particles solution, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 DEG C of preservations are stand-by.
2.3 latex particle marks
According to the quality of latex particle, latex after 2.2 processing is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, add 1mg EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M PH7.4 phosphate buffer, add the monoclonal antibody 0.5mg PGII-8103 of different antigenic determinants and the potpourri of 0.5mg PGII-8101 for PGⅡ surface, antibody is in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle uses confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up 4 DEG C of preservations.
Use above-mentioned flow process respectively the latex of particle diameter 70nm and 170nm to be distinguished to mark, after mark finishes, described 70nm latex and described 170nm latex are mixed, making 70nm latex concentration is 0.691%(w/v), making 170nm latex concentration is 0.191%(w/v).
5, determine extension rate
Get antibody labeling residue supernatant liquid and use respectively OD
260and OD
280measure its absorbance, utilize measurement result and calculate the concentration and the labeling effciency that remain antibody in supernatant according to following formula; Finally determine extension rate according to labelled antibody amount.
Antibody concentration=1.45*A
280-0.74*A
260
Protein content in labelled antibody amount=mark total protein concentration-supernatant
Labeling effciency=(protein content in mark total protein concentration-supernatant)/mark total protein concentration * 100%
6, latex particle dilution
According to antibody excess principle, the antibody of mark is diluted with dispersion liquid according to fixed required labelled antibody concentration.
Dilution process: the emulsion reagent that mark is completed dilutes, dilution ratio is 1:8; 1:9; 1:10, uses respectively the emulsion reagent after dilution to carry out calibration test, and choosing linear optimal is best dilution ratio.
7, packing
By detect qualified present latex particulate working fluid carry out packing, labelled after, be placed in 2～8 DEG C of preservations.
8, calibration object and quality-control product preparation
Get 6, sizeable container, use standard diluent preparing concentration point is followed successively by the calibration object each point of 0,5.4,15.0,31.0,64.5,107.8 μ g/L, and the quality-control product of 10 μ g/L, and each concentration point will mix with the raw material strong solution adding successively.
9, packing
Through after the assay was approved, the standard items that prepare and quality-control product are divided and installed in 1ml plastic bottle, 1.0ml/ bottle.
The object of quality-control product: after calibration completes, first test quality-control product, its concentration should be within the scope indicating, thinks that as exceeded the unsuccessful or kit of kit calibration lost efficacy.In the present embodiment, quality-control product concentration is 10 μ g/L, if detectable concentration is between 9-12 μ g/L, thinks that kit is effective, exceeds this scope and assert that measurement result is invalid.
Two, the preparation of pepsinogen I detection kit:
1 damping fluid configuration
The preparation of 1.1 dilutions (R1)
Reagent in following table 10 is weighed and puts into clean container, add purified water and mix, measuring pH value is 7.4, constant volume 1000ml.
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 2.88g |
| Sodium dihydrogen phosphate | 2.4g |
| Sodium chloride | 116.88g |
| PEG-6000 | 2g |
| BSA | 10.0g |
| Tween-20 | 0.5ml |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
1.2 the preparation of standard dilution
Reagent in following table 11 is weighed and puts into clean container, add purified water dissolving and mix, measuring pH value is 7.4, is settled to 1000ml.
The preparation of table 11 standard dilution
| Reagent | Every liter of volume |
| Sodium hydrogen phosphate | 1.44g |
| Potassium dihydrogen phosphate | 1.36g |
| Sodium chloride | 8g |
| Potassium chloride | 0.2g |
| BSA | 10g |
| Tween-20 | 0.5mL |
| Proclin-300 | 0.5ml |
| Add purified water extremely | 1000ml |
2 preparations of the emulsion reagent (reagent 2) containing pepsinogen I antibody
The configuration of 2.1 damping fluids
As shown in table 12.
The configuration of table 12 damping fluid
2.2 latex pre-treatments
2.2.1 get the unmarked latex particle of 1ml, be that 1ml10% Properties of Polystyrene Nano Particles solution adds in 10ml0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml 0.02M pH7.4 phosphate buffer is resuspended, 4 DEG C of preservations are stand-by.
2.3 latex particle marks
According to the quality of latex particle, latex after 2.2 processing is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, add 1mg EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02M PH7.4 phosphate buffer, add 0.5mg PGI-8003 antibody and 0.5mg PGI-8015 antibody in 10mg/ml 2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle uses confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, and precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up 4 DEG C of preservations.
Use above-mentioned flow process respectively the latex of particle diameter 75nm and 167nm to be distinguished to mark, after mark finishes, described 75nm latex and described 167nm latex are mixed, making 75nm latex concentration is 0.679%(w/v), making 167nm latex concentration is 0.178%(w/v).After mixing, preserve.
2.4 determine extension rate
Get antibody labeling residue supernatant liquid and use respectively OD
260and OD
280measure its absorbance, utilize concentration and the labeling effciency of antibody in measurement result basis formula calculating once residue supernatant; Finally determine extension rate according to labelled antibody amount.
Antibody concentration=1.45*A
280-0.74*A
260
Protein content in labelled antibody amount=mark total protein concentration-supernatant
Labeling effciency=(protein content in mark total protein concentration-supernatant)/mark total protein concentration * 100%
Calculating protein content in supernatant through measurement is 0.367mg, and labelled antibody amount is 1.633mg, and labeling effciency is 81.65%.
2.5 latex particle dilutions
According to antibody excess principle, the antibody of mark is diluted with dispersion liquid according to fixed required labelled antibody concentration.
Dilution process: the emulsion reagent that mark is completed dilutes, dilution ratio is 1:8; 1:9; 1:10, uses respectively the emulsion reagent after dilution to carry out calibration test, and choosing linear optimal is best dilution ratio.
2.6 packing
By detect qualified present latex particulate working fluid carry out packing, labelled after, be placed in 2～8 DEG C of preservations.
3 calibration objects and quality-control product
3.1 calibration objects and quality-control product compound method
3.1.1 adopt the preparation of pointwise application of sample method;
3.1.2A be standard dilution;
3.1.3 get 6, sizeable container, be followed successively by A, B, C, D, E, the F calibration object each point of 0,13.2,33.3,67.0,150.5,245.0 μ g/L by standard diluent preparing concentration point, and the quality-control product of 30 μ g/L, the raw material strong solution that each concentration point will add successively mixes.
3.2 packing
Through after the assay was approved, the standard items that prepare and quality-control product are divided and installed in 1ml plastic bottle, 1.0ml/ bottle.
The object of quality-control product: after calibration completes, first test quality-control product, its concentration should be within the scope indicating, thinks that as exceeded the unsuccessful or kit of kit calibration lost efficacy.In the present embodiment, quality-control product concentration is 30 μ g/L, if detectable concentration is between 26-36 μ g/L, thinks that kit is effective, exceeds this scope and assert that measurement result is invalid.
Embodiment 5 is for the application of three joint inspection kits of diagnosis of gastrosis or cancer of the stomach
By detecting pepsinogen I, pepsinogen I I and helicobacter pylori antibody content in blood, thereby to the comprehensive reaction gastric mucosal state of analyzing of three results.At the bottom of pepsinogen I mainly reflects stomach stomach and the gastric mucosa situation of body of stomach, its main manifestations as shown in Figure 3; Pepsinogen I I mainly reflects the gastric mucosa situation of stomach stomach hole and stomach angular position, and its main manifestations as shown in Figure 4; Helicobacter pylori is the Main Pathogenic Bacteria that disease of stomach occurs, and the detection of HP antibody has very important effect to the situation of reflection stomach entirety gastric mucosa.
Inspection principle: adopt immunoturbidimetry, taking Immunoturbidimetry as measuring principle.Respectively the helicobacter pylori antibody in sample, PG I and PG II are measured, helicobacter pylori antibody, PG I and PG II respectively with mark the latex particle of Heliobacter pylori antigen and anti-PG I antibody, anti-PG II antibody there is specific antigen-antibody reaction, form immune complex, thereby cause the variation of absorbance.Helicobacter pylori antibody, PG I and PG II concentration in variation and the sample of this absorbance are proportional.Make working curve with helicobacter pylori antibody, PG I and the PG II standard items of concentration known respectively, from this curve, can calculate helicobacter pylori antibody, PG I and PG II content in sample.
Condition of storage and the term of validity: kit should be at 2-8 DEG C kept dry, when storage, avoid weight and should protection against the tide, lucifuge, be Protected from Heat; The term of validity: 12 months.
Be suitable for instrument: the Biochemical Analyzers such as Toshiba (TBA-40FR), Di Rui (CS-T300), Laura (FAITH-1000), Olympus (AU-1000), Mai Rui (BS-200).
Sample requires: patient specimen is without special processing, adopts conventional medical technology to collect whole blood sample, after centrifuging, draw serum for detection of.Test serum, as used within 24 hours, can, in 2～8 DEG C of preservations, if need long-term storage should be kept at below-20 DEG C, and be avoided multigelation.Please do not use significant hemolysis, piarhemia or jaundice sample.
Mensuration to the helicobacter pylori antibody in sample, PG I and PG II is carried out respectively in accordance with the following methods:
The method of inspection:
1. reagent configuration: reagent i.e. use, and damping fluid and emulsion reagent will fully mix before mensuration;
2. test condition:
Serum specimen: 5ul
R1:200ul R2:30ul wavelength: 578nm temperature of reaction: 37 DEG C
First add R1 reagent 200ul, then add serum sample 5ul, 37 DEG C of reactions added R2 reagent 30ul to read OD after 5 minutes, reacted after 5 minutes reading out data under the wavelength of 578nm.
3, result is calculated: do typical curve with standard items concentration, the antigen concentration in sample is read by typical curve.
4, testing result
Adopt kit of the present invention according to the methods analyst of embodiment 5 without 280 of stomach trouble crowds (after health check-up confirm without disease of digestive tract, liver, kidney disease, without stomachache history crowd) and stomach trouble group 329 examples (all through gastrocopy, pathology is made a definite diagnosis.Be divided into 5 groups: duodenal bulbar ulcer group 75 examples; Gastric ulcer group 55 examples; Atrophic gastritis group 61 examples; Cancer of the stomach group 113 examples; Cardia cancer group 5 examples.The results are shown in Table 13:
Table 13 testing result
According to clinical test results, in conjunction with literature research, kit of the present invention adopts following diagnosis index, and this evaluation is using 1-10X as evaluating cancer of the stomach degree of risk, and wherein 1X is minimum, and 10X is the highest:
pG I>=67ng/ml, PG I/PG II>=7.5, HP≤12AU/ml, without disease of stomach, cancer of the stomach risk index low (1X);
pG I>=67ng/ml, PG I/PG II≤7.5,12AU/ml≤HP≤29AU/ml, duodenal bulbar ulcer or gastric ulcer, cancer of the stomach risk index low (1X)
35ng/ml≤PG I≤67ng/ml, PG I/PG II≤3.0, HP>=30AU/ml, is diagnosed as atrophic gastritis, cancer of the stomach risk index high (7-9X), suggestion endoscopy.
pG I <35ng/ml, PG I/PG II≤1.5, HP>=60AU/ml is diagnosed as cancer of the stomach, cancer of the stomach risk index (9-10X), suggestion endoscopy is to make a definite diagnosis.
2, the result that adopts kit of the present invention to diagnose model case
Case 1
Case 2
Case 3
Case 4
The sensitivity of the latex of test example 1 single particle size and the mixing latex of two kinds of particle diameters and range of linearity comparison
Test the biochemical instruments that uses be the auspicious CS-T300 of enlightening.
Sensitivity comparison experiment: use latex after single 75nm particle diameter mark and 75nm and the latex mixing after 167nm mark null value calibration object to be carried out to the turbidity changing value of replication 20 times, calculating mean value, more sluggishness is higher for turbidity changing value.
Range of linearity contrast experiment: use latex after single 167nm particle diameter mark and 75nm and the latex mixing after 167nm mark 50AU/ml calibration object to be carried out to the turbidity changing value of replication 20 times, calculating mean value, the larger range of linearity of turbidity changing value is larger.Measurement result is as shown in table 14.
Table 14 sensitivity and range of linearity measurement result
Single use particle diameter occurs that lower than the latex of 100nm detection sensitivity does not reach requirement, the range of linearity is less for the latex that single use particle diameter is 200nm left and right, cannot take into account detection sensitivity and the range of linearity therefore use single particle size latex simultaneously, use compounded latex of the present invention to carry out after mark, can improve detection sensitivity, can widen again the detection range of linearity, to disease, particularly the detection of stomach trouble or cancer of the stomach have detect fast, susceptibility is high, high specificity and the advantage such as accuracy is good.
Claims (9)
1. for measuring the latex enhancing immune of helicobacter pylori antibody than a turbid kit, it is characterized in that comprising: the emulsion reagent of dilution, Heliobacter pylori antigen and blank solution;
Described dilution is the damping fluid that contains increased response agent, and described damping fluid is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid; Described increased response agent is any one or several in PEG-300, PEG2000 or PEG6000;
The emulsion reagent of described Heliobacter pylori antigen is the potpourri that has adsorbed the latex particle of two kinds of different-grain diameters of Heliobacter pylori antigen, and described latex particle is polystyrene microsphere;
Described emulsion reagent prepares in accordance with the following methods:
(1) latex pre-treatment
Getting the unlabelled latex particle of 1ml adds in 10ml0.02M pH7.4 phosphate buffer, the centrifugal 20min of hydro-extractor 10000rpm, remove supernatant, use the resuspended particle of 10ml0.02M pH7.4 phosphate buffer again, the centrifugal 20min of hydro-extractor 10000rpm, removes supernatant and use 5ml0.02M pH7.4 phosphate buffer is resuspended, 4 DEG C of preservations are stand-by;
(2) latex particle mark
After step (1) is processed, latex is diluted to 10mg/ml concentration with 0.02M pH6.1MES damping fluid by latex particle, then add 1mg EDC, mix activation 15 minutes, centrifugal 8000rpm supernatant discarded, and resuspended with 0.02MpH7.4 phosphate buffer, add 1mg Heliobacter pylori antigen in 10mg/ml2ml latex fluid, room temperature mixes 2-4 hour, centrifugal 15 minutes of hydro-extractor 8000rpm, supernatant is separated to other containers for future use, particle uses confining liquid resuspended, and room temperature mixes 1 hour, the centrifugal 15min of hydro-extractor 8000rpm, precipitation is redissolved in dispersion liquid, the latex particle that mark is completed is distributed into 1ml/ and props up, 4 DEG C of preservations,
(3) use above-mentioned steps respectively the latex of particle diameter 60-90nm and 160-200nm to be distinguished to mark, after mark finishes, described 60-90nm latex and described 160-200nm latex are mixed, by quality percent by volume, the concentration that makes 60-90nm latex is 0.673-0.732%, making the concentration of 160-200nm latex is 0.135-0.323%, after mixing, preserves;
Described confining liquid is the phosphate buffer of the 0.02M pH7.4 that contains 5g/L BSA;
Described dispersion liquid is by BSA, Tween-20, PVP-K30 and biological preservative are formulated by the phosphate buffer of 0.02M pH7.4, wherein BSA concentration is 1g/L, the concentration of volume percent of Tween-20 is 0.05%, PVP-K30 concentration is 2g/L, and the concentration of volume percent of biological preservative is 0.05%.
2. according to kit claimed in claim 1, it is characterized in that: described dilution is the damping fluid that contains increased response agent, wherein, described damping fluid is phosphate buffer; Described increased response agent is PEG6000; In described dilution, also contain the sodium chloride of 2mol/L.
3. according to kit claimed in claim 1, it is characterized in that using above-mentioned flow process respectively the latex of particle diameter 70-90nm and 160-180nm to be distinguished to mark, after mark finishes, described 70-90nm latex and described 160-180nm latex are mixed, by quality percent by volume, making 70-90nm latex concentration is 0.708-0.732%, making 160-180nm latex concentration is 0.135-0.235%, after mixing, preserves.
4. according to kit claimed in claim 1, it is characterized in that: described antigen comprises one or more in helicobacter pylori whole cell proteantigen, helicobacter Pylori urease antigen, Cytotoxin-associated gene-A antigen or helicobacter pylori vacuolate cytotoxin Staphylococal Protein A.
5. according to kit claimed in claim 1, it is characterized in that: also contain standard dilution, calibration object and quality-control product, described standard dilution is any in Tris/HCl damping fluid, phosphate buffer, HE PES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid or HEPPS damping fluid.
6. according to kit claimed in claim 5, it is characterized in that: described standard dilution is phosphate buffer.
7. according to kit claimed in claim 5, it is characterized in that: described calibration object, quality-control product is a kind of helicobacter pylori antibody solution, comprising PBS damping fluid, stabilizing agent, antiseptic and helicobacter pylori specific antibody;
Described stabilizing agent is selected from one or more in protein, amino acid, inorganic salts, surfactant;
Described antiseptic is selected from one or more in 0.1% Sodium azide, Proclin-300, gentamicin.
8. the application of the kit described in claim 1-7 any one in formation determination helicobacter pylori antibody reagent.
9. for three joint inspection kits of diagnosis of gastrosis or cancer of the stomach, it is characterized in that comprising the kit described in claim 1-7 any one, also comprise pepsinogen I detection kit and pepsinogen I I detection kit.
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