CN105277722A - Protein chip for typing detection on helicobacter pylori infection - Google Patents
Protein chip for typing detection on helicobacter pylori infection Download PDFInfo
- Publication number
- CN105277722A CN105277722A CN201510711577.8A CN201510711577A CN105277722A CN 105277722 A CN105277722 A CN 105277722A CN 201510711577 A CN201510711577 A CN 201510711577A CN 105277722 A CN105277722 A CN 105277722A
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- China
- Prior art keywords
- protein chip
- antigens
- helicobacter pylori
- detection
- antigen
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
Abstract
The invention provides a protein chip for typing detection on helicobacter pylori infection. The protein chip comprises a basement membrane and antigens which are respectively dotted on the basement membrane in the form of dot matrix, wherein the antigens include three types, i.e. CagA (cytotoxin-associated gene) antigens, VacA (Vacuolating cytotoxin A) antigens and Ure (ureaplasma urealyticum) antigens. Due to the fact that the detection antigens used by the protein chip are three single proteins, i.e. Ure, CagA and VacA, specific IgG antibodies for the three antigens in the blood serum of a patient can be detected at the same time, I-type infection or II-type infection can be judged, and clinic treatment is better facilitated; furthermore, the protein chip has the advantages of high detection sensitivity, quick, simple and convenient detection, instant readability, small using amount of antigen and the like.
Description
Technical field
The present invention relates to a kind of protein chip detecting helicobacter pylori infections for somatotype, belong to biochip technology field.
Background technology
Helicobacter pylori (Hp) can in people's tumor growth, breeding, and can get rid of external through ight soil, saliva again, broadcast by mouth-oral instructions, the approach such as the propagation of fecal-oral transmission, endoscope, close contact are propagated in crowd.In developing country, nearly 80% children less than 10 years old and the adult more than 90% infected.
Body can be stimulated to produce strong immune response through studying the albumen such as the CagA+ Hp (CagA), vacuolate cytotoxin A albumen (VacA) and the urease (Ure) that find Hp for many years, can corresponding antibody be detected in the serum of Hp infected patient.Joint-detection is carried out to the antibody of these antigens, susceptibility, specificity that helicobacter pylori is detected can not only be improved, and more pathogen infection relevant information can be obtained, for the Diagnosis and Treat of clinician is offered help.
Different according to toxinferous situation, Hp can be divided into can toxigenic I type bacterial strain and can not toxigenicly be II type bacterial strain.The disease that I type Hp strain infection causes mainly comprises: multiple disease of digestive tract such as chronic superficial gastritis, atrophic gastritis, chronic gastritis and duodenal ulcer and cancer of the stomach etc., also may cause halitosis, other relevant diseases such as property disease, skin disease etc.In addition, I type Hp infects or a key factor of gastric mucosa associated lymphoid tissue lymthoma development, closely related with the generation of MALT lymthoma and cancer of the stomach.II type Hp bacterial strain is not owing to producing cytotoxin, and virulence is low, generally only causes chronic superficial gastritis after infecting.
After human infection Hp, in blood and gastric juice, produce specific IgG, IgM and IgA antibody, can be used for the detection of plasma diagnosis that Hp infects.
Clinical or the laboratory diagnostic method of helicobacter pylori mainly contains:
13c and
14the experiment of C urea breath, direct smear, bacteria distribution cultivation, RUT experiment (RUT), pathological tissue section statining, detection of plasma method etc.Wherein
13c and
14the experiment of C urea breath is for detecting " goldstandard " of " helicobacter pylori ", but the method can only detect and whether infects helicobacter pylori and cannot judge infection conditions and testing cost is higher; Direct smear, bacteria distribution are cultivated, RUT tests (RUT), pathological tissue section statining needs to be drawn materials by gastroscope, belongs to traumatic, detect patient painful large, not easily accept, exist and sample the inaccurate false positive caused; Detection of plasma method mainly contains enzyme linked immunosorbent assay (ELISA), complement combined techniques, Western blot, latex agglutination test and hemagglutination test, wherein conventional with ELISA method.ELISA method accuracy rate is up to 90% ~ 95%.Generally believe at present, ELISA detect peripheral blood anti-HpIgG highly sensitive, specificity is high, reproducible, be easy to apply, but once can only detect a detection, detection time is longer; Latex agglutination test, detect the antibody in Trace Blood with the Hp be combined on latex particle, advantage does not need instrument, and blood sample requirement is few, and result needs experience, and accuracy is unsatisfied with; Western-blot is mainly used in analyzing the antibody production to Hp plurality of antigens component in the infected's blood, antigen preparation quality can be controlled and analyze with the cross reaction of other similar bacterium but complicated operation, technical difficulty is large and unstable, does not make routine diagnostic method.
Obtained at present listing with product similar mainly contain helicobacter pylori antibody detection kit (colloidal gold method); Helicobacter Pylori urease antibody assay kit (colloidal gold method); Helicobacter pylori IgG Antibodies detection agent box (colloidal gold method).The detectable antigens that the said goods uses or be single Ure albumen (as Gold standard), only can detect the anti-Ure antibody in patients serum, therefore positive rate is limited; Or whether infect for hybrid antigen can only detect helicobacter pylori, cannot judge it is that I type and II type infect.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of protein chip detecting helicobacter pylori infections for somatotype is provided.
The protein chip detecting helicobacter pylori infections for somatotype of the present invention, comprise basement membrane and put respectively at epilamellar antigen with the form of dot matrix, described antigen has three kinds: CagA antigen, VacA antigen and Ure antigen.
Described basement membrane is also fixed with negative control and reaction contrast.
Containing the kit detecting the protein chip of helicobacter pylori infections for somatotype of the present invention, comprising:
1) cleansing solution: by TBE and saturated ammonium sulfate solution composition;
2) developer: staphylococcal protein A colloid gold label thing;
3) protein chip.
This chip take patients serum as sample, can detect experimenter simultaneously and whether infect CagA, VacA and Ure3 kind antibody, thus be judged as that a few type infects, be more conducive to the Diagnosis and Treat of clinician.
Be different from obtained at present listing with product similar mainly contain helicobacter pylori antibody detection kit (colloidal gold method); Helicobacter Pylori urease antibody assay kit (colloidal gold method); Helicobacter pylori IgG Antibodies detection agent box (colloidal gold method).The difference of the present invention and mentioned reagent box is: the detectable antigens that the said goods uses or be single Ure albumen (as Gold standard), and only can detect the anti-Ure antibody in patients serum, therefore positive rate is limited; Or whether infect for hybrid antigen can only detect helicobacter pylori, cannot judge it is that I type and II type infect.And the detectable antigens that the present invention uses is Ure, CagA, VacA tri-kinds of single albumen, can detect simultaneously in patients serum for the specific IgG antibodies of three kinds of antigens, thus can judge it is that I type or II type infect, more be conducive to clinical treatment, in addition also to have detection sensitivity high for this product, fast and convenient, vertical etc. readable, the advantages such as antigen consumption is few.
Accompanying drawing explanation
Fig. 1 is the schematic diagram detecting protein chip 16 dot pattern of helicobacter pylori infections for somatotype of the present invention.0 is reaction contrast probe, and 1 is CagA detection, and 2 is VacA detection, and 3 is Ure detection, and 4 is negative control probe, and the common negative control probe of detection is the BL21 bacterium lysate of pRSET-A plasmid transfection.
Embodiment
Embodiment 1 prepares protein chip
Specifically comprise the steps:
1. prepare NC diaphragm
(1) be that the NC film of 0.45 μm first uses distilled water immersion by aperture, take out airing, airing after soaking with 0.05mol/L carbonate buffer solution.
(2) with semi-automatic sanction film machine, NC film is cut into the square piece of 0.9cm × 0.9cm, for subsequent use.
(3) get the CagA antigen of the 0.5mg/ml diluted, the VacA antigen of 1.0mg/ml, the Ure antigen of 1.0mg/ml puts into the corresponding sample box of point sample instrument respectively.
(4) under temperature control (≤10 DEG C) condition, nitrocellulose filter is flatly positioned in point sample groove, with MicroGrid-2 point sample instrument by antigenic solution, location solution and Quality Control contrast solution with the form of dot matrix point on nitrocellulose filter, every 3 points are one group.Point sample diameter should control at 0.8 ± 0.1mm, and dot spacing is 0.8mm.Product for matrix, adopts 16 dot pattern point samples, as shown in Figure 1 with cellulose nitrate (NC) film.
(5), after point sample, with 2%BSA lock solution Seal treatment NC film, three times are cleaned with distilled water, each 10min, drier, for subsequent use.
2. assemble protein chip
(1) housing screening: check that chip surface of shell has no marking, incompleteness or distortion, whether paint is even, and the housing if any this type of phenomenon puts into substandard products basket without exception.Housing without quality problems is placed in certified products Special-purpose basket for subsequent use.
(2) pad pasting: be fixed on the glue limit of chip case inside in the NC diaphragm front of making, makes it be in Process window center.
(3) absorbent material is filled: first a lens wiping paper front is close to the NC diaphragm back side and places, and then reverse side absorbent material being close to lens wiping paper keeps flat.
(4) buckle closure: chip upper shell and lower house are linked closely, makes housing four gaps evenly detain reality.
(5) label the name of an article: by the label consistent with the NC diaphragm name of an article, be pasted in the name of an article groove in chip housing front.
(6) chip purification: the dust on surface of shell and NC diaphragm and filament are blown away with ear washing bulb, keeps face clean.
7) chip envelope: the chip after purified treatment is neatly loaded the valve bag posting respective identification, and be placed in 4 DEG C of constant temperature preservations.
Embodiment 2 detects the kit of the protein chip of helicobacter pylori infections for the preparation of somatotype
Kit is made up of the protein chip of cleansing solution, developer and embodiment 1.
1. prepare cleansing solution
(1) measure 500mlTBE damping fluid with 1000ml graduated cylinder, pour 2000ml beaker into;
(2) measure 100ml saturated ammonium sulfate solution with 100ml graduated cylinder, pour 2000ml beaker into;
(3) open magnetic stirring apparatus switch, adjusting rotary speed can stir to liquid;
(4) measure 20mlTween-20 with 100ml graduated cylinder, pour 2000ml beaker into, continue to stir;
(5) 500ml deionized water is measured with 500ml graduated cylinder;
(6) pour the graduated cylinder filling Tween-20 into and be about 100ml, the residual Tween-20 of shake graduated cylinder cleaning, then pour 2000ml beaker into;
(7) repeating to wash 3 times, residue deionized water is all poured into 2000ml beaker, being settled to after 1000ml fully mixes, by often propping up 7ml packing.
Note: TBE (1L): 54gTris, 27.5gH
3bO
3, 10ml0.5MpH8.0EDTA
2. prepare collaurum and immune colloid gold developer
Prepare collaurum: the gold chloride 300ul of 0.4g/mL is joined 800ml deionized water and is then heated to boil, add rapidly 1g/mL trisodium citrate 32ml under agitation, continue heating 10min, liquid is that wine red is aurosol, adds and steam water to original volume after cooling.
Prepare immune colloid gold developer:
(1) 800ml collaurum is got, with the K of 0.1M
2cO
3solution adjusts pH to 6.0;
(2) under the condition stirred, dropwise add the SPA of 5ml1mg/ml, continue to stir 20min, guarantee to mix but non-foaming foam;
(3) under the condition stirred, drip the BSA of 10ml10%, continue to stir 5min and fully mix;
(4) with the centrifuge tube of 70ml, in 4 DEG C of centrifugal 10min of 3000rpm, supernatant is got;
(5) by supernatant in 4 DEG C of centrifugal 40min of 14000rpm, abandon supernatant;
(6) precipitation is suspended in the suspending liquid of 800ml, by often propping up 4ml packing.
Embodiment 3 detection validation
Get each 5 parts of the serum of normal person and infection Patients with H. pylori respectively, be divided into control group and experimental group.Adopt the kit of embodiment 2, in strict accordance with the following step operation, carry out the testing result contrasting and verify protein chip of the present invention.
Detecting step:
1) film is moistened: get cleansing solution 160 μ l (4) and be added on chip detection window face, uniform wet face;
2) application of sample: get the serum to be checked 200 μ l handled well, be added on chip detection window face;
3) washing: after sample fully infiltrates, for ensureing clean result, washing in two steps: get cleansing solution 120 μ l (3), be added on chip detection window face, after cleansing solution infiltrates, then get cleansing solution 120 μ l (3), be added on chip detection window face;
4) develop the color: after cleansing solution fully infiltrates, get developer 360 μ l (6), be added on chip detection window face;
5) wash: after developer fully infiltrates, for ensureing clean result, wash in two steps: get cleansing solution 120 μ l (3), be added on chip detection window face, after cleansing solution fully infiltrates, get cleansing solution 120 μ l (3) again, be added on chip detection window face;
6) detect: after cleansing solution fully infiltrates, read gray-scale value with biological chip reading apparatus.
Found that, time on the protein chip that normal human serum detects, on protein chip, except reaction contrast probe has colour developing, the colourity of all the other detections does not all change; And during Virus monitory protein chip with Patients with H. pylori, on protein chip, reaction contrast probe and detection all have colour developing, concrete outcome is see table 1.
Table 1
Experimental result shows that the colourity of each detection changes along with the difference of the content of Helicobacter pylori IgG Antibodies in serum, the gray-scale value of detection increases progressively along with the increase of Helicobacter pylori IgG Antibodies content in serum within the specific limits, when in serum, the content of Helicobacter pylori IgG Antibodies reaches a critical value, its gray-scale value reaches maximal value no longer to be increased.And experiment shows that the accuracy of its diagnosis of 3 albumen joint-detection is obviously better than a Protein Detection item; And 3 albumen joint-detection are more fast and convenient relative to the method for 3 detections, 3 albumen.
Claims (3)
1. detect a protein chip for helicobacter pylori infections for somatotype, it is characterized in that, comprise basement membrane and put respectively at epilamellar antigen with the form of dot matrix, described antigen has three kinds: CagA antigen, VacA antigen and Ure antigen.
2. the protein chip detecting helicobacter pylori infections for somatotype according to claim 1, is characterized in that, described basement membrane is also fixed with negative control and reaction contrast.
3., containing, for example the kit that for somatotype detect the protein chip of helicobacter pylori infections of claim 1-2 described in any one, it is characterized in that, comprising:
1) cleansing solution: by TBE and saturated ammonium sulfate solution composition;
2) developer: staphylococcal protein A colloid gold label thing;
3) protein chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510711577.8A CN105277722A (en) | 2015-10-28 | 2015-10-28 | Protein chip for typing detection on helicobacter pylori infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510711577.8A CN105277722A (en) | 2015-10-28 | 2015-10-28 | Protein chip for typing detection on helicobacter pylori infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105277722A true CN105277722A (en) | 2016-01-27 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510711577.8A Pending CN105277722A (en) | 2015-10-28 | 2015-10-28 | Protein chip for typing detection on helicobacter pylori infection |
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| CN (1) | CN105277722A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867698A (en) * | 2016-12-27 | 2017-06-20 | 泰州欣康基因数码科技有限公司 | A kind of biochip cleaning fluid |
| CN106932585A (en) * | 2017-04-26 | 2017-07-07 | 蔡长春 | Helicobacter pylori collaurum parting test strip and kit |
| CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
| CN113804879A (en) * | 2020-06-15 | 2021-12-17 | 北京康美天鸿生物科技有限公司 | Helicobacter pylori IgM and IgG antibody joint detection kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1139544A (en) * | 1995-07-06 | 1997-01-08 | 中国预防医学科学院流行病学微生物研究所 | Quick diagnose reagent box for pylorus helicobacterium infection |
| CN2554170Y (en) * | 2002-07-01 | 2003-06-04 | 西安联尔生物技术有限公司 | Bio-chip for detecting pylorus spirochete bacillus index |
-
2015
- 2015-10-28 CN CN201510711577.8A patent/CN105277722A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1139544A (en) * | 1995-07-06 | 1997-01-08 | 中国预防医学科学院流行病学微生物研究所 | Quick diagnose reagent box for pylorus helicobacterium infection |
| CN2554170Y (en) * | 2002-07-01 | 2003-06-04 | 西安联尔生物技术有限公司 | Bio-chip for detecting pylorus spirochete bacillus index |
Non-Patent Citations (3)
| Title |
|---|
| 孟祥坤等: "蛋白芯片检测幽门螺杆菌抗体谱与胃十二指肠疾病的关系", 《宁夏医学杂志》 * |
| 李光艳: "金标免疫斑点法快速诊断儿童幽门螺旋杆菌感染", 《实用医药杂志》 * |
| 李红涛等: "蛋白芯片对幽门螺杆菌感染的诊断", 《解放军预防医学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867698A (en) * | 2016-12-27 | 2017-06-20 | 泰州欣康基因数码科技有限公司 | A kind of biochip cleaning fluid |
| CN106932585A (en) * | 2017-04-26 | 2017-07-07 | 蔡长春 | Helicobacter pylori collaurum parting test strip and kit |
| CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
| WO2019011125A1 (en) * | 2017-07-11 | 2019-01-17 | 深圳市伯劳特生物制品有限公司 | Composition for elisa kit and kit for detecting spectrum of helicobacter pylori antibody and preparation method thereof |
| CN108279302B (en) * | 2017-07-11 | 2020-05-26 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, helicobacter pylori antibody spectrum detection kit and preparation method thereof |
| CN113804879A (en) * | 2020-06-15 | 2021-12-17 | 北京康美天鸿生物科技有限公司 | Helicobacter pylori IgM and IgG antibody joint detection kit |
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