CN102636641A - Detection kit of helicobacter pylori emulsion method and preparation process thereof - Google Patents
Detection kit of helicobacter pylori emulsion method and preparation process thereof Download PDFInfo
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- CN102636641A CN102636641A CN2012100826012A CN201210082601A CN102636641A CN 102636641 A CN102636641 A CN 102636641A CN 2012100826012 A CN2012100826012 A CN 2012100826012A CN 201210082601 A CN201210082601 A CN 201210082601A CN 102636641 A CN102636641 A CN 102636641A
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Abstract
The invention relates to the field of the emulsion method immune chromatography, and particularly relates to a detection kit of a helicobacter pylori emulsion method. The detection kit of the helicobacter pylori emulsion method comprises a reagent strip, wherein the reagent strip comprises a substrate, filter sample paper, a sensitized emulsion polyester film, an immune cellulose nitrate film and absorbent paper, wherein the filter sample paper, the sensitized emulsion polyester film, the immune cellulose nitrate film, and the absorbent paper are orderly connected end to end, and are fixed on the substrate, the immune cellulose nitrate film is covered with helicobacter pylori resisting urea enzyme antibodies I marked by colorful emulsion particles, and the immune cellulose nitrate film is provided with a detection line coated with helicobacter pylori resisting urea enzyme antibodies II and a quality control line coated by goat anti-rabbit serum IgG. The detection kit of the helicobacter pylori emulsion method, which is provided by the invention, has the advantages of convenience in detection, stability in detection results and cost conservation.
Description
Technical field
The present invention relates to the immunochromatography technique field, be specifically related to a kind of stomach Helicobacter pylori emulsion process detection kit and preparation technology thereof.
Background technology
Stomach Helicobacter pylori Helicobacter Pylori (being called for short HP) is a kind of spiral fashion gramnegative bacterium in the human stomach mucous membrane.This bacterium is the arch-criminal who causes chronic gastritis, gastric ulcer and duodenal ulcer even cancer of the stomach.It is generally acknowledged that the CC that stomach Helicobacter pylori infects is such: the stomach Helicobacter pylori per os is settled down infection after arriving gastric mucosa; Cause chronic, superficial gastritis through several weeks or several months; Develop into duodenal ulcer, gastric ulcer, lymphocytic hyperplasia property gastric lymphoma, atrophic gastritis etc. behind several years or the many decades, and the latter is the factor that causes cancer of the stomach the most dangerous.Brainstrust thinks that stomach Helicobacter pylori infects and makes the danger that gets a cancer of the stomach increase 2.7-12 doubly, if there is not Helicobacter pylori infection, has at least the cancer of the stomach of 35%-89% can not take place.
Urease is the nickel ion dependent enzyme of a kind of catalyzing urea hydrolysis ammonification and carbonic acid; Be the rich in protein of expression in the helicobacter pylori; Account for the 5-10% of HP total protein; Urease through in help bacterium settling down and ammonia is provided in stomach with hydrochloric acid in gastric juice for bacterioprotein is synthetic, the parasitism of HP in stomach all played an important role with pathogenic.The urease molecular weight is about 550KD, and monomer whose is made up of A, two subunits of B, and A, two subunit molecular of B are respectively about 30KD and 66KD; Wherein the urease B subunit is a urease activity subunit, in each HP bacterial strain, has good conservative property, is acknowledged as the desirable antigen of HP vaccine; HP vaccine to urease A subunit can be obtained good protective action equally, and therefore, urease A, B subunit all are the desirable antigen of HP Antibody Preparation.
Stomach Helicobacter pylori all has infection in the crowd of the different races in the world, different regions, can be described as chronic bacillary infection the most widely among the adult.Infection rate increases with the age and rises, and developing country is about 80%, and developed country is about 40%.20 years old-40 years old infection rate of China is 45.4%-63.6%, and the infection rate more than 70 years old is up to 78.9%.Therefore, whether exist HP antibody to infect in the detection human blood and have the very clinical meaning of reality for diagnosis HP.
The method that detects HP at present mainly contains:
(1) EUSA and western blot test: confirm the existence of HP in the human body through detecting HP antibody in the serum, this kind method specificity is high, and can carry out quantitative test, but can not differentiate previous infection and eqpidemic disease people at present.
(2) urea breath test and PCR detect test: the metabolin through measuring the urease that HP produces in vivo or the method for gene are confirmed the existence of HP; This kind method is highly sensitive; But complicated operation, apparatus expensive are inappropriate for basic unit's testing, have promoted the use of limitation.
(3) microbe growth and other pathology detection methods etc., these methods also generally have advantage highly sensitive, high specificity, but also exist to cultivate and the testing conditions of pathology method is strict, complicated operation, the limitation that needs the professional to operate.
So; Be badly in need of at present a kind of quick, accurate, highly sensitive, method that just can detect HP without any need for main equipment; The establishing of this kind method is beneficial to stomach Helicobacter pylori and detects in the popularizing and applying of basic unit, and chronic gastritis, gastric ulcer and duodenal ulcer even Prevention and Treatment of Stomach Cancer that stomach Helicobacter pylori is caused have great clinical meaning.
The latex chromatography is to be the immunochromatographic method of label with the color latex, and its detection principle and colloidal gold immunity chromatography are similar, and it does not need instrument; Direct sentence read result with the naked eye has simple to operately, quick, and reagent stability is good; Be prone to preserve; Single part independent packaging, advantage such as reagent loss is few is fit to the rapid screening of samples such as clinical patients, health examination person.
Summary of the invention
The objective of the invention is to overcome the prior art defective,, a kind of detection sensitivity height, high specificity are provided, detect stomach Helicobacter pylori emulsion process detection kit fast from improving detection sensitivity.
One aspect of the present invention discloses a kind of stomach Helicobacter pylori emulsion process detection kit; Comprise reagent strip; Said reagent strip comprises substrate, filter appearance paper, sensitization latex polyester film, immune nitrocellulose filter and thieving paper; Said filter appearance paper, sensitization latex polyester film, immune nitrocellulose filter and thieving paper is tandem array and being fixed on the said substrate successively; Be coated with the anti-stomach Helicobacter pylori urease antibody I of color latex particle mark on the said sensitization latex polyester film, immune nitrocellulose filter is provided with detection line that has encapsulated anti-stomach Helicobacter pylori urease antibody II and the nature controlling line that has encapsulated goat anti-rabbit igg.
More excellent, said color latex particle is the granules of polystyrene of particle diameter 280～310nm.
More excellent, the color of said color latex particle is red.
More excellent, said anti-stomach Helicobacter pylori urease antibody I is to be antigenic substance with fusion Ure-BA, the polyclonal antibody that immune animal prepares, and said fusion Ure-BA sequence is seen SEQ ID NO:5.
More excellent, the said animal that is used to prepare polyclonal antibody is a rabbit.
The anti-stomach Helicobacter pylori urease antibody I of sensitization latex polyester film marked chromatic colour latex particle of the present invention is a polyclonal antibody, and the anti-stomach Helicobacter pylori urease antibody II that immune nitrocellulose filter detection line (T line) encapsulates is a monoclonal antibody.
That summarizes says, these many anti-preparation processes of the anti-stomach Helicobacter pylori urease antibody of the present invention I comprise:
1) the PCR method is cloned UreB respectively
138And UreA
65Gene, and be equipped with the Ure-BA fusion through overlap extension PCR legal system;
2) make up the recombinant plasmid that contains step 1) Ure-BA fusion nucleotide sequence;
3) with step 2) the middle recombinant plasmid transformed host cell for preparing;
4) host cell expression recombinant protein obtains purified recombinant urease antigen coalescence protein after separation and purification;
5) the recombinant urease antigen coalescence protein immune animal that obtains with step 4) prepares anti-stomach Helicobacter pylori urease polyclonal antibody.
The preparation of fusion of the present invention, the production of fusion, purifying, and Polyclonal Antibody Preparation all can be with reference to prior art.
Height is tired and the polyclonal antibody of high specific in order to prepare, must the desirable immunogene of preparation.The present invention analyzes through albumen water wettability, antigenicity and structure function territory etc. to urease B subunit, A subunit; Particular segment in a particular segment in the urease B subunit and the urease A subunit is connected through specific connection peptides obtains fusion Ure-BA; This fusion Ure-BA has immunogenicity and the reactionogenicity of UreB and UreA simultaneously; The polyclonal antibody that adopts this fusion protein immunization animal to produce; Can with stomach Helicobacter pylori urease generation specificity association reaction, affinity is strong; Further, the reagent strip that the polyclonal antibody that adopts this fusion to produce prepares is highly sensitive.Said fusion Ure-BA sequence is seen SEQ ID NO:5.
Recombinant antigen therefore of the present invention selects the functional fragment of protein molecular to substitute the full-length proteins molecule, and the immune response that both can induce the host specifically is convenient to the successful preparation of fusion again.
Another aspect of the present invention provides a kind of preparation method of stomach Helicobacter pylori emulsion process detection kit, may further comprise the steps:
1) preparation of sensitization latex particle:, obtain the sensitization latex particle with the carboxylated anti-stomach Helicobacter pylori urease antibody of polystyrene mark I;
2) preparation of sensitization latex polyester film: the sensitization latex particle that obtains with step 1) encapsulates polyester film, obtains sensitization latex polyester film;
3) will resist stomach Helicobacter pylori urease antibody II and goat anti-rabbit antibody to be sprayed on respectively on the position of nitrocellulose filter detection line (T line) and nature controlling line (C line), dry for standby makes immune nitrocellulose filter;
4) will filter appearance a paper, sensitization latex polyester film, immune nitrocellulose filter, thieving paper sticks on the PVC sheet successively, cutting makes reagent strip;
5), obtain stomach Helicobacter pylori emulsion process detection kit with the reagent strip plastic casing of packing into.
More excellent, being prepared as of said step 1) sensitization latex particle:
A. get the carboxylated polystyrene of certain volume, with the carbonate buffer solution of 2～3 times of volume 20mM pH 6.0 repeatedly wash, centrifugal, abandon supernatant, obtain carboxylated granules of polystyrene deposition;
B. the carboxylated granules of polystyrene deposition that obtained with a last step of the phosphate buffer solution suspension of 1～1.5 times of volume 20mM pH6.0; And add the water-soluble charing diimine of 1～1.5 times of volume; Stirred under the room temperature 15～40 minutes; At last the borate buffer solution with 2～3 times of volume 0.01M pH 8.0 repeatedly wash, centrifugal, abandon supernatant, obtain thing to be marked;
C. the borate buffer that in the thing said to be marked that step b obtains, adds 2～3 times of volume 0.2M pH 7.0 is processed the latex suspension, and in said latex suspension, adds anti-stomach Helicobacter pylori urease antibody I;
D. fully reaction is spent the night, and adds the terminator cessation reaction, obtains the sensitization latex particle.
In the preparation process of sensitization latex particle; The water-soluble charing diimine that adds, the volume of each buffer solution are benchmark with the volume of the initial carboxylated polystyrene of being got all; Add by volume multiple of the present invention: the volume like the carboxylated granules of polystyrene got is 1ml; The phosphate buffer solution volume of pH 6.0 20mM that then need add can be 1～1.5ml, and the volume of the borate buffer of pH 7.0 0.2M is 2～3ml.
More excellent, carbonate buffer solution is the carbonate buffer solution that 20mM pH 6.0 contains 0.01%SDS among the said step a.The carbonate buffer solution that is added with SDS is the surface-active substance of flush away latex better, and cleaning performance is better.
More excellent, carboxylated polystyrene concentration is 5～15w/v% among the said step a.
More excellent, the centrifugal condition among the said step a is 12000～14000rpm, centrifugal 10min.
More excellent, the concentration of water-soluble charing diimine is 8～12mg/ml among the said step b.
More excellent, the centrifugal condition among the said step b is 12000～14000rpm, centrifugal 10min.
More excellent, the anti-stomach Helicobacter pylori urease antibody I among the said step c is to be antigenic substance with fusion Ure-BA, the polyclonal antibody that immune animal prepares, and said fusion Ure-BA sequence is seen SEQ ID NO:5.
More excellent, the amount of the carboxylated anti-stomach Helicobacter pylori urease antibody of granules of polystyrene coupling I is in the said step c latex suspension: the anti-stomach Helicobacter pylori urease antibody of 0.5mg I/10mg granules of polystyrene.
More excellent, terminator is the Tris-HCl of 50mM pH 8.2 in the said steps d.
Among the present invention, v% refers to percent by volume; W/v% refers to percent weight in volume, is like 1w/v% to contain the 1g material in the 100ml solution.
More excellent, said step 2) the preparation concrete steps of sensitization latex polyester film are in:
A. with latex damping fluid dilution sensitization latex particle, obtain the sensitization latex solution of 0.2～0.6w/v%;
B. the sensitization latex solution spraying polyester film for preparing with steps A, drying makes sensitization latex polyester film.
More excellent, the latex buffer formulation in the said steps A is following: 8～12v%1.0M Tris liquid, 0.2～0.4w/v% Macrogol 2000 0; 0.15～0.25w/v% bovine serum albumin(BSA); 0.1～0.3w/v% skim milk, 0.25～0.35w/v% casein and 0.02～0.08w/v% sodium azide; Regulate pH to 8.0 ± 0.05 with hydrochloric acid, surplus is a water.
Optimum; Latex buffer formulation in the said steps A is following: 10v%1.0M Tris liquid, 0.3w/v% Macrogol 2000 0,0.2w/v% bovine serum albumin(BSA); 0.2w/v% skim milk, 0.3w/v% casein; With the 0.05w/v% sodium azide, regulate pH to 8.0 ± 0.05 with hydrochloric acid, surplus is a water.
More excellent, with sensitization latex solution spraying polyester film, spraying 1.28ml sensitization latex solution on every 261mm * 220mm polyester film among the step B, drying makes sensitization latex polyester film.
More excellent; In the said step 3); The concentration that is sprayed on the goat anti-rabbit igg on the nitrocellulose filter nature controlling line (C line) is 1.0～2.0mg/ml, and the anti-stomach Helicobacter pylori urease antibody II concentration that is sprayed on the nitrocellulose filter detection line (T line) is 1.0～2.0mg/ml.
More excellent, the long nitrocellulose filter of every 50m is coated with the goat anti-rabbit igg of 6ml and the anti-stomach Helicobacter pylori urease antibody II solution of 6ml respectively, and the spacing of detection line and nature controlling line is 4.0～6.0mm.
The present invention has prepared a kind of high-quality antigen through the method for gene fusion, and the particular segment through intercepting urease B subunit and A subunit also is connected one section connection peptides, prepares stomach Helicobacter pylori urease fusion.This fusion has better space structure and higher immunogenicity and reactionogenicity, with its as the antigenic substance immune animal can obtain high to tire, the anti-stomach Helicobacter pylori urease polyclonal antibody of high specific; And the stomach Helicobacter pylori emulsion process detection kit of the present invention preparation is highly sensitive, and high specificity can detect the urease of 1ng/ml in the blood sample; Kit of the present invention in addition also has simple to operate, and price is low, advantages such as storage and convenient transportation.
The method of application of stomach Helicobacter pylori emulsion process detection kit of the present invention:
1. the processing of sample: at random from the different parts sampling of patient's ight soil, sampling amount is as the criterion with the small circle ring that is stained with special-purpose sampling rod front end with special-purpose sampling rod; Prepare a sample processing tube, vertically be put in and handle on the pipe holder, and in sample hose, add 0.6ml distilled water; The distilled water that the sample of getting on the special-purpose sampling rod is inserted in the sample hose stirs, and is subsequent use; The sample that suggestion is handled well in time detects, and can store 48 hours down at 2～8 ℃ as not detecting the sample of handling well immediately.
2. the detection of sample: draw sample liquid 2-3 with suction pipe and be added dropwise in the sample cell, sample to be checked to thieving paper end chromatography, passes through polyester film, nitrocellulose filter successively under capillary action.After arriving polyester film; Fully discerned and combine by the polyclonal antibody of latex mark; Continue chromatography to nitrocellulose filter, immune response takes place, show corresponding red lines with the anti-stomach Helicobacter pylori urease monoclonal antibody-II that encapsulates on the nitrocellulose filter.
3. result's interpretation: 8-15 minute sentence read result after dripping sample.
Positive: red lines respectively appear in detection line and nature controlling line position at the detectable bar, and the existence of stomach Helicobacter pylori urease is arranged in the expression sample.
Negative: as red lines only to occur, the existence of no stomach Helicobacter pylori urease in the expression sample in the nature controlling line position.
Invalid: the nature controlling line position does not have red line and occurs, and ecbatic is invalid, answers retry.
Beneficial effect of the present invention is: the present invention passes through the fusion of gene recombination technology preparation as the antigenic substance immune animal; The polyclonal antibody that can prepare better quality is used for kit of the present invention; The stomach Helicobacter pylori emulsion process detection kit of preparation is highly sensitive; High specificity can detect the urease of 1ng/ml in the blood sample; Kit of the present invention in addition also has simple to operate, and price is low, advantages such as storage and convenient transportation.
Description of drawings
Fig. 1: stomach Helicobacter pylori emulsion process detection kit reagent strip synoptic diagram (1. filter appearance paper 2. sensitization latex polyester films 3. immune nitrocellulose filter 4. thieving papers 5. detection lines 6. nature controlling lines)
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to explain the present invention, and unrestricted scope of the present invention.
The preparation of embodiment 1 anti-stomach Helicobacter pylori urease antibody
1. the design of antigen coalescence protein
To the immunologic mechanism of stomach Helicobacter pylori and the immune property of stomach Helicobacter pylori urease epitope, select urease UreB particular segment UreB according to body
138The particular segment UreA of (UreB amino acid sequence GenBank:AAU21200.1) and UreA
65(UreA amino acid sequence GenBank:AAK69750.1) is used for the structure of Hp antigen coalescence protein.UreB
138And UreA
65Between be inserted with connection peptides fragment RWYCR, and urease UreB
138Fragment merges at PROTEIN C end, UreA
65Fragment merges at albumen n end.
2. the structure of recombinant expression carrier
(1) primer is synthetic
Utilize Primer Premier V5.0 design UreB
138Gene and UreA
65The primer of gene is following, and the underscore of P1 partly is a BamH I restriction enzyme site, and P1 and P3 partly include the connection peptides sequence, and the underscore of P4 partly is the EcoRI restriction enzyme site:
UreB
138Upstream primer P1:
CGGATCCGGACACTTTGAATGAAGC
Downstream primer P2:CCTGCAGTACCACCTAAATTCTTTCTTG
UreA
65Upstream primer P3:AGGTGGTACTGCAGGTCACACTTCCATTTC
Downstream primer P4:
GAATTCGTCATCGCTTTTAGCGCC
(2) PCR obtains target gene fragment
Genomic DNA with stomach Helicobacter pylori is a template, respectively with primer P1 and P2, and P3 and P4 amplification UreB
138And UreA
65Genetic fragment, set up following reaction system in 500 μ l microcentrifugal tubes:
UreB
138Genetic fragment:
Upstream primer P1:CGGATCCGGACACTTTGAATGAAGC (SEQ ID NO:1)
Downstream primer P2:CCTGCAGTACCACCTAAATTCTTTCTTG (SEQ ID NO:2)
Pcr amplification program: 94 ℃ of preparatory sex change 5min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ are extended 10min; 4 ℃ of 60min.
The PCR reaction finishes laggard row agarose gel electrophoresis, and the size of PCR product conforms to the base-pair size of expectation, the dna fragmentation that amplifies is reclaimed kit with glue reclaim.
UreA
65Genetic fragment
Upstream primer P3:AGGTGGTACTGCAGGTCACACTTCCATTTC (SEQ ID NO:3)
Downstream primer P4:GAATTCGTCATCGCTTTTAGCGCC (SEQ ID NO:4)
Pcr amplification program: 94 ℃ of preparatory sex change 5min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ are extended 10min; 4 ℃ of 60min.
The PCR reaction finishes laggard row agarose gel electrophoresis, and the size of PCR product conforms to the base-pair size of expectation, the dna fragmentation that amplifies is reclaimed kit with glue reclaim.
Ure-BA fusion fragment
With P1 and P4 is primer, with the UreB of abovementioned steps preparation
138And UreA
65Genetic fragment is mixed as template, carries out overlap extension PCR reaction, amplification Ure-BA fusion, and pcr amplification system and program are following:
Pcr amplification program: 94 ℃ of preparatory sex change 5min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 2min, totally 30 circulations; 72 ℃ are extended 10min; 4 ℃ of 60min.
The PCR product is carried out agarose gel electrophoresis, and the size of PCR product conforms to the expectation size.
(3) construction of recombinant plasmid
Be connected with pMD-19T reclaiming the Ure-BA fusion fragment that obtains, obtain recombinant plasmid pMU-BA, concrete steps are following:
1) uses the plasmid extraction kit, extract DNA according to the kit explanation.
2) with conventional agarose gel electrophoresis method isolated plasmid dna, 1.0% Ago-Gel, 1 * TAE damping fluid, 120-150mA, electrophoresis 20-40 minute.
3) coupled reaction: with the purified product (Ure-BA fusion) that reclaims with BamH I and the processing of EcoRI double digestion, with same double digestion processing pMD-19T be connected, the coupled reaction system is following, 22 ℃ of coupled reaction 16h.
3. the structure of the engineering bacteria of expressed fusion protein
(1) preparation (CaCl of competent cell
2Method)
Plate streak activation Escherichia coli E.coli DH 5 α, 37 ℃ of LB plating mediums are cultivated 12h, and single Escherichia coli colony inoculation is in 100ml LB nutrient culture media on the picking flat board; 37 ℃ of shaking tables are cultivated 12h, and rotating speed is 300rpm, obtain the Escherichia coli seed liquor.
Under the aseptic condition, the Escherichia coli seed liquor that obtains is inoculated in the LB nutrient culture media with 1% ratio, 3h are cultivated in 37 ℃ of concussions, and the centrifugal 5min of 8000g collects thalline.The 5mL 0.1M CaCl that in the thalline of collecting, adds precooling
2Suspend and precipitate ice-water bath 3h; 4 ℃ of centrifugal 5min of 8000g abandon supernatant.The 0.1M CaCl that adds 100 μ L precoolings
2The resuspension deposition, ice-water bath 1h, subsequent use.
Draw 100 μ L competence bacteria liquid with the aseptic suction nozzle of precooling, add and connect product, ice-water bath 45min, 42 ℃ of water-bath heat shock 90s place ice-water bath 2min rapidly.Add 100 μ L LB nutrient culture media, 37 ℃ of shaking tables are cultivated 1h.The centrifugal 5min of 8000g, discard 100 μ L supernatants after, mixing deposition also coating to contain the LB of 100 μ g/ml ampicillins dull and stereotyped; Behind 37 ℃ of cultivation 16h; Shake bacterium behind the picking list bacterium colony, the plasmid extraction kit extracts the recombinant plasmid after the amplification, and handles with BamH I and EcoRI double digestion; The result shows that the endonuclease bamhi size is consistent with design, tentatively proves the construction of recombinant plasmid success; Further order-checking is confirmed, reclaims fragment and contains the sequence shown in the SEQ ID NO:6, and sequencing result is correct, confirms that fusion prepares successfully.
(2) efficiently express the screening of fusion reorganization bacterium
Select the correct plasmid transformation escherichia coli BL21 (DE3) of order-checking (day root biochemical technology company limited) to be inoculated in the LB nutrient solution; Cultivate 16h for 37 ℃; The bacterium of will recombinating after cultivation finishes is transferred in the LB nutrient solution in 1% ratio, and 37 ℃ of shaking tables are cultivated 3h, is that the IPTG of 0.5mM induces 5h with the final concentration; SDS-PAGE detects Expression of Fusion Protein form and expression, and the screening efficient expression strain is used for Expression of Fusion Protein.Experimental result shows that fusion protein expression rate of the present invention reaches as high as 31%.
4. Expression of Fusion Protein and purifying
Collect thalline, extract inclusion body: the ultrasonication damping fluid of cell precipitation and precooling suspends with 1: 10 (w/v) ratio, and ultrasonication thalline in ice bath, ultrasonic power are 280W, ultrasonic 3s, and 3s carries out 40min altogether at interval.The centrifugal 30min of thalline solution 500g with after the fragmentation abandons deposition; With the centrifugal 40min of supernatant 15000g, abandon supernatant, collecting precipitation.Use A liquid and B liquid washing precipitation twice respectively with the ratio of 1: 10 (w/v), each wash conditions is: 4 ℃ are stirred 20min, the centrifugal 40min of 15000g, collecting precipitation; To precipitate with the ratio mixing of solubilization of inclusion bodies liquid with 1: 10 (w/v), 4 ℃ are stirred 3h, and the centrifugal 50min of 15000g gets supernatant.
Ultrasonication damping fluid: 50mmol/L NaH
2PO
4, 300mmol/L NaCl, pH8.0; A liquid: 5mmol/L EDTA, 20mmol/L Tris, 1%Triton X-100, pH8.0; B liquid: 20mmol/L Tris, 2mol/L urea, pH8.0; Solubilization of inclusion bodies liquid: 20mmol/L Tris, 1mmol/L EDTA, 2mol/L urea, pH8.0.
Metal ion-chelant chromatography: select affinity column Chelating separose to carry out purifying; The affinity purification filler is Chelating Sepharose HP, adopts 20mmol/L Tris, 5mmol/L EDTA; PH8.0 carries out purifying to destination protein, adopts the imidazoles gradient elution.
The anion column purifying: select anion column HiTrap Q to carry out purifying, negative ion purifying filler is that Q Sepharose FF uses 20mmol/L Tris, 5mmol/L EDTA, and pH8.0 carries out purifying to destination protein, adopts the NaCl gradient elution.
The desalination of Superdex gel permeation chromatography: after the target protein that a last step is obtained carries out concentrating in the glucose PEG bag filter, with solvent resistant column Superdex filtration desalination, removed urea and imidazoles, the gel permeation chromatography post is Superdex 200.
Purified target protein is carried out SDS-PAGE, identifies its purity greater than 95%, and the result shows that the fusion reorganization bacterium that the present invention makes up can efficiently express the stomach Helicobacter pylori urease albumen of fusion.
5. stomach Helicobacter pylori urease Polyclonal Antibody Preparation
Fusion Ure-BA with the purifying of present embodiment preparation carries out immunity as immunizing antigen to rabbit; Inject the stomach Helicobacter pylori urease fusion of 150 μ g purifying first; The stomach Helicobacter pylori urease fusion of per subsequently two weeks injection, 200 μ g purifying is injected 3 times altogether and is carried out immunity.
Immunity is got serum after finishing, and 3000rpm obtains antiserum after centrifugal 15 minutes; The purifying antiserum: using the Hitrap Protein A HP affinity purification post of GEHealthcare company, with this post of combination liquid balance of 10 times of bed volumes, is 20mM Na in conjunction with the liquid composition earlier
3PO
4, pH is 7.0, passes through this post through the antiserum that extracts, and again with this post of combination liquid flushing of 5 times of bed volumes, uses the eluent wash-out of 5 times of bed volumes then, the eluent composition is 0.1M Na
3C
6H
5O
7, pH is 3.0, detects through UV (254nm) and collects eluting peak.The product that obtains behind the antiserum process Hitrap Protein A HP affinity purification post purifying is effectively removed abundant seralbumin; Obtain highly purified stomach Helicobacter pylori urease polyclonal antibody, be anti-stomach Helicobacter pylori urease antibody I.
The preparation of embodiment 2 stomach Helicobacter pylori emulsion process detection kit
One, reagent source:
Goat anti-rabbit igg polyclonal antibody, anti-stomach Helicobacter pylori urease antibody II (available from MAXMEDLABORATORIES INC.)
Cellulose nitrate film, polyester film, granules of polystyrene (available from the SARTORIUS of Sartorius AG) granules of polystyrene (available from WHATMAN company)
Two, preparation process:
Method 1:
1. the preparation of sensitization latex particle:
A. get 0.4ml 10% carboxylated polystyrene latex and put into centrifuge tube, add the carbonic acid buffer flushing twice that 1ml pH 6.0 contains 0.01%SDS 20mM, the centrifugal 10min of 14000g/min abandons supernatant, obtains carboxylated granules of polystyrene deposition.
B. the carboxylated granules of polystyrene deposition that obtained with a last step of the phosphate buffer solution suspension of 0.5ml pH 6.0 20mM; And adding 0.5ml 10% water-soluble charing diimine; Stirred 30 minutes under the room temperature; At last with the borate buffer solution of 0.8ml 0.01M pH 8.0 flushing three times, centrifugal, abandon supernatant, obtain thing to be marked.
C. the borate buffer that in the thing to be marked that step b obtains, adds 1ml 0.2M pH 7.0 is processed the latex suspension, and in this latex suspension, adds the anti-stomach Helicobacter pylori urease antibody of 2mg I.
D. after fully reacting 12 hours, the Tris-HCl that adds 50mM pH8.2 obtains the sensitization latex particle as the terminator cessation reaction.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex damping fluid: get the 800ml purified water, add 100ml 1.0M Tris liquid inward, accurately take by weighing 3.0g Macrogol 2000 0,2.0g bovine serum albumin(BSA); 2.0g skim milk; 3.0g casein solution and 0.5g sodium azide add in the solution, fully dissolving mixes; Regulate pH to 8.0 with hydrochloric acid, add purified water to cumulative volume 1000ml.
(2) the latex damping fluid of using a step dilutes the sensitization latex particle, obtains the sensitization latex solution of 0.4w/v%.
(3) get 1.28ml sensitization latex solution with some latex machine specking on pretreated polyester film, placed under 37 ℃ the temperature oven dry 2 hours, keep in Dark Place under sealing 4-30 ℃, make sensitization latex polyester film.
3. the preparation of immune nitrocellulose filter:
2.0mg/ml goat anti-rabbit antibody and the anti-stomach Helicobacter pylori urease antibody of 1.5mg/ml II are sprayed on respectively on the position of nitrocellulose filter nature controlling line and detection line; Dried 2 hours down for 37 ℃; Keep in Dark Place under sealing 4-30 ℃, make immune nitrocellulose filter.The long nitrocellulose filter of every 50m is coated with goat anti-rabbit igg and the anti-stomach Helicobacter pylori urease antibody II solution of 6ml respectively, and the spacing of detection line and nature controlling line is 5mm.
4. will filter appearance a paper, sensitization latex polyester film, immune nitrocellulose filter, thieving paper sticks on the PVC sheet successively, cutting makes reagent strip; The reagent strip plastic casing of packing into is made detection kit, obtain stomach Helicobacter pylori emulsion process detection kit.(as shown in Figure 1).
The threshold value of the stomach Helicobacter pylori emulsion process detection kit of the present invention's design is 1ng/ml.
Method 2:
1. the preparation of sensitization latex particle:
A. get 1ml 5% carboxylated polystyrene latex and put into centrifuge tube, add 2ml pH 6.0 carbonic acid buffers flushing three times, the centrifugal 10min of 12000g/min abandons supernatant, obtains carboxylated granules of polystyrene deposition.
B. the carboxylated granules of polystyrene deposition that obtained with a last step of the phosphate buffer solution suspension of 1.5ml pH 6.020mM; And the water-soluble charing diimine of adding 1.5ml%; Stirred 40 minutes under the room temperature; At last with the borate buffer solution of 3ml 0.01M pH 8.0 flushing three times, centrifugal, abandon supernatant, obtain thing to be marked.
C. the borate buffer that in the thing to be marked that step b obtains, adds 2ml pH 7.0 0.2M is processed the latex suspension, and in this latex suspension, adds the anti-stomach Helicobacter pylori urease antibody of 2.5mg I.
D. after fully reaction is spent the night, add the terminator cessation reaction, obtain the sensitization latex particle.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex damping fluid: get the 800ml purified water, add 80ml 1.0M Tris liquid inward, accurately take by weighing 4.0g Macrogol 2000 0,2.5g bovine serum albumin(BSA); 1.0g skim milk; 3.5g casein solution and 0.2g sodium azide add in the solution, fully dissolving mixes; Regulate pH to 8.0 with hydrochloric acid, add purified water to cumulative volume 1000ml.
(2) the latex damping fluid of using a step dilutes the sensitization latex particle, obtains the sensitization latex solution of 0.2w/v%.
(3) get 1.28ml sensitization latex solution with some latex machine specking on pretreated polyester film, placed under 37 ℃ the temperature oven dry 2 hours, keep in Dark Place under sealing 4-30 ℃, make sensitization latex polyester film.
3. the preparation of immune nitrocellulose filter:
1.5mg/ml goat anti-rabbit antibody and the anti-stomach Helicobacter pylori urease antibody of 2.0mg/ml II are sprayed on respectively on the position of nitrocellulose filter nature controlling line and detection line; Dried 2 hours down for 37 ℃; Keep in Dark Place under sealing 4-30 ℃, make immune nitrocellulose filter.The long nitrocellulose filter of every 50m is coated with goat anti-rabbit igg and the anti-stomach Helicobacter pylori urease antibody II solution of 6ml respectively, and the spacing of detection line and nature controlling line is 5mm.
4. with method 1.
The threshold value of the stomach Helicobacter pylori emulsion process detection kit of the present invention's design is 1ng/ml.
Method 3:
1. the preparation of sensitization latex particle:
A. get 0.5ml 15% carboxylated polystyrene latex and put into centrifuge tube, add the carbonic acid buffer flushing twice that 1.5ml pH 6.0 contains 0.01%SDS20mM, the centrifugal 10min of 13000g/min abandons supernatant, obtains carboxylated granules of polystyrene deposition.
B. the carboxylated granules of polystyrene deposition that obtained with a last step of the phosphate buffer solution suspension of 0.5ml pH 6.0 20mM; And the water-soluble charing diimine of adding 0.5ml%; Stirred 15 minutes under the room temperature; At last with twice of the borate buffer solution of 1.5ml 0.01M pH8.0 flushing, centrifugal, abandon supernatant, obtain thing to be marked.
C. the borate buffer that in the thing to be marked that step b obtains, adds 1.5ml pH 7.0 0.2M is processed the latex suspension, and in this latex suspension, adds the anti-stomach Helicobacter pylori urease antibody of 3.75mg I.
D. after fully reaction is spent the night, add the terminator cessation reaction, obtain the sensitization latex particle.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex damping fluid: get the 800ml purified water, add 120ml 1.0M Tris liquid inward, accurately take by weighing 2.0g Macrogol 2000 0,1.5g bovine serum albumin(BSA); 3.0g skim milk; 2.5g casein solution and 0.8g sodium azide add in the solution, fully dissolving mixes; Regulate pH to 8.0 with hydrochloric acid, add purified water to cumulative volume 1000ml.
(2) the latex damping fluid of using a step dilutes the sensitization latex particle, obtains the sensitization latex solution of 0.6w/v%.
(3) get 1.28ml sensitization latex solution with some latex machine specking on pretreated polyester film, placed under 37 ℃ the temperature oven dry 2 hours, keep in Dark Place under sealing 4-30 ℃, make sensitization latex polyester film.
3. the preparation of immune nitrocellulose filter:
1.0mg/ml goat anti-rabbit antibody and the anti-stomach Helicobacter pylori urease antibody of 1.0mg/ml II are sprayed on respectively on the position of nitrocellulose filter nature controlling line and detection line; Dried 2 hours down for 37 ℃; Keep in Dark Place under sealing 4-30 ℃, make immune nitrocellulose filter.The long nitrocellulose filter of every 50m is coated with goat anti-rabbit igg and the anti-stomach Helicobacter pylori urease antibody II solution of 6ml respectively, and the spacing of detection line and nature controlling line is 4mm.
4. with method 1.
The threshold value of the stomach Helicobacter pylori emulsion process detection kit of the present invention's design is 1ng/ml.
The specificity experiment of embodiment 3 stomach Helicobacter pylori emulsion process detection kit
Detection method:
1. get the stomach Helicobacter pylori emulsion process detection kit of embodiment 2 preparations, kit is placed on the level table.
2. the preparation of testing sample: press four types of experimental subjectss of table 1, at random from the different parts sampling of tester's ight soil, sampling amount is as the criterion with the small circle ring that is stained with special-purpose sampling rod front end with special-purpose sampling rod; Prepare a sample processing tube, vertically be put in and handle on the pipe holder, and in sample hose, add 0.6ml distilled water; The distilled water that the sample of getting on the special-purpose sampling rod is inserted in the sample hose stirs as testing sample.
3. every type of experimental subjects is 100 people, testing result such as table 1:
Table 1 stomach Helicobacter pylori emulsion process detection kit specificity test findings
Can be known that by last table stomach Helicobacter pylori emulsion process detection kit of the present invention is to Escherichia coli and the equal no cross reaction of shigella dysenteriae, specificity is good.
The sensitivity experiment of embodiment 4 stomach Helicobacter pylori emulsion process detection kit
Detection method:
1, gets the stomach Helicobacter pylori emulsion process detection kit that embodiment 2 makes, kit is placed on the level table.
2, test sample: configuration stomach Helicobacter pylori urease concentration be the Quality Control of 0.6ng/ml, 0.8ng/ml, 1.0ng/ml, 1.2ng/ml, 1.4ng/ml with reference to article as standard items.
3, observations: draw sample liquid 2-3 with suction pipe and be added dropwise in the sample cell, 8-15 minute sentence read result after dripping sample.
Table 2 kit sensitivity result
Table 1 result confirms that the stomach Helicobacter pylori emulsion process detection kit sensitivity that the present invention makes is 1ng/ml, the coincidence detection requirement.
The repeatability and the stability experiment of embodiment 5 stomach Helicobacter pylori emulsion process detection kit
One, kit batch in and batch between repeated experiment
1. experimental technique:
Will be with criticizing the stomach Helicobacter pylori urease standard items that detect 0.8ng/ml, 1.0ng/ml, 1.2ng/ml, 1.4ng/ml with the stomach Helicobacter pylori emulsion process detection kit of different batches respectively, each concentration repeats 5 times, observes the repeatability of kit.
2. experimental result: empirical tests, stomach Helicobacter pylori emulsion process detection kit batch in and batch between repeatability be 100%, false positive rate and false negative rate are 0.
Two, the stability experiment of kit
1. experiment purpose:
Stomach Helicobacter pylori emulsion process detection kit sealing is preserved, and deposit under 4 ℃ and the room temperature (about 25 ℃), observe of the influence of different storage temperatures kit stability.
2. experimental technique:
The kit that is stored in 4 ℃ takes out 4 boxes weekly, detects the stomach Helicobacter pylori urease standard items of 0.8ng/ml, 1.0ng/ml, 1.2ng/ml, 1.4ng/ml respectively; The kit that is stored in room temperature (25 ℃) took out 4 boxes in per 3 days, detected the stomach Helicobacter pylori urease standard items of 0.8ng/ml, 1.0ng/ml, 1.2ng/ml, 1.4ng/ml respectively.
3. experimental result:
Empirical tests, paper box can be preserved 24 months under 4 ℃, at room temperature can preserve 18 months; In the preservable time limit, kit all can reach the detection sensitivity of 1.0ng/ml.
The clinical testing of embodiment 6 stomach Helicobacter pylori emulsion process detection kit
Through special-purpose sampling rod patient's ight soil is taken a sample; Gather 302 parts of patients' fecal sample altogether; Wherein 171 parts is the sample that has infected stomach Helicobacter pylori; 131 parts is normal person's fecal sample, uses stomach Helicobacter pylori emulsion process detection kit of the present invention respectively sample to be detected.Drip 2-3 at the kit well respectively and drip urine sample, 8-15 minute sentence read result behind the dropping sample.The result shows that 166 parts of patients' fecal sample is positive, and 131 parts of normal persons' testing result is all negative, the kit of colour developing do not occur not having.
Testing result with urea breath test serves as that reference is added up clinical test results, and statistics is seen table 3:
Table 3 clinical test results summary sheet
Urea breath examination experiment detectable is reference, and the clinical test results of stomach Helicobacter pylori emulsion process detection kit of the present invention is following:
Sensitivity=166/ (166+5) * 100%=97.1%
Specificity=131/131 * 100%=100%
The predictive value of positive test=166/166 * 100%=100%
The predictive value of negative test=131/ (131+5) * 100%=96.3%
Coincidence rate (crude agreement)=(166+126)/(166+5+5+126) %=96.7%
In 302 duplicate samples of having investigated, sensitivity is for being 97.1%, and specificity is 100%, positive test predictive value 100%, and negative test predictive value 96.3%, coincidence rate is 96.7%.
This shows; " stomach Helicobacter pylori emulsion process detection kit " and existing ripe stomach Helicobacter pylori detection technique according to the present invention's preparation have good consistance aspect sensitivity, the specificity; It is simple to operate; Detect rapidly, be suitable for medical institutions or individual and be used for diagnosing whether infect stomach Helicobacter pylori.
Claims (12)
1. stomach Helicobacter pylori emulsion process detection kit; Comprise reagent strip; Said reagent strip comprises substrate, filter appearance paper, sensitization latex polyester film, immune nitrocellulose filter and thieving paper; Said filter appearance paper, sensitization latex polyester film, immune nitrocellulose filter and thieving paper is tandem array and being fixed on the said substrate successively; It is characterized in that, be coated with the anti-stomach Helicobacter pylori urease antibody I of color latex particle mark on the said sensitization latex polyester film, immune nitrocellulose filter is provided with detection line that has encapsulated anti-stomach Helicobacter pylori urease antibody II and the nature controlling line that has encapsulated goat anti-rabbit igg.
2. kit as claimed in claim 1 is characterized in that, said color latex particle is the granules of polystyrene of particle diameter 280～310nm.
3. kit as claimed in claim 1; It is characterized in that; Said anti-stomach Helicobacter pylori urease antibody I is to be antigenic substance with fusion Ure-BA, the polyclonal antibody that immune animal prepares, and said fusion Ure-BA sequence is seen SEQ ID NO:5.
4. the preparation method of the described stomach Helicobacter pylori emulsion process of the arbitrary claim of claim 1-3 detection kit may further comprise the steps:
1) preparation of sensitization latex particle:, obtain the sensitization latex particle with the carboxylated anti-stomach Helicobacter pylori urease antibody of polystyrene mark I;
2) preparation of sensitization latex polyester film: the sensitization latex particle that obtains with step 1) encapsulates polyester film, obtains sensitization latex polyester film;
3) will resist stomach Helicobacter pylori urease antibody II and goat anti-rabbit antibody to be sprayed on respectively on the position of nitrocellulose filter detection line and nature controlling line, dry for standby makes immune nitrocellulose filter;
4) will filter appearance a paper, sensitization latex polyester film, immune nitrocellulose filter, thieving paper sticks on the PVC sheet successively, cutting makes reagent strip;
5), obtain stomach Helicobacter pylori emulsion process detection kit with the reagent strip plastic casing of packing into.
5. preparation method as claimed in claim 4 is characterized in that, being prepared as of said step 1) sensitization latex particle:
A. get the carboxylated polystyrene of certain volume, with the carbonate buffer solution of 2～3 times of volume 20mM pH 6.0 repeatedly wash, centrifugal, abandon supernatant, obtain carboxylated granules of polystyrene deposition;
B. the carboxylated granules of polystyrene deposition that obtained with a last step of the phosphate buffer solution suspension of 1～1.5 times of volume 20mM pH6.0; And add the water-soluble charing diimine of 1～1.5 times of volume; Stirred under the room temperature 15～40 minutes; At last the borate buffer solution with 2～3 times of volume 0.01M pH 8.0 repeatedly wash, centrifugal, abandon supernatant, obtain thing to be marked;
C. the borate buffer that in the thing said to be marked that step b obtains, adds 2～3 times of volume 0.2M pH 7.0 is processed the latex suspension, and in said latex suspension, adds anti-stomach Helicobacter pylori urease antibody I;
D. fully reaction is spent the night, and adds the terminator cessation reaction, obtains the sensitization latex particle.
6. preparation method as claimed in claim 5 is characterized in that, carbonate buffer solution is the carbonate buffer solution that 20mM pH 6.0 contains 0.01%SDS among the said step a.
7. preparation method as claimed in claim 5 is characterized in that, carboxylated polystyrene concentration is 5～15w/v% among the said step a, and the concentration of water-soluble charing diimine is 8～12mg/ml among the said step b.
8. preparation method as claimed in claim 5; It is characterized in that; Anti-stomach Helicobacter pylori urease antibody I among the said step c is to be antigenic substance with fusion Ure-BA, the polyclonal antibody that immune animal prepares, and said fusion Ure-BA sequence is seen SEQ ID NO:5.
9. preparation method as claimed in claim 5; It is characterized in that the amount of the carboxylated anti-stomach Helicobacter pylori urease antibody of granules of polystyrene coupling I is in the said step c latex suspension: the anti-stomach Helicobacter pylori urease antibody of 0.5mg I/10mg granules of polystyrene.
10. preparation method as claimed in claim 4 is characterized in that, said step 2) in the preparation concrete steps of sensitization latex polyester film be:
A. with latex damping fluid dilution sensitization latex particle, obtain the sensitization latex solution of 0.2～0.6w/v%;
B. the sensitization latex solution spraying polyester film for preparing with steps A, drying makes sensitization latex polyester film.
11. preparation method as claimed in claim 10 is characterized in that, the latex buffer formulation in the said steps A is following: 8～12v% 1.0M Tris liquid; 0.2～0.4w/v% Macrogol 2000 0,0.15～0.25w/v% bovine serum albumin(BSA), 0.1～0.3w/v% skim milk; 0.25～0.35w/v% casein; With 0.02～0.08w/v% sodium azide, regulate pH to 8.0 ± 0.05 with hydrochloric acid, surplus is a water.
12. the application of the described kit of the arbitrary claim of claim 1-3 in preparation stomach Helicobacter pylori detectable.
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