CN102565391A - Immunoassay method for helicobacter pylori in gastric mucosa sample - Google Patents
Immunoassay method for helicobacter pylori in gastric mucosa sample Download PDFInfo
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Abstract
An immunoassay method for Helicobacter pylori (H. pylori) in a gastric mucosa sample comprises (a) suspending the gastric mucosa sample doubted about containing the H. pylori into sample diluent; (b) enabling the gastric mucosa sample in the diluent to be contacted with a first polyclonal antibody of an antigen resisting to the H. pylori to form a compound of the antibody and the antigen; (c) separating the sample and the compound; (d) enabling the compound to be exposed on a second polyclonal antibody of the antigen, enabling one part of the antibody to react with the compound, combining one of the first polyclonal antibody and the second polyclonal antibody onto a solid carrier, and marking the other one of the first polyclonal antibody and the second polyclonal antibody through a detection agent; and (e) assaying volume of the marked antibody and sequentially assaying existence of H. pylori antibodies in the gastric mucosa sample. The immunoassay method for the H. pylori in the gastric mucosa sample has the advantages of being low in cost, visual and accurate in detection result and fast in reaction speed.
Description
Technical field
The present invention relates to the immunoassay of helicobacter pylori in a kind of stomach lining sample.
Background technology
(Helicobacter pylori is the bacterium that a kind of upper gastro-intestinal tract the people is found H.pylori) to helicobacter pylori, has been found that it is relevant with other disease with gastroduodenal disease such as peptic ulcer, gastritis.By sources belong to campylobacter to this bacterium, then according to dividing at Helicobacterium again about its more detailed data of ultrastructure and fatty acid component aspect.
The detection method of helicobacter pylori is a lot, and common method has: (1) bacterial cultivation.This method is diagnosing helicobacter pylori " golden standard ".This method can directly prove the existence of H.pylori, and non-false positive occurs, and also can do drug sensitive test simultaneously.Different strains is done gene research.But the separation and Culture technical conditions are higher.And need the certain culture time, can have false negative to occur, still fail extensively to carry out.(2) urease test.Helicobacter pylori can produce very highly active in a large number urease, and the rare so high urease activity of other bacteriums.Based on this principle, adopt pH＜6.0 more, the reagent color is constant generally speaking.If there is H.pylori to infect in the stomach, when sample is put in people's reagent, the urease that H.pylori produced then decomposing urea produces ammonia, and pH is raise, and causes the phenol red variable color-pink of agent.Simple and practical, the rapid sensitive of urease test method.Can be in 1 minute judged result, and the intact back of microscopy just can in time be treated.(3) the histopathological examination method is more, and methods such as HE dyeing, Warthin-Starry dyeing, Giemsa dyeing, carbolfuchsin dyeing, Gimenez dyeing, acridine orange dyeing, indirect immune light method, phase microscope observation, unmarked antibody PAP decoration method, bispecific antibody enzyme immunostaining, oligonucleotide probe inspection are arranged.With Warthin-Starry dyeing, the Gimenez Color is best and use always.But this detection method is loaded down with trivial details time-consuming, needs certain technology, and certain false positive is arranged.(4) breath test.Infect the oral isotope-labeled urea liquid of patient of H.pylori.The isotope CO that then produces after the urea decomposition
2Breathe out from lung.
For the stomach lining biopsy specimen of doing behind the gastrointestinal endoscopy that is obtained, the helicobacter pylori of mainly adopting two kinds of methods inspections of RUT method and pathology detection to exist.Generally speaking, these two detection methods have higher consistance, still, are not to replace each other, and inconsistent place is still arranged.Inconsistent reason can have: be distributed with difference in (1) H.pylori stomach, the RUT method is to detect a block organization.The problem that exists the sample sampling to get, and pathology is several sections of a block organization, the problem of sampling is more outstanding.(2) urease activity and H.pylori's is inconsistent.Though H.pylori can produce very highly active in a large number urease, and the rare high urease activity like this of other bacteriums.But find that at present pressing down sour medicine, some antibiotic etc. can influence urease test, might take for the patient and press down sour medicine.(3) pH method, because other ureases that mix, there is false positive in the collimation error of color judgment etc.(4) interference such as other similar helicobacters, vibrios can be arranged on the Pathomorphology.Exactly because this reason; In April, 1999, China medical association digested disease credit meeting proposition in " the some problem common recognitions of helicobacter pylori suggestion "; H.pylori cultivate positive or following 4 in wantonly 2 positive persons, be diagnosed as the H.pylori positive: (1) H.pylori morphology (smear, histological stain or immune group method dye); (2) urease relies on test (rapid urease test); (3) serological test (ELISA or western blot test etc.); (4) special PCR detects.The clinical diagnosis standard that H.pylori infects, wantonly 1 positive person in following 2 is diagnosed as the H.pylori positive: (1) H.pylori morphology (smear or histological stain); (2) urease dependence test (RUT examination, 13C-UBT).
Summary of the invention
The object of the present invention is to provide a kind of with low cost, the immunoassay of helicobacter pylori in the intuitive and accurate stomach lining sample of testing result.
The objective of the invention is to realize like this: it may further comprise the steps:
(a) make the sample diluting liquid of being with detection material;
(b) contact the stomach lining sample in this dilution with first kind of polyclonal antibody of anti-helicobacter pylori antigen to form antibody-antigenic complex;
(c) separate the sample of step (b) with the resulting antibody-antigenic complex of step (b);
(d) antibody-antigenic complex after the separation that obtains step to step (c) is exposed to second kind of polyclonal antibody of anti-helicobacter pylori antigen and reacts the part of second kind of polyclonal antibody of anti-helicobacter pylori antigen and complex; Wherein a kind of polyclonal antibody in wherein said first kind of polyclonal antibody and the second kind of polyclonal antibody is combined on a kind of solid carrier, and another kind of polyclonal antibody carries out mark with a kind of detection agent;
(e) amount of the antibody of mensuration mark.
It is characterized in that: shown in the sample diluting liquid production method be suspended in based in dilution protein, that contain alkaline matter for a kind of stomach lining sample that contains helicobacter pylori to suspection.
The invention has the advantages that: 1 is with low cost, methods such as existing relatively urease test, bacterial cultivation, and the present invention makes the with low cost of sample diluting liquid, and it is comparatively cheap that each reagent belongs to product and the price obtained easily.2 testing results are directly perceived, accurate.If mark is horseradish peroxidase etc.; Can be through adding colour developing liquid; Reacted result can directly find out through chromogenic reaction; The reacted sample diluting liquid that contains helicobacter pylori presents blueness or light blue, and the reacted sample diluting liquid that does not contain helicobacter pylori presents water white transparency; If mark is collaurum, then can the Direct observation result.3 reaction velocitys are fast; General 5～30 minutes can display result; 4, through test, particularly to having taken antiacid, bismuth agent or microbiotic patient ELISA test and helicobacter pylori quantitative fluorescent PCR, the two has and likewise detects usefulness; And the former to have a latter unexistent cheap, convenient and swift, the advantage that detection speed is fast.
Embodiment
Concrete steps of the present invention are following:
(a) make the sample diluting liquid of being with detection material; Production method is suspended in based in dilution protein, that contain alkaline matter for a kind of stomach lining sample that contains suspection helicobacter pylori.Protein wherein is to refer to the wherein a kind of protein that is selected from hyclone, normal goats serum, GPS, horse serum, casein, albumin, gelatin and the bovine serum albumin(BSA), and it is non-specific in order to improve to increase protein.Be the contained acidic liquid of antagonism stomach lining sample; Sample buffer should contain alkaline matter, helps to reduce the change to reacting liquid pH value of stomach lining material, helps to improve the pH value; Reduce the contained protease activities of stomach lining, and other materials are to the interference of reaction.
Wherein sample diluting liquid is to contain the sample diluting liquid that is selected from a kind of albumen in hyclone, normal goats serum, GPS, horse serum, casein, albumin, gelatin and the bovine serum albumin(BSA).
The alkaline matter that sample diluting liquid contains comprises but is not limited to Pehanorm base propane sulfonic acid, N; One or more combinations in two (2-hydroxyethyl) glycocoll of N-, trishydroxymethylaminomethane, N-three-(methylol) methyl aminoacetic acid, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid half sodium salt, N-three (methylol) methyl-2-tarine, 3-(the non-quinoline of N-) ethyl sulfonic acid, sodium tetraborate damping fluid, sodium carbonate-sodium bicarbonate buffer liquid, the glycine buffer, wherein by its pH of damping fluid of alkaline matter preparation greater than 8.The method of configuration alkaline matter is that those skilled in the art of the present technique are familiar with.
After a kind of stomach lining sample that will contain helicobacter pylori was suspended in based on sample diluting liquid protein, that contain alkaline matter, the pH of sample diluting liquid was advisable 6～10, and wherein pH=8 the time, the effect of detection is best.
(b) contact the stomach lining sample in the sample diluting liquid with first kind of polyclonal antibody of anti-helicobacter pylori antigen to form antibody-antigenic complex; In general the present invention has used the antibody of anti-helicobacter pylori, can be two kinds of monoclonal antibody and polyclonal antibodies, but commonly used at present be polyclonal antibody.These antibody can obtain from the serum of the animal of sensitization.At present both at home and abroad existing many companies can provide the antibody of commercial anti-helicobacter pylori, and price that can less expensive obtains.Chinese patent (application number 97103112.6) has been described the preparation method of helicobacter pylori antibody in detail.Only be summarized as follows at this: be expelled to Heliobacter pylori antigen in mammal such as the bodies such as rabbit, sheep, the antigen of the suitable dosage of multiple injection, the demonstration test animal has produced helicobacter pylori antibody, gets experimental animal serum, purifiedly can obtain required antibody.Many sample diluting liquids contain Triton X-100 and/or Tween 20 with scope concentration between 0.05% to 2%, add the ionic strength that 0～2.9%NaCL can change buffer system.These weak or nonspecific reactions that change through reducing in forming cause specificity preferably.
(c) separate the sample of step (b) with the resulting antibody-antigenic complex of step (b);
(d) antibody-antigenic complex after the separation that obtains step to step (c) is exposed to second kind of polyclonal antibody of anti-helicobacter pylori antigen and reacts the part of second kind of polyclonal antibody of anti-helicobacter pylori antigen and complex; Wherein a kind of polyclonal antibody in wherein said first kind of polyclonal antibody and the second kind of polyclonal antibody is combined on a kind of solid carrier, and another kind of polyclonal antibody carries out mark with a kind of detection agent; Wherein solid carrier is enzyme reaction plate or cellulose membrane (like nitrocellulose filter, cellulose mixture film) etc., can certainly be the common solid carriers of other field of immunology.
Above-mentioned detection agent is selected from one or more combinations in alkaline phosphatase and beta galactosidase, horseradish peroxidase, radiomaterial (like iodine-125), collaurum, colloidal solid (gold, selenium etc.), color latex microballoon, the disperse dyes granule.One of the most frequently used enzyme labeling thing is HRPO (HRP) and alkaline phosphatase.Is the technology of antibodies on this type mark substance well known to those of ordinary skill in the art.
In sample diluting liquid, also contain pepsin inhibitor, described pepsin inhibitor comprises but is not limited to one or more the potpourri in Pepstatin, the thimerosal.
After wherein antibody-antigenic complex is exposed to the part reaction of second kind of polyclonal antibody, can reduces the cross reaction degree or otherwise improve this complex of the specific damping fluid washing of this determination method with a kind of.Described damping fluid is a phosphate buffer.
(e) amount of the antibody of mensuration mark; Wherein also possibly be provided with in the step (e) is added with colour developing liquid.Whether interpolation colour developing liquid can determine that if the antibody of mark is horseradish peroxidase, substrate is tetramethyl benzidine (TMB) according to the detection agent of mark, if the antibody of mark is alkaline phosphatase, substrate is a para-nitro-pheneye phosphate; If mark is collaurum or coloured dye particle, then do not need substrate, through the direct result of determination of color.
A kind of method of measuring helicobacter pylori in the stomach lining sample, it comprises:
Wherein be used for to be fixed on holder commonly used any immunoassay from the used unlabelled polyclonal antibody of method of underproof stomach lining sample extraction antigenic substance.Spendable in these holders is filter paper, nitrocellulose filter, plastic bead, tygon, polystyrene, polypropylene or other suitable test tube, enzyme reaction plate etc.Is the technology of antibodies on this type material well known to those of ordinary skill in the art.
Wherein after step (d) mesocomplex is exposed to the part reaction of second kind of polyclonal antibody, can reduces the cross reaction degree or otherwise improve this complex of the specific damping fluid washing of this determination method with a kind of.In the preparation of antibody-solutions that is used for determination method and cleansing solution, also can embody the cross reaction degree.This antibody can combine above-mentioned a kind of albumen urea of mentioning and be provided in damping fluid.Control the cross reaction degree through adding salt and surfactant and can prepare and cushion cleansing solution used in this determination method.
Configuration through part reagent, detection agent and and the ELISA experiment the present invention will be described:
The following example two explanation determination methods of a kind of being called " forward " wherein at first contact the antibody that is combined on the holder through forming a kind of antibody/antigen complex and from sample, extracting antigen to the method that this complex contacts with a kind of labelled antibody of known quantity with sample to be tested.But those of ordinary skills also can expect carrying out immunoassays with being called " simultaneously " or " oppositely " determination method.Simultaneously determination method comprises simultaneously and to be added to the single incubation step in the sample to be tested to the antibody and the antibody of mark that are attached on the solid support.After accomplishing incubation, the flushing solid support is removed the remaining sample and the antibody of compound token not.Measure the existence of the labelled antibody that links to each other with solid support then.Reverse determination method comprises that elder generation is added to the antibody-solutions of mark in the fecal specimens, then adds the step that is combined in the unlabelled antibody on the holder.Incubation makes it not contain remaining sample and unreacted labelled antibody with conventional this holder of washing lotion flushing after one second.
Because damping fluid; Every kind of solvent in the detection agent equal solvent all relates to and uses multiple material; At concrete parameter aspect aspect the configuration solvent bigger difference is arranged; Below in conjunction with specific embodiment (during laboratory experiment, parameter when adopting various materials and collocation method), step of the present invention is described as solvent.
One, the solution preparation that relates to of this test:
(1), common alkaline solution preparation
The compound method of the employing alkalescence biological buffer that the present invention relates to is following:
1, hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid
HEPES molecular formula: C
8H
18N
2O
4S molecular weight: 238.3
Add 2～5 gram HEPES in every 1000ml deionized water, wait to dissolve the back and transfer between pH to 8.2～9.15 scopes, use behind the EK with lN NaOH.
2, borax-lime chloride damping fluid
Get borax 0.3～2g and lime chloride 2～5g, add the about 800ml of water dissolving after, to 8.0-9.5, thin up promptly gets to 1000ml with the about 2.5ml adjusting of 1mol/L hydrochloric acid solution pH value.
3, boric acid-potassium chloride damping fluid (pH8.0～9.5)
Get boric acid 2.7～5.0g, add 0.1mol/L Klorvess Liquid 500ml and make dissolving, add 0.1mol/L sodium hydroxide solution 210ml again, promptly get.
4, glycine buffer preparation
Glycine buffer: 6～8 gram glycocoll, 9～12 gram sodium chloride are dissolved in distilled water, regulate between pH to 8.2～9.15 scopes with 1mol/L NaOH, and distilled water complements to 1 liter.
(2), the preparation of sample diluting liquid
The compound method of sample diluting liquid is following: add 0.1～2.0g bovine serum albumin(BSA) (BSA) at above-mentioned akaline liquid 100ml, add an amount of (final concentration is 0.01%～0.02%) thimerosal, add 0.05～2% Triton X-100 and/or Tween-20.
(3), encapsulating damping fluid is used for ELISA test helicobacter pylori antibody is encapsulated microwell plate
0.05mol/L carbonate buffer solution (pH9.6): 0.75g sodium carbonate, the 1.46g soda mint adds deionized water and is settled to 500ml.
(4), the sealing damping fluid is used to encapsulate back minimizing nonspecific reaction
0.05mol/L carbonate buffer solution (pH9.6) (the same)+2.0%BSA:2g bovine serum albumin(BSA) (BSA) adds the above-mentioned 0.05mol/L carbonate buffer solution (pH9.6) for preparing of 100ml
(5), 0.02mol/L phosphate buffer (PBS) (pH7.4)
0.2g potassium dihydrogen phosphate, the 2.90g sodium hydrogen phosphate, 8g sodium chloride adds deionized water and is settled to 1000ml.
(6), antibody diluent is used to dilute various antibody etc.
0.02mol/L PBS (pH7.4)+0.2%BSA:0.2g bovine serum albumin(BSA) (BSA) adds among the above-mentioned above-mentioned 0.02mol/L PBS (pH7.4) for preparing that prepares of 100ml.
(7), lavation buffer solution is used for unnecessary unconjugated antigen of wash-out and antibody etc.
0.02mol/L PBS (pH7.4)+0.05% Tween-20 (Tween-20): 50 microlitre Tween-20 are added among the above-mentioned 100ml 0.02mol/L PBS (pH7.4) for preparing the concussion mixing.
(8), colour developing liquid
TMB-carbamide peroxide solution
A liquid 3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine (TMB) 20mg, absolute ethyl alcohol (or DMSO) 10ml adds distilled water to 100ml.
B liquid (receive, pH5.0-5.4): Na by 0.1ml/L citric acid-0.2ml/L phosphoric acid hydrogen two
2HPO
414.60g, citric acid 9.33g, 0.75% carbamide peroxide 6.4ml adds deionized water to 1000ml, transfers to pH5.0-5.4.
Be TMB-carbamide peroxide solution after A liquid and B liquid mixed by 1: 1
(9), stop buffer: 2mol/L H
2SO
4Solution
10ml 98% concentrated sulphuric acid adds in the 60ml deionized water, is settled to 100ml, room temperature preservation.
Stop buffer generally adds the back at colour developing liquid and added in 5～10 minutes.
(10) dilution of enzyme labeling helicobacter pylori antibody
The helicobacter pylori antibody of (HRP) enzyme labeling is diluted to working concentration with antibody diluent according to proper proportion.What the present invention used is anti-people's helicobacter pylori antibody of the horseradish peroxidase-labeled of U.S. KPL company, and method to specifications is diluted to 0.1mg/ml.
Embodiment one: the ELISA test
(1), ELISA test alkaline solution preparation
The compound method of the employing alkalescence biological buffer that the present invention relates to is following:
1, hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid
HEPES molecular formula: C
8H
18N
2O
4S molecular weight: 238.3
Add 2.38 gram HEPES in every 1000ml deionized water, wait to dissolve the back and transfer pH to 8.2, use behind the EK with lN NaOH.
2, the preparation of sample diluting liquid
The compound method of sample diluting liquid is following: 100ml adds 1g bovine serum albumin(BSA) (BSA) at hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid, adds an amount of (final concentration is 0.02%) thimerosal, adds 0.1% Tween-20.
3, encapsulating damping fluid is used for ELISA test helicobacter pylori antibody is encapsulated microwell plate
0.05mol/L carbonate buffer solution (pH9.6): 0.75g sodium carbonate, the 1.46g soda mint adds deionized water and is settled to 500ml.
4, the sealing damping fluid is used to encapsulate back minimizing nonspecific reaction
0.05mol/L carbonate buffer solution (pH9.6) (the same)+2.0%BSA:2g bovine serum albumin(BSA) (BSA) adds the above-mentioned 0.05mol/L carbonate buffer solution (pH9.6) for preparing of 100ml
5, lavation buffer solution is used for unnecessary unconjugated antigen of wash-out and antibody etc.
0.02mol/L PBS (pH7.4)+0.05% Tween-20 (Tween-20): 50 microlitre Tween-20 are added among the above-mentioned 100ml 0.02mol/L PBS (pH7.4) for preparing the concussion mixing.
6, colour developing liquid
TMB-carbamide peroxide solution
A liquid 3 ', 3 ', 5 ', 5 ' ,-tetramethyl benzidine (TMB) 20mg, absolute ethyl alcohol (or DMSO) 10ml adds distilled water to 100ml.
B liquid (receive, pH5.0-5.4): Na by 0.1ml/L citric acid-0.2ml/L phosphoric acid hydrogen two
2HPO
414.60g, citric acid 9.33g, 0.75% carbamide peroxide 6.4ml adds deionized water to 1000ml, transfers to pH5.0-5.4.
Be TMB-carbamide peroxide solution after A liquid and B liquid mixed by 1: 1
7, stop buffer: 2mol/L H
2SO
4Solution
10ml 98% concentrated sulphuric acid adds in the 60ml deionized water, is settled to 100ml, room temperature preservation.
Stop buffer generally adds the back at colour developing liquid and added in 5～10 minutes.
8, the dilution of enzyme labeling helicobacter pylori antibody
The helicobacter pylori antibody of (HRP) enzyme labeling is diluted to working concentration with antibody diluent according to proper proportion.What the present invention used is anti-people's helicobacter pylori antibody of the horseradish peroxidase-labeled of U.S. KPL company, and method to specifications is diluted to 0.1mg/ml.
Two, the encapsulating and sealing of enzyme reaction plate (process that promptly detects the b～d of step), this process is directly directly being done as a whole the processing with step b～d.
Encapsulate with the above-mentioned damping fluid that encapsulates first kind of polyclonal antibody of anti-helicobacter pylori antigen is diluted to debita spissitudo; What first kind of polyclonal antibody of anti-helicobacter pylori antigen used is anti-people's helicobacter pylori antibody of U.S. KPL company, and method to specifications is diluted to 10 μ g/ml.Every hole adds the good antibody of above-mentioned dilution 100 μ l, wraps with film to be put in 4 degree and to preserve and to spend the night.
Sealing next day dries above-mentioned ELISA Plate, and every hole adds the aforementioned sealing damping fluid for preparing of 100 μ l, wraps with film to be put in 4 degree and to preserve and spend the night.Dry ELISA Plate next day, dry, and sealing, subsequent use.
Three, detect the sample diluting liquid of band detection material
1, after stomach lining to be measured mark article and positive control and negative control were done dilution in 1: 4 with aforesaid sample diluting liquid, every hole added 100ul in the aforementioned ELISA Plate that has encapsulated, put in the wet box 37 ℃ and acted on 30 minutes.
The taking-up ELISA Plate dries, and with the aforementioned lavation buffer solution washing for preparing, washs each 3-5 minute 3-5 time.
2, every hole in the above-mentioned anti-people's helicobacter pylori antibody ELISA Plate that has diluted good horseradish peroxidase-labeled is added 100ul, put in the wet box 37 ℃ of effects 30 minutes.
The taking-up ELISA Plate dries, and with the aforementioned lavation buffer solution washing for preparing, washs each 3-5 minute 3-5 time.Button is done.
3, colour developing.Every hole adds aforementioned colour developing liquid A liquid and the B liquid of having prepared of 50ul in ELISA Plate.
4, detect.Put in the warm box 37 ℃ of effects 5-15 minute, directly the visual inspection result; Perhaps every hole adds aforementioned stop buffer 100ul, the absorbance when on ELIASA, reading wavelength and being 450 nanometers.Criterion is positive with ratio P/N >=2.1 of known negative contrast stomach lining (N) with stomach lining to be checked (P).
Embodiment two dot-ELISA test
(1), this tests required solution solution preparation
1, the preparation of alkaline buffer is the same, explains with borax-lime chloride damping fluid (pH8.0).
Borax-lime chloride damping fluid (pH8.0)
Get borax 0.572 and lime chloride 2.94g, add the about 800ml dissolving of water after, with the about 2.5ml adjusting of 1mol/L hydrochloric acid solution pH value to 8.0, thin up promptly gets to 1000ml.
2, the preparation of sample diluting liquid
The compound method of sample diluting liquid is following: add 1g bovine serum albumin(BSA) (BSA) at borax-lime chloride damping fluid (pH8.0) 100ml, add an amount of (final concentration is 0.01%) thimerosal, add 0.1% Tween-20.
3, encapsulate damping fluid
0.05mol/L carbonate buffer solution (pH9.6): 0.75g sodium carbonate, the 1.46g soda mint adds deionized water and is settled to 500ml.
4, the sealing damping fluid is used to encapsulate back minimizing nonspecific reaction
0.05mol/L carbonate buffer solution (pH9.6) (the same)+2.0%BSA:2g bovine serum albumin(BSA) (BSA) adds the above-mentioned 0.05mol/L carbonate buffer solution (pH9.6) for preparing of 100ml
5,0.02mol/L phosphate buffer (PBS) (pH7.4)
0.2g potassium dihydrogen phosphate, the 2.90g sodium hydrogen phosphate, 8g sodium chloride adds deionized water and is settled to 1000ml.
6, antibody diluent is used to dilute various antibody etc.
0.02mol/L PBS (pH7.4)+0.2%BSA:0.2g bovine serum albumin(BSA) (BSA) adds among the above-mentioned above-mentioned 0.02mol/L PBS (pH7.4) for preparing that prepares of 100ml.
7, lavation buffer solution is used for unnecessary unconjugated antigen of wash-out and antibody etc.
0.02mol/L PBS (pH7.4)+0.05% Tween-20 (Tween-20): 50 microlitre Tween-20 are added among the above-mentioned 100ml 0.02mol/L PBS (pH7.4) for preparing the concussion mixing.
8, substrate solution:
3,3 diaminobenzidine 50mg, 0.05mol/L, pH value 7.6TrisHCl damping fluid 100ml, filter the dissolving back, and 4 ℃ keep in Dark Place.Face with preceding adding H
2O
2Making final concentration is 0.03%.
The dilution of 9 enzyme labeling helicobacter pylori antibodies
The helicobacter pylori antibody of (HRP) enzyme labeling is diluted to working concentration with antibody diluent according to proper proportion.What the present invention used is anti-people's helicobacter pylori antibody of the horseradish peroxidase-labeled of U.S. KPL company, and method to specifications is diluted to 0.1mg/ml.
10, carrier: nitrocellulose filter 022～045 μ m.Nitrocellulose filter (the NC film is produced by Huangyan, Zhejiang people chemical plant, specification: 20cm * 20cm).
11, the goat-anti people helicobacter pylori antibody of rabbit helicobacter pylori antibody and horseradish peroxidase-labeled is provided by U.S. KPL company.
Two, pre-treatment (promptly detecting the process of the b～d of step), this process is directly directly being done as a whole the processing with step b～d.
1, nitric acid is tieed up the suitably big or small nitrocellulose filter of processing clip of plain film, places distilled water to soak 2～10 minutes, takes out to be put on the clean filter paper, and air dry impresses with the light face of diameter 4～6mm card punch in film at interval, and is subsequent use.
2, encapsulate.With the above-mentioned damping fluid that encapsulates helicobacter pylori antibody is diluted to debita spissitudo.What the present invention used is anti-people's helicobacter pylori antibody of U.S. KPL company, and method to specifications is diluted to 10 μ g/ml.Every hole adds the good antibody of above-mentioned dilution 100 μ l, covers and is incubated overnight at room temperature.With the lavation buffer solution flushing once.At room temperature it was sealed 1 hour with the sealing damping fluid.Again once with the lavation buffer solution flushing.
3, the dilution of enzyme labeling helicobacter pylori antibody
The helicobacter pylori antibody of (HRP) enzyme labeling is diluted to working concentration with antibody diluent according to proper proportion.What the present invention used is anti-people's helicobacter pylori antibody of the horseradish peroxidase-labeled of U.S. KPL company, and method to specifications is diluted to 0.01mg/ml.
(3), dot-ELISA test
1, diluted stomach lining sample to be measured, the positive and negative control by 1: 4 with sample diluting liquid.Then, respectively add 100 μ l and go in the hole of test paper, cover and incubation 1 hour at room temperature.
2, wash plate 5 times with lavation buffer solution.
The rabbit anti-helicobacter pylori that 3, will dilute good HRPO mark adds 100 μ l to each hole.Hide plate, and incubation 1 hour at room temperature.
4, wash plate 5 times with lavation buffer solution.
5, at room temperature add the substrate solution that embodiment 2 disposes in the solution allocation steps then.
6, visual observations result is dried in flushing.
Selection can produce the dilution of peak signal and minimum background and make desirable dilution.
The determination method that is called sandwich according to the present invention also can be used in the helicobacter pylori of measuring in the stomach lining sample.Three step determination methods known in the state of the art and this basic skills can be used in the helicobacter pylori of measuring in the stomach lining sample.Three step determination methods generally are performed such: the stomach lining sample dispersion that contains suspection helicobacter pylori joins in the insolubilized antibody of a kind of helicobacter pylori that from the animal of first kind of generation antibody, has obtained in a kind of sample diluting liquid of minimum cross reaction degree and the sample of dilution.This sample incubation, form antibody-antigenic compound.After the excess sample of flushing on the solid support, produced the antibody animal resulting and add in this antibody-antigenic compound also as the known helicobacter pylori antibody of primary antibody that incubation forms a kind of antibody-antigen-antibody complex a kind of from second kind.After forming this compound and removing unreacted antibody, this compound and a kind of known antibodies reaction as secondary antibody, secondary antibody wherein is the immunoglobulin (Ig) of a kind of generation secondary antibody kind like anti-(rabbit, ox or goat).With conventional method (generally using enzyme) mark secondary antibody and with antibody-antigen-antibody complex incubation, form three antibody complexes or sandwich.After removing unreacted secondary antibody, measure antigen with conventional method.In using enzyme labeling, in the compound of a kind of substrate this antigen of adding and three kinds of antibody and the reaction of controlling substrate and ligase measure the antigen amount that is present in the sample.In this sandwich determination method as the fundamental measurement method, if necessary, preparation or buffering washing lotion and antibody-solutions are controlled the cross reaction degree.Through describing the present invention in detail and through with reference to preferred embodiment, all will be conspicuous not exceeding in the equivalent structures improvement possible in institute's restricted portion and changing to those of ordinary skills.
Claims (10)
1. the immunoassay of helicobacter pylori in the stomach lining sample, it comprises:
(a) make the sample diluting liquid of being with detection material;
(b) contact the stomach lining sample in the sample diluting liquid with first kind of polyclonal antibody of anti-helicobacter pylori antigen to form antibody-antigenic complex;
(c) separate the sample of step (b) with the resulting antibody-antigenic complex of step (b);
(d) antibody-antigenic complex after the separation that obtains step (c) is exposed to second kind of polyclonal antibody of anti-helicobacter pylori antigen and reacts the part of second kind of polyclonal antibody of anti-helicobacter pylori antigen and complex; Wherein a kind of polyclonal antibody in wherein said first kind of polyclonal antibody and the second kind of polyclonal antibody is combined on a kind of solid carrier, and another kind of polyclonal antibody carries out mark with a kind of detection agent;
(e) amount of the antibody of mensuration mark;
It is characterized in that: shown in the sample diluting liquid production method be suspended in based in dilution protein, that contain alkaline matter for a kind of stomach lining sample that contains helicobacter pylori to suspection.
2. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1; It is characterized in that: described sample diluting liquid is a kind of dilution based on protein, and sample diluting liquid is to contain the sample diluting liquid that is selected from a kind of albumen in hyclone, normal goats serum, GPS, horse serum, casein, albumin, gelatin and the bovine serum albumin(BSA).
3. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1, it is characterized in that: in sample diluting liquid, also contain the alkaline matter that contains that the contained acid solution of promising reduction stomach lining adds the influence that detects.Described alkaline matter comprises but is not limited to Pehanorm base propane sulfonic acid, N; One or more combinations in two (2-hydroxyethyl) glycocoll of N-, trishydroxymethylaminomethane, N-three-(methylol) methyl aminoacetic acid, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid half sodium salt, N-three (methylol) methyl-2-tarine, 3-(the non-quinoline of N-) ethyl sulfonic acid, sodium tetraborate damping fluid, sodium carbonate-sodium bicarbonate buffer liquid, the glycine buffer, wherein by formulated its pH of damping fluid of alkaline matter greater than 8.
4. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1; It is characterized in that: after antibody-antigenic complex is exposed to the part reaction of second kind of polyclonal antibody in the step (d), can reduces the cross reaction degree or otherwise improve this complex of the specific damping fluid washing of this determination method with a kind of.
5. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 4, it is characterized in that: described damping fluid is a phosphate buffer.
6. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1, it is characterized in that: the detection agent in the step (d) is selected from one or more combinations in alkaline phosphatase and beta galactosidase, horseradish peroxidase, radiomaterial (like iodine-125), collaurum, colloidal solid (gold, selenium etc.), color latex microballoon, the disperse dyes granule.
7. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1, it is characterized in that: solid carrier is enzyme reaction plate or cellulose membrane.
8. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1, it is characterized in that: the PH of sample diluting liquid is 6～10, and wherein optimal value is PH=8.
9. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1; It is characterized in that: in sample diluting liquid, also contain pepsin inhibitor, described pepsin inhibitor comprises but is not limited to one or more the potpourri in Pepstatin, the thimerosal.
10. according to the immunoassay of helicobacter pylori in the stomach lining sample of claim 1, it is characterized in that: also be added with colour developing liquid in the step (e).
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| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
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