CN102393460A - Rapid detection device for helicobacter pylori - Google Patents
Rapid detection device for helicobacter pylori Download PDFInfo
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- CN102393460A CN102393460A CN2011102411090A CN201110241109A CN102393460A CN 102393460 A CN102393460 A CN 102393460A CN 2011102411090 A CN2011102411090 A CN 2011102411090A CN 201110241109 A CN201110241109 A CN 201110241109A CN 102393460 A CN102393460 A CN 102393460A
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Abstract
The invention discloses a rapid detection device for helicobacter pylori. The device comprises construction of a helicobacter pylori pairing monoclonal antibody, test paper comprising the monoclonal antibody, and a detection device. A method for manufacturing the device comprises the following steps of: inoculating recombinant helicobacter pylori to a mouse for immunizing; constructing a tumor strain by fusing SP20 cells for screening; injecting tumor cells into the abdominal cavity of an BALB/c mouse to make the BALB/C mouse produce ascites; extracting the ascites of the mouse for purifying and screening to obtain two strains of helicobacter pylori pairing monoclonal antibodies with high specificity; marking the monoclonal antibodies as Hp-1 and Hp-2; and applying the pairing monoclonal antibodies to test paper to obtain a detection device with high specificity and high sensitivity specific to the helicobacter pylori.
Description
Technical field
The present invention is a kind of test paper that utilizes the colored particle immunization method to detect helicobacter pylori.
Background technology
Helicobacter pylori (Helicobacter pylori Hp) is one of human modal gastrointestinal pathogen, and China general population's infection rate is 50%-80%, and still with annual 1%-2% speed increment.Helicobacter pylori infection not only with the substantial connection that has of Type B gastritis and peptic ulcer; And important relationship is also arranged with non-ulcer dyspepsia, MALT lymthoma and cancer of the stomach, so listed in first kind procarcinogen by international cancer research institution of the World Health Organization (WHO).
Detection methods such as the microbe growth of generally acknowledging at present, breath test (UBT), tissue staining; Though have high sensitivity and specificity; Can be used as Hp Infect And Diagnose standard, big, the required instrument of said method misery is expensive, complex operation step is time-consuming, need the professional.Helicobacter pylori is grown under little aerobic environment, and condition of culture requires high, is difficult for survival, microbe growth is done clinical diagnosis cause very big difficulty, is difficult for promoting.And breath test is still unresolved to the pollution of patient and medical worker and surrounding environment.
Utilize immunological method two kinds of approach to be arranged for the detection of helicobacter pylori, the one, detect antibody, the 2nd, detect antigen.Detecting antibody needs serum as test sample, and detecting the antigen noninvasive method then can be through detecting ight soil antigen as the means that detect.Existing at present a lot of documents are reported helicobacter pylori ight soil antigen, detect ight soil antigen and have Clinical detection meaning and value equally, can be used as clinical diagnosis foundation and result of treatment and observe.The ELISA method detects ight soil antigen to be had than high specific and susceptibility by clinical approval, but needs professional and instrument, and the running time is long, and cost is high, and kit needs refrigeration, and it is inconvenient to transport.Chinese patent 01223042 pylori antigen fast detecting box; And in this patent specification the 3rd page of the 10th row said be with polyclone Hp antibody as solid-phase capture antibody with detect antibody; Polyclonal antibody has cross reactivity usually, and performance is relatively poor on specificity.
According to existing research; The positive Hp of cytotoxin-associated protein (CagA) is a virulent strain; Closely related with the disease of digestive tract that atrophic gastritis, cancer of the stomach and digestive tract ulcer etc. are common, the expression that is positive of the helicobacter pylori clinical isolates strain CagA more than 90%.Therefore, the present invention adopts the monoclonal antibody of anti-CagA, utilizes the colored particle immunization method to realize the ight soil detection of antigens, the method high specificity, and highly sensitive, cost is low, and is easy and simple to handle, is convenient to store and transportation.
Summary of the invention
The present invention has overcome the shortcoming that exists in the prior art, and a kind of helicobacter pylori device for fast detecting is provided, adopt anti-CagA monoclonal antibody, process with double antibodies sandwich method principle, be utilize the Hp monoclonal antibody as solid-phase capture antibody with detect antibody.Characteristics such as monoclonal antibody has high specificity, and is highly sensitive, and difference between batch is little.
In order to solve the problems of the technologies described above, the present invention realizes through following technical scheme:
The present invention includes helicobacter pylori pairing monoclonal antibody foundation, comprise the detection test paper and the pick-up unit of this monoclonal antibody.
Set up helicobacter pylori pairing monoclonal antibody, adopt the recombinant C agA that handled to be inoculated in mouse and carry out immunity, set up the knurl strain with the SP20 Fusion of Cells and screen, get oncocyte and be injected in the BALB/c mouse abdominal cavity, make it produce ascites.Extract the screening of mouse ascites purifying, finishing screen is selected two strains and is had strong specificity pairing Hp monoclonal antibody, is designated as Hp-1 and Hp-2.
The pairing monoclonal antibody is applied to detect test paper after by colloid gold label; Collaurum is a kind of of metal colloid particles in the colored particle; Method involved in the present invention and device remove and adopt the colloid gold particle labelled antibody; Also can adopt other similar colored particle mark, promptly can be a kind of metal colloid particles, a kind of marking particle, includes but are not limited to collargol particle, iron particle, magnetic-particle, dye granule, latex particle, fluorescent grain.
Utilize colloidal gold immunity chromatography to process the detection test paper; Realize through following method: detect test paper and form by base plate, water sucting plate, nitrocellulose filter, helicobacter pylori monoclonal antibody gold mark pad, sample liquid-adsorption layer, Max line; The base plate middle part is a nitrocellulose filter; A test wire and a polyclonal antibody control line are arranged on the nitrocellulose filter, and base plate one end termination is a water sucting plate, and other end termination is the sample liquid-adsorption layer; The nitrocellulose filter two ends overlap each other with helicobacter pylori monoclonal antibody gold mark pad with water sucting plate respectively and are connected, and on helicobacter pylori monoclonal antibody gold mark pad, are pressed with the sample liquid-adsorption layer; The sample liquid-adsorption layer is made up of the trilaminate material stack; Ground floor is that certain specification nonwoven layer, the second layer are that glass layer, the 3rd layer are the certain specification nonwoven layer; Above-mentioned substance all need pass through surfactant damping fluid immersion treatment, and dry back is subsequent use, and said apparatus is except that having the capillarity principle; Also have the syphonic effect principle, accelerate the suction translational speed greatly.By known technology base plate, water sucting plate, nitrocellulose filter and monoclonal antibody gold mark pad pad are assembled.
Detect test paper utilization immunity double antibodies sandwich method and process, helicobacter pylori is matched monoclonal antibody be applied to detect test paper, Hp-1 is used to encapsulate test wire, and another strain Hp-2 monoclonal antibody is used for colloid gold label.Control line is to be encapsulated by the sheep anti mouse polyclonal antibody to form, and when the helicobacter pylori monoclonal antibody of sample through colloid gold label moved to sheep anti mouse polyclonal antibody control line, control line just developed the color.
Can above-mentioned detection test paper sample liquid-adsorption layer end directly be inserted and detect (sample liquid must not surpass test paper Max line) in the testing sample, can also process pick-up unit and detect.
Check-out console comprises plate body and plate lid two parts, and check-out console is processed by the moderate material of hardness, and there are well and display window as a result in plate body front, detects test paper and places between plate body and the plate lid, and the plate body covers with plate and is connected through the buckle mode.Be connected last time when the plate body covers with plate through the buckle mode, the sample liquid-adsorption layer is closed within the plate lid, and the nitrocellulose filter of colloid gold test paper is corresponding with the display window as a result of plate body.
Gather the ight soil test sample; Dilute with dilution, draw sample and splash in the device well, because the capillarity sample will move along test strips water accepting layer end; When moving to helicobacter pylori monoclonal antibody II gold mark pad; Helicobacter pylori in the sample and helicobacter pylori monoclonal antibody gold mark probe generation specific bond, when moving to when being fixed with helicobacter pylori monoclonal antibody I test wire, the helicobacter pylori in the sample again with test wire in the helicobacter pylori monoclonal antibody combine; Therefore its collaurum is stranded on the test wire, and the colour developing of test wire place is positive; If do not have helicobacter pylori in the opposite sample; Helicobacter pylori monoclonal antibody gold mark probe just not can with the helicobacter pylori monoclonal antibody I generation specific bond on the test wire; Do not have collaurum to be detained, promptly only have a control line negative, double antibodies sandwich method principle that Here it is.According to this principle, two lines are positive, and line is negative to draw judgement.
When moving to sheep anti mouse polyclonal antibody control line, no matter have or not helicobacter pylori to be checked in the sample, the gold mark probe of mark all can combine with the sheep anti mouse polyclonal antibody that has configured to be detained, and control line is developed the color.Therefore control line does not have colour band and produces that then the representative operation is wrong, during detection the sample liquid level surpass the MAX line or test paper expired.
According to the helicobacter pylori device for fast detecting of such scheme realization, have quick, sensitive, accurate, advantages of simple operation, have simultaneously and preserve conveniently, the characteristics of term of validity length.
Description of drawings
Fig. 1 is an outside drawing of the present invention.
Fig. 2 is a cut-open view of the present invention.
Fig. 3 is the outside drawing of test paper.
Fig. 4 is the exploded view of test paper.
Fig. 5 is the negative findings figure of test paper.
Fig. 6 is the positive findings figure of test paper.
1. plate lid, 2. plate body, 3. well, 4. display window as a result, 5. groove 6. detects test paper, 7. base plate, 8. water sucting plate, 9. nitrocellulose filter, 10. monoclonal antibody gold mark pad, 11. sample liquid-adsorption layers, 12. test wires, 13. control lines, 14.MAX line.
Specific embodiment
Specific embodiment 1: helicobacter pylori MONOCLONAL ANTIBODIES SPECIFIC FOR
1,HP cultivates, and prepares amplification template, the design primer
:
1) designs 2 pairs of primers altogether: the N end of amplification CagA, C end
cagA-N-F：ACGTGGATCCGAATTC?ATCGTTGATAAGAATGATAGG（136-156bp）
cagA-N-R：ACGTGTCGACCTCGAGTAGGCTACCTTGAAGAGTGG?（1472-1491bp）
The big or small 1356bp that increases, 452 amino acid of encoding, molecular weight is 50.947kDa, called after antigens c-N.
cagA-C-F：ACGTGGATCCGAATTC?ACCAACGCCTCCAAGAGTCCTG（1537-1558bp）
cagA-C-R：ACGTGTCGACCTCGAGAGCGTAATTGTCCAATTTCGC?（3115-3135bp）
Amplification size 1599,533 amino acid of encoding, molecular weight is 58.455kDa, called after antigens c-C.
2) PCR amplification: each 10 μ l of primer, HP bacterium liquid supernatant (template) 1 μ l, Taq E 0.5 μ l, damping fluid 5 μ l,
redistilled water 23.5 μ l; 94 ℃ of 5min, 94 ℃ of 30S, 56 ℃ of 30S, 72 ℃ of 120S (30 circulation).
2, the detection of amplified production detects amplified production with the agarose gel electrophoresis that reclaims with 1 %, and the fragment that size is correct reclaims kit with glue and reclaims.
3, connection and conversion are carried out double digestion (BamH I/ XhoI with the encoding proteins C-N that reclaims, the dna sequence dna of C-C respectively; 37 ℃ of 3h), and with carrier pET32a carry out double digestion (BamH I/ XhoI, 37 ℃ of 3h); After glue reclaimed, the purpose fragment was connected with carrier.In 16 ℃ of connections 6 hours, be transformed into
E.coliDH5 α is coated with LB flat board (AMP
r), 37 ℃ of overnight incubation.The dna sequence dna of coding U albumen connects with carrier pET32a double digestion (EcoR I/ XhoI) back with EcoR I/ XhoI double digestion.
4, enzyme is cut or PCR identifies that choose 6 clones respectively extracts plasmids, carries out that enzyme is cut or PCR identifies that positive colony is seen order-checking off.
5, the correct clone of expression and purification order-checking extracts plasmid and changes the expression bacterial classification over to
E.coliBL21 DE3, sample is groped to express temperature, is used the IPTG abduction delivering, and 12% SDS-PAGE identifies.After expressing successfully, with 1L LB antigen expressed, the ultrasonication thalline is confirmed expressive site, carries out purifying with the Ni post.
6, the recombinant C agA that handled is inoculated in mouse and carries out immunity, sets up the knurl strain with the SP20 Fusion of Cells and screens, and gets oncocyte and is injected in the BALB/c mouse abdominal cavity, makes it produce ascites.Extract the screening of mouse ascites purifying, finishing screen is selected two strains and is had strong specificity pairing Hp monoclonal antibody, is designated as Hp-1 and Hp-2.
Specific embodiment 2: the preparation of the preparation of collaurum and the anti-pylori spiral bacilli antibody II solution of gold mark
The preparation of colloidal gold solution: prepare 1% chlorogold solution, 2% citric acid three sodium solution; Add hot deionized water to 80 ℃; Add 1% chlorogold solution 30ml, carry out heated and stirred, add 2% citric acid trisodium 18ml; Observation continues to boil 10min by purple to red the variation, transfers to 4 ℃ of preservations in the brown bottle after the cooling.
The preparation of the anti-pylori spiral bacilli antibody II solution of gold mark: weighing gold liquid 250ml adds 0.2MK
2CO
32.5ml stirred 5 minutes, Hp-2 adds in the 250ml gold liquid and stirs, and adds 2.5ml 1%PEG20000, and is centrifugal, abandon supernatant, deposition is diluted to 50ml with PH8.1 citric acid-Tris damping fluid.
The collaurum liquid of antibody labeling, on the adsorbing fiber material, dry back is subsequent use.
Specific embodiment 3: the preparation of antibody labeling nitrocellulose filter
Use special film-making machine system film, the nitrocellulose filter test wire is used Hp-1 and is made, and the nitrocellulose filter control line is processed by the sheep anti mouse polyclonal antibody.
Specific embodiment 4: detect test paper and device
Fig. 1 has represented outside drawing of the present invention in detail, and as can be seen from the figure the present invention is made up of check-out console (1) and detection test paper (8) two parts.
(1) check-out console
Visible by Fig. 1, check-out console comprises plate lid (1) and plate body (2) two parts.There are well (3) and display window (4) as a result in plate lid front.Plate body (2) is provided with and test strips corresponding groove (5).Plate lid (1) is connected through the buckle mode with plate body (2).
(2) colloidal gold fast detecting test paper
Fig. 4 is the exploded view of test paper; Be fixed in plate body (2) inside by the visible colloid gold test paper (6) of figure; Form by base plate (7), water sucting plate (8), nitrocellulose filter (9), monoclonal antibody gold mark pad (10), sample liquid-adsorption layer (11), MAX line (14); Base plate (7) middle part is nitrocellulose filter (9); A test wire (12) and a polyclonal antibody control line (13) are arranged on the nitrocellulose filter (9), and base plate one end termination is water sucting plate (8), and other end termination is sample liquid-adsorption layer (11); Nitrocellulose filter (9) two ends overlap each other with monoclonal antibody gold mark pad (10) with water sucting plate (8) respectively and are connected, and on monoclonal antibody gold mark pad (10), are pressed with sample liquid-adsorption layer (11).
Method of application: gather the ight soil test sample; Dilute with dilution, draw sample and splash in the device well, because the capillarity sample will move along test strips water accepting layer end; When moving to helicobacter pylori monoclonal antibody II gold mark pad; Helicobacter pylori in the sample and helicobacter pylori monoclonal antibody gold mark probe generation specific bond, when moving to when being fixed with helicobacter pylori monoclonal antibody I test wire, the helicobacter pylori in the sample again with test wire in the helicobacter pylori monoclonal antibody combine; Therefore its collaurum is stranded on the test wire, and the colour developing of test wire place is positive; If do not have helicobacter pylori in the opposite sample, helicobacter pylori monoclonal antibody gold mark probe just not can with the helicobacter pylori monoclonal antibody I generation specific bond on the test wire, do not have collaurum to be detained, promptly only have a control line negative.
The result judges: when in the ight soil pylori antigen being arranged, the colour developing district of test paper has two lines to show, and promptly test wire and control line all become redness, and be positive.When in the ight soil during no pylori antigen, the colour developing district of test paper only has a control line to become redness, and is negative.When having color, the colour developing district of test paper do not show that represent that then misoperation or test paper are expired, this result is invalid
Claims (9)
1. helicobacter pylori device for fast detecting, comprise helicobacter pylori pairing monoclonal antibody foundation, comprise the detection test paper and the pick-up unit of this monoclonal antibody, it is characterized in that: adopt the recombinant C agA that handled to be inoculated in mouse and carry out immunity; Setting up the knurl strain with the SP20 Fusion of Cells screens; Get oncocyte and be injected in the BALB/c mouse abdominal cavity, make it produce ascites, extract the screening of mouse ascites purifying; Finishing screen is selected two strains and is had strong specificity helicobacter pylori pairing monoclonal antibody; Be designated as Hp-1 and Hp-2, the monoclonal antibody that should match is applied to detect test paper, and then processes to helicobacter pylori high specificity, highly sensitive pick-up unit.
2. a kind of helicobacter pylori device for fast detecting according to claim 1 is characterized in that being applied to detect test paper after said pairing monoclonal antibody is by colloid gold label, and collaurum is a kind of of metal colloid particles in the colored particle.
3. a kind of helicobacter pylori device for fast detecting according to claim 1; It is characterized in that this device removes employing colloid gold particle labelled antibody; Also can adopt other similar colored particle mark; Promptly can be a kind of metal colloid particles, a kind of marking particle, include but are not limited to collargol particle, iron particle, magnetic-particle, dye granule, latex particle, fluorescent grain.
4. a kind of helicobacter pylori device for fast detecting according to claim 1 is characterized in that said detection test paper utilizes colloidal gold immunity chromatography to process, and realizes through following method:
Detecting test paper is made up of base plate, water sucting plate, nitrocellulose filter, helicobacter pylori monoclonal antibody gold mark pad, sample liquid-adsorption layer, Max line; The base plate middle part is a nitrocellulose filter; A test wire and a polyclonal antibody control line are arranged on the nitrocellulose filter; Base plate one end termination is a water sucting plate; Other end termination is the sample liquid-adsorption layer, and the nitrocellulose filter two ends overlap each other with helicobacter pylori monoclonal antibody gold mark pad with water sucting plate respectively and are connected, and on helicobacter pylori monoclonal antibody gold mark pad, are pressed with the sample liquid-adsorption layer;
The sample liquid-adsorption layer is made up of trilaminate material stack, and ground floor is that certain specification nonwoven layer, the second layer are that glass layer, the 3rd layer are the certain specification nonwoven layer, and above-mentioned substance all need pass through surfactant damping fluid immersion treatment, and is dry afterwards subsequent use.
5. a kind of helicobacter pylori device for fast detecting according to claim 1; It is characterized in that detecting test paper utilizes the double antibodies sandwich method to process; Utilize the double antibodies sandwich principle; Helicobacter pylori is matched monoclonal antibody be applied to detect test paper, Hp-1 is used to encapsulate test wire, and another strain antibody Hp-2 is used for colloid gold label.
6. a kind of helicobacter pylori device for fast detecting according to claim 1 is characterized in that described control line is to be encapsulated by the sheep anti mouse polyclonal antibody to form, and when sample moved to sheep anti mouse polyclonal antibody control line, control line just showed colored ribbon.
7. according to claim 1 or 5 described a kind of helicobacter pylori device for fast detecting, it is characterized in that utilizing the test wire of the detection test paper that the double antibodies sandwich method processes to show colored ribbon, i.e. positive when colour developing district is two lines; Negative when having only a control line.
8. a kind of helicobacter pylori device for fast detecting according to claim 1; It is characterized in that described pick-up unit is that check-out console comprises plate body and plate lid two parts; Check-out console is processed by the moderate material of hardness; There are well and display window as a result in plate body front, detects test paper and places between plate body and the plate lid, and the plate body covers with plate and is connected through the buckle mode.
9. be connected last time when the plate body covers with plate through the buckle mode, the sample liquid-adsorption layer is closed within the plate lid, and the nitrocellulose filter of colloid gold test paper is corresponding with the display window as a result of plate body.
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| CN2011102411090A CN102393460A (en) | 2011-08-22 | 2011-08-22 | Rapid detection device for helicobacter pylori |
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| CN2011102411090A CN102393460A (en) | 2011-08-22 | 2011-08-22 | Rapid detection device for helicobacter pylori |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614522A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Helicobacter pylori antibody detection kit, detection method and application |
| WO2015151128A1 (en) * | 2014-04-04 | 2015-10-08 | Over S.R.L. | Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor |
| CN106290855A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of pylori antigen near-infrared fluorescent detection kit and application thereof |
| RU2642588C1 (en) * | 2017-03-14 | 2018-01-25 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятский государственный университет" | Immunochromatographic test system for pathogenic helicobacter pylori strains detection |
| CN108020665A (en) * | 2016-10-31 | 2018-05-11 | 联华生技股份有限公司 | Set group and sampling gimmick are tested in upper and lower digestive system cancer screening |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015151128A1 (en) * | 2014-04-04 | 2015-10-08 | Over S.R.L. | Method for rapid detection of infection from caga positive helicobacter pylori and diagnostic kit therefor |
| CN104614522A (en) * | 2015-02-10 | 2015-05-13 | 深圳市新产业生物医学工程股份有限公司 | Helicobacter pylori antibody detection kit, detection method and application |
| CN106290855A (en) * | 2015-06-01 | 2017-01-04 | 上海凯创生物技术有限公司 | A kind of pylori antigen near-infrared fluorescent detection kit and application thereof |
| CN108020665A (en) * | 2016-10-31 | 2018-05-11 | 联华生技股份有限公司 | Set group and sampling gimmick are tested in upper and lower digestive system cancer screening |
| RU2642588C1 (en) * | 2017-03-14 | 2018-01-25 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Вятский государственный университет" | Immunochromatographic test system for pathogenic helicobacter pylori strains detection |
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Application publication date: 20120328 |