CN1207394C - Pylorospirobacillus antigen in blood - Google Patents
Pylorospirobacillus antigen in blood Download PDFInfo
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Abstract
The present invention relates to a method for discovering and detecting H. pylori antigens in infected individual blood. The H. pylori antigens are ingredients for H. pylori cells, and comprise (not only comprise) DNA, RNA, nucleotide, protein and peptide fragments. The DNA, the RNA and nucleotide fragment antigens of the H. pylori are detected by polymerase chain reaction (PCR) or ligase chain reaction (LCR) or a DNA hybridization method or other amplification methods. The protein or the peptide or other antigen ingredients of the H. pylori are detected through immunity detection or an immunoblotting method by H. pylori antigens antibodies, and the H. pylori antigens antibodies are preferably antibodies purified through affinity columns.
Description
Technical field
The present invention relates in blood (comprising whole blood, blood plasma and serum) discovery to helicobacter pylori (H.pylori) antigen and antigen fragment thereof.The Heliobacter pylori antigen that is found in the blood includes but not limited to helicobacter pylori DNA, RNA or its fragment or helicobacter pylori protein/peptide or its other antigen component.Helicobacter pylori DNA or its fragment are detected by polymerase chain reaction (PCR), ligase chain reaction (LCR), DNA hybridization, branched DNA signal amplification detection or other method for amplifying signal.Its helicobacter pylori RNA is detected by PCR, hybridization or other signal amplification detecting process.Helicobacter pylori protein or peptide or its other antigen component use the affinity purification antibody to helicobacter pylori to detect by immunodetection or immunoblot experiment.The Heliobacter pylori antigen that the invention still further relates to by detecting in the blood comes diagnosing helicobacter pylori infection.
Background technology
Helicobacter pylori (H.pylori) is a gram negative bacterium, and it infects stomach mucous membrane and is the inducement of most peptic ulcer disease (PUD).Up to now, the maldigestion of ulcer and other form is considered to relevant with stress reaction level or food habits.Recently, medical circle has confirmed that helicobacter pylori is the inducer that the gastropathy of some form comprises ulcer and cancer of the stomach.Eradicate helicobacter pylori has promoted ulcer healing and has greatly reduced cancer and the sickness rate of PUD.
Helicobacter pylori causes most of gastric duodenal ulcer and peptic ulcer disease (PUD).The contact of helicobacter pylori and PUD was found (Lancet I:1311-1344) by Australian doctor Warren and Marshall at first in 1984.Now, the most commonly encountered diseases that helicobacter pylori infection is counted as gastritis because of, and from the nosetiology angle, it is relevant with former B cell lymphoma with stomach ulcer, duodenal ulcer, adenocarcinoma of stomach.
Confirm that now PUD is recoverable, and quite easy.The cause of disease of most PUD is a helicobacter pylori infection.But helicobacter pylori infection can not routine diagnosis, and this may be can not make the doctor particularly the attending doctor is satisfied because detect the method (being the invasive examination of living tissue) of helicobacter pylori.Therefore, the attending doctor tends to treat the patient that disease is sent out with the secretion inhibitor agent.
The doctor needs a kind of method of simple, accurate and cheap diagnosing helicobacter pylori infection, when treats patient and when patient is introduced to the gastroenterologist so that they are known.But present helicobacter pylori detects, and can classify as invasive inspection and non-intrusive inspection, and they all can not make us very satisfied.
The invasive inspection needs to use endoscope to carry out examination of living tissue then earlier.To the tissue samples that obtains by biopsy method, analyze by cultivation, histology or RUT experiment again.
Though the cultivation to biopsy sample provides the most reliable result for the helicobacter pylori check, but the success ratio of reporting in laboratory preferably is (Han between 70% to 80% only, S.W. etc., Eur.J.Clin.Microbiol.Infect.Dis. (1995), 14:349-352).Can provide direct evidence for acute or chronic inflammatory mucomembranous cell and damage to the inspection of the biopsy sample of specific stain.But, this need endoscope doctor and pathologist cooperation (Genta, R.M. etc., Hum.Pathol. (1994), 25:221-226).The pH that the ammonia that quick urase inspection is produced by the helicobacter pylori urase causes raises, and this urase is decomposed into ammonia and carbonic acid gas with urea.But it needs highdensity bacterium, and any incident that reduces this amount of bacteria all can cause false negative result (Cutler, A.F, Am.J.Med. (1996), 100:35S-39S).
Since nineteen ninety, developed the existence that some Noninvasive methods of inspection are checked helicobacter pylori infection.For example, the urea respiration test is based on the urease activity of this organism, and this urase will be used
13C or
14The urea of C mark is decomposed into on-radiation
13CO
2Or radioactivity
14CO
2This urea respiration test is extensively recommended to be used for to confirm the elimination to helicobacter pylori in treatment 4 all backs.
United States Patent (USP) 5,716,791,5,871,942 and 5,932,430 disclose the immunologic detection method that detects Heliobacter pylori antigen in the fecal sample with polyclonal antibody, wherein the polyclonal antibody animal of helicobacter pylori cell (being atcc strain 43504) sensitization of must using by oneself.This antibody is by DEAE (DEAE-cellulose) column purification.Though this ight soil antigen check it was reported it is gratifying, find that the collection of fecal sample and processing are difficulties and undesirable.A lot of patients are unwilling to provide fecal sample to the doctor, are because undesirable smell and shortage are collected utensil easily.
With ELISA the serum helicobacter pylori antibody being carried out serum test is another kind of widely used experiment.The example of state-of-the-art technology is seen United States Patent (USP) 5262156 and EP 0329570.Identify several main antigens and be used for checking the immunologic detection method of helicobacter pylori antibody.But, these detection methods do not have specificity that serum diagnosis requires and susceptibility (Newell, D.G. etc., Serodian.Immunother.Infec.Dis., (1989), 3:1-6).One of problem is caused by cross reaction.This is because the dominance antigen (for example, molecular weight is the flagellin of the hypothesis of 60Da) in the helicobacter pylori is not specific concerning helicobacter pylori.Some such antigens are found in other bacterium such as C.jeuni and C.coli.Second problem that runs in the design pylorospirobacillus immune detects is the strain variation.In the not homophyletic of helicobacter pylori, observed antigenic essence difference.These problems have hindered with single antigen design detection method.Specificity and susceptibility that helicobacter pylori antibody detects are improved, promptly used the antigen mixture that derives from different helicobacter pylorus strains, this mixture is rich in some antigen fragment.A kind of ELISA reagent that detects helicobacter pylori antibody in the serum is commercially available.This full cell lysate that detects the use bacterium is as antigen.
The ELISA that use exists with helicobacter pylori antibody in the Detection of antigen serum also has other shortcoming.Specifically, in the long period of infecting after having treated in (reaching 12 months in the certain situation) human serum this antibody titers still keep high level.So the positive findings that obtains with this ELISA might not illustrate that this patient is infected at present and need treat helicobacter pylori infection.When in the face of positive ELISA, before the beginning antibiotic therapy, the treatment doctor often requires gastric biopsy to confirm existing of this bacterium.Therefore, can not get rid of needs based on antigenic ELISA to invasive method.
Therefore, the objective of the invention is to design Noninvasive and diagnostic method split hair caccuracy to helicobacter pylori infection.During studying, found the Heliobacter pylori antigen in the blood, they are with DNA or its fragment, or the form of albumen/peptide or its other antigen component is present in the blood (comprising whole blood, blood plasma and serum).Therefore, designed the special methods that detects these Heliobacter pylori antigens, be present in evidence in the blood so that the Heliobacter pylori antigen fragment to be provided.These methods include but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR) and DNA hybridization, with derive from the helicobacter pylorus bacteria strain, to the specific primer of helicobacter pylori tool or oligonucleotide and/or dna probe, detect the nucleic acid fragment of helicobacter pylori.In addition, also develop immunodetection and immunoblot experiment, used the albumen/peptide or any antigen component that detect helicobacter pylori at the affinity purification antibody of helicobacter pylori.
At present, still not about there being the report of Heliobacter pylori antigen in the blood.First has proved that Heliobacter pylori antigen does not exist only in the blood the present invention, and can detect by the method that lower part provides.
Summary of the invention
The invention provides the Heliobacter pylori antigen that is present in the blood samples of patients, and can provide polymerase chain reaction (PCR), ligase chain reaction (LCR), DNA hybridization, RNA hybridization, branched DNA detection, immunoblotting and immunologic detection method to detect.The present invention also provides the method for coming diagnosing helicobacter pylori infection by the Heliobacter pylori antigen that detects in the blood.
The invention provides a kind of method of utilizing antibody directly to detect Heliobacter pylori antigen in the blood, this method can more accurately be known the infection of helicobacter pylori.
Particularly, wherein a kind of method that detects Heliobacter pylori antigen in the blood provided by the invention, be to use the antigenic antibody of anti-helicobacter pylori, utilize immunological method to detect the Heliobacter pylori antigen that exists with the antigen-antibody complex form in the blood sample, and the antigenic antibody of the described anti-helicobacter pylori antibody of affinity purification preferably.
Immunological detection method of the present invention includes but not limited to immunoblotting and immunoassays method (detecting as the sandwich detection in basis, three sandwich detections or immune chromatograph).
The term that uses among the present invention " antigen " broadly comprised can directly or indirectly cause under proper condition specific immune response and with any material of the product of this reaction reaction (promptly with the T lymphocyte reaction of specific antibody or specificity sensitization, or the two haves both at the same time).The example of these materials includes but not limited to albumen/peptide, polysaccharide, lipid and many or oligonucleotide.
The Heliobacter pylori antigen that can detect in blood has specific two types.The first kind relates to polynucleotide or oligonucleotide, and they are chromosomal DNA, RNA or its fragment of helicobacter pylori.This type of helicobacter pylori can pass through polymerase chain reaction (PCR), ligase chain reaction (LCR) and hybridization (preferred point-like DNA hybridization) method or other amplification method detects.
PCR method of the present invention need be used a pair of primer of specific detection helicobacter pylori.Term " primer " refers to oligonucleotide herein, no matter be natural or synthetic, when place cause with nucleotide chain complementary primer product synthetic condition under, promptly under the proper temperature in the presence of four different IPs thuja acid triphosphates and suitable enzyme, it can cause point as the nucleic acid synthetic.Term " oligonucleotide " is defined as the molecule of being made up of two or more (preferred more than three) deoxyribonucleotides and/or ribonucleotide herein.Its actual size will depend on multiple factor, and it depends on the final function or the purposes of this oligonucleotide successively.This oligonucleotide can derive from synthetic or the clone.
The preparation of this primer is according to the conserved sequence of finding in the consensus sequence fragment of helicobacter pylorus bacteria strain such as atcc strain 43504,43571,43629 and 49053.Preferred primer scope is long 15 to 25 base pairs (bps), most preferably about 20bps.When the identical and same Nucleotide component of two kinds of primers (forward primer and reverse primer) length is basic identical, can obtain to increase preferably.The blood sample that preferably is used for PCR is a blood plasma.
LCR method provided by the invention need be used a kind of dna ligase and helicobacter pylori specific two is overlapped oligonucleotide.Preferred dna ligase is the Pfu dna ligase, and it is an isolating heat-stable DNA ligase enzyme from Pyrococcus furiosus, and can be purchased.The two cover oligonucleotide that LCR uses are preferably long than the primer of PCR.As if the PCR primer is the same, the LCR oligonucleotide derives from the helicobacter pylorus bacteria strain, as the conserved sequence in the consensus sequence fragment of atcc strain 43504,43571,43629 and 49053.
LCR finishes, it is the recirculation of the thermally denature of the dna profiling by containing target sequence, with unique mode first cover is annealed at two of the target DNA sequence adjacent oligonucleotide probes, and the second cover complementary oligonucleotide probe and the sequence opposite with the target DNA sequence are hybridized.Term " target sequence " refers to be found in " chromosomal DNA or its fragment " in the blood sample herein.After this, dna ligase can covalently bound every pair of adjacent probe, as long as these two adjacent probe are complementary fully on connecting.
Hybridizing method need prepare the helicobacter pylori dna probe.This helicobacter pylori dna probe prepares by cutting the extract behind helicobacter pylori nucleic acid agarose gel electrophoresis and extraction dna fragmentation.This probe generally has at least about 25 bases, 30 bases of having an appointment at least usually, and can be no more than about 5000 bases usually up to about 10000 bases or more.Then, this dna fragmentation is digested with restriction enzyme, and be connected to form construction of recombinant plasmid with the host, it can transfection eucaryon and prokaryotic host cell.This dna fragmentation can be bred and be separated in these host cells.Then, can with propagation after dna fragmentation with radio isotope (as
32P,
3H,
14C or the like) mark or fluorescent substance mark (as combining with fluorescence detection method with digoxigenin and biotinylated DNA probe) are also as dna probe.
This hybridizing method is by with sex change agent treated blood, serum preferably, sample of nucleic acid carry out so that be present in the DNA sex change of solid phase carrier such as nitrocellulose filter.Preferred sex change reagent includes but not limited to basic solution, organic reagent (for example, alcohol, acid amides, amine, urea, phenol and sulfoxide) or some mineral ion (for example, thiocyanate ion and perchlorate) or realizes by heating up.Then, the dna probe with mark joins in the DNA point-like film of sex change.Then, detect the mark characteristic of the DNA crossbred that exists in this film.If this mark is radioactive, this film can be used for x-ray film, if mark is epipolic, this film can be used the fluorescent microscope direct viewing.
The second class antigen relates to helicobacter pylori protein and/or the peptide in the blood, or contains any material of epitope, and it can detect by immunoblotting or immunoassay, preferably uses the antibody of the antigenic affinity purification of anti-helicobacter pylori.One-level or secondary antibody all may be that Heliobacter pylori antigen is needed in detection or the mensuration blood, and this depends on the kind of method therefor in the detection.One-level antibody is the antibody that produces at antigen (being Heliobacter pylori antigen in this case).Secondary antibody is the kind antibody (as goat-anti-rabbit igg) at the immunoglobulin (Ig) generation of one-level antibody.The blood sample that is preferred for detecting is a serum.
Immunoblotting is by analyzing blood sample with SDS-PAGE earlier.Behind the electrophoresis, the proteins/peptides band is transferred on the nitrocellulose filter, then the helicobacter pylori antibody of itself and capacity is hatched.Produce anti-helicobacter pylori, can join on the nitrocellulose filter at a kind of enzyme mark secondary antibody of the immunoglobulin (Ig) of animal species.The preferred enzyme of this method is an alkaline phosphatase, and wherein this reaction can detect by adding 5-bromo-4-chloro-3-indyl phosphoric acid ester.Another the preferred enzyme labelling thing that is used for this method is a horseradish peroxidase, its can by add 4-chloro-1-naphthols or 3,3 '-colored to produce, the insoluble product of diaminobenzidine so that observe detects.
The immunoassays method includes but not limited to basic sandwich calibrating (basic sandwich assay), three sandwich calibratings (triple sandwich assay) and immune chromatograph calibrating.
In the sandwich calibrating in basis, need two one-level antibody, one of them combines with solid carrier and another uses the detection reagent mark.This three sandwich calibrating need be united the one-level antibody (that is, first antibody and second antibody) that uses two anti-helicobacter pyloris and the secondary antibody of an anti-animal species IgG, this secondary antibody detection reagent mark.Have only needs to be bonded on the solid carrier in the described one-level antibody, this animal species produces unconjugated one-level antibody, finally forms the mixture of anti-antibody-antigen-antibody complex.
The solid carrier of sandwich calibrating can be plastic bead, polyethylene, polystyrene, polypropylene etc.Detection reagent can be that enzyme labelling thing (as alkaline phosphatase or horseradish peroxidase), fluorescence or luminescence reagent (as fluorescent yellow, Luo Mingdan, or europium, luminol,3-aminophthalic acid cyclic hydrazide or acridine pigment (acridium)), labelled with radioisotope are (as I
125), perhaps coloured particle (as gold and silver, blue emulsion or selenium).
Basis sandwich and three sandwich immunoassay calibrating all needs the interaction of first one-level antibody and helicobacter pylori with the formation antigen-antibody complex, then with the antibody of second anti-helicobacter pylori therewith antigen-antibody complex contact.
This immune chromatograph experiment also needs to unite the one-level antibody that uses two anti-helicobacter pyloris.Compare with three sandwich calibratings with the basis, first antibody (being first antibody that contacts with biological specimen) is used the coloured particle mark, second antibodies solid carrier such as nitrocellulose (nitrocellulose derivative) film, nylon membrane, polyester film, filter paper, agarose or cross-linked glucose gel.The preferred solid carrier that is used for this immune chromatograph calibrating is a nitrocellulose filter.Optionally, add at sample near the end of the reciprocal chromatographic band of point, the secondary antibody that produces the anti-animal species of first antibody can be added and/or in conjunction with on the solid carrier so far.This secondary antibody is caught unconjugated coloured particle in contrast as the end that carries out in chromatogram.Therefore, if this sample does not contain Heliobacter pylori antigen, first kind of antibody of coloured particle mark will pass by second kind of antibody and debond be not because there is antibody-antigen-antibody complex to form.But, because secondary antibody is the anti-animal IgG that produces first kind of antibody, when first antibody walk out-of-date its will be in conjunction with this first kind of antibody, no matter whether this first kind of antibody forms mixture with sample.The associative list light colour spectrum of first kind of antibody and secondary antibody has proceeded to terminal point.
The method according to this invention, described blood sample can be serum, and one of problem relevant with serum sample to be the patient that infects helicobacter pylori have in the serum of being everlasting helicobacter pylori antibody of himself.These serum helicobacter pylori antibodies can form immunocomplex with the serum Heliobacter pylori antigen, and this can impact the accuracy of immunoassays of the present invention.The method of dissociating this pylorospirobacillus immune mixture includes but not limited to, with the reagent that dissociates this mixture that dissociates, or examines and determine under condition at sample solution.
The example of this reagent that dissociates, include but not limited to, high salt concentration (for example, at least the sodium-chlor of 1M or Repone K), washing composition (as, at least 1% Sodium dodecylbenzene sulfonate (SDS), polysorbas20, octyl glucoside, deoxycholate salt or Triton X-100), chaotropic agent (for example, Guanidinium hydrochloride, urea or KSCN), organic solvent (for example, dioxan or ethylene glycol), enzyme (for example, proteolytic enzyme such as trypsinase, Chymotrypsin, stomach en-, V8 proteolytic enzyme and subtilisin) or lipase is (for example, derive from the lipoprotein lipase of milk, and derive from the lipase of wrinkle Zhe candiyeast).Sample solution includes but not limited to high pH (for example, pH 〉=9) or low pH (for example, pH≤3) from the example of condition, with and/or elevated temperature (for example, at least 50 ℃).
Attempt though reported some, they detect for the patient who infects helicobacter pylori provides quantitatively and qualitatively, and none is the helicobacter pylori that directly detects in the blood sample.Its major cause is also not have the researchist to suppose that Heliobacter pylori antigen can be found in the blood flow.
But in table (that is, table 1-3), all evidences shown in the embodiment 9 (table 1) and 11 (table 2 and 3) of back will show in helicobacter pylori infection patient's serum sample and can and have been found that Heliobacter pylori antigen.
Find based on these, the objective of the invention is to utilize in the blood detectable Heliobacter pylori antigen as the instrument of diagnosing helicobacter pylori infection.This diagnostic method can be used for detecting dissimilar Heliobacter pylori antigens, and it includes but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), branched DNA amplification calibrating, hybridization calibrating, immunoblotting, immunoprecipitation, flow cytometry, immunoelectrophoresis and immunoassays (for example enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immune chromatograph).
PCR is the technology of high-level efficiency amplification specific dna sequence.Use polysaccharase, for example repetition, primer annealing and the extension of the sex change of thermophilic enzyme Taq polysaccharase causes the exponential increase of required dna sequence dna.Every kind of dna sequence dna can separate with agarose gel electrophoresis, and then carries out nucleotide sequencing.PCR is a blood plasma with the blood sample preferred type, and this is that the haemachrome molecule that derives from corpuscular hemoglobin may disturb pcr amplification because if haemolysis takes place.
Ligase chain reaction (LCR) is a kind of DNA cloning technology, and it can be used to detect the trace level of known nucleotide sequence.LCR comprises the round-robin two-step reaction: (1) high temperature step of unwinding, its double center chain target DNA unwinds and becomes strand, and (2) cooling step, wherein two covers adjacent, annealed complementary oligonucleotide is the strand target molecule and links together with dna ligase.The product that connects from first circulation is as the template of next round-robin ligation, and LCR causes connecting the exponential amplification of product, and its mode is similar to the exponential amplification of template in the PCR reaction.
PCR and LCR need to find that helicobacter pylori Auele Specific Primer or oligonucleotide are to cause this nucleotide chain reaction.Because at gene level helicobacter pylorus bacteria strain is highly multifarious (Fujimoto etc., J.Clin.Microbiol., (1994), 32:331-334), and individuality can be by more than one strain infections, therefore, according to segmental conserved sequence design primer of the consensus sequence of finding in multiple helicobacter pylorus bacteria strain or oligonucleotide, this is a kind of means of dealing with problems.
Found that the helicobacter pylorus mycetocyte that derives from ATCC bacterial strain 43504 is particularly conducive to the one-level antibody (seeing United States Patent (USP) 5716791) for preparing helicobacter pylori in the anti-ight soil.This is because the antibody that produces with the cell sensitization that derives from bacterial strain 43504 can cross-region and striden the diet group and detect this organism.Other helicobacter pylorus bacteria strain has shown similar antigenicity as ATCC43571,43629,49053.Therefore, the fragment of searching consensus sequence is valuable in these bacterial strains.This can followingly carry out: by will deriving from the extraction nucleic acid digestion of above-mentioned helicobacter pylorus bacteria strain with identical restriction enzyme (one or more), the helicobacter pylori nucleic acid fragment that then will digest carries out agarose gel electrophoresis.This consensus sequence fragment can excise and extract.Can analyze the segmental nucleotide sequence of consensus sequence.Then, the segmental conserved sequence of this consensus sequence can be used to design PCR or LCR with primer or oligonucleotide.
Except PCR or LCR, can detect the existence of Heliobacter pylori antigen in the blood sample with dna hybrid probe.The preferred nucleic acid hybridization probe is no more than about 5000 bases.This probe sequence is preferred at least basically and the segmental nucleotide sequence complementation of the consensus sequence between the helicobacter pylorus bacteria strain.Except the consensus sequence fragment of finding in multiple helicobacter pylorus bacteria strain, this probe can derive from messenger RNA(mRNA), cDNA, and the latter is by obtaining with the messenger RNA(mRNA) reverse transcription or by the genome cracking with reversed transcriptive enzyme.To required probe separate with qualitative after, the dna fragmentation of probe can be cloned in host cell or breed.Then, the probe of this propagation can be with atom or inorganic group mark, the most frequently used radionuclide of this mark, but also can be with heavy metal or fluorescent substance.Use can specificity in conjunction with the antibody of the probe of the single stranded DNA hybridization of Heliobacter pylori antigen, be feasible.In this example, this antibody is labeled so that detect.Be used for the marker of the same-type of probe also can be according to known technique antibodies therewith.
Radioactively labelled substance as
32P,
3H,
14C etc. can be used for label probe, though other radioactively labelled substance also can use, need only them the suitable signal with enough transformation period can be provided.Other marker comprises part, its can be used as the traget antibody fluorescent agent specific combination unit, chemoluminescence agent, enzyme, can be used as the right antibody of tagged ligand specific combination etc.In immunoassays, also can use multiple marker.The selection of marker be by marker to the speed of hybridization and probe in conjunction with the influence of sample DNA decision.Need this probe to provide enough susceptibilitys for detecting DNA hybridization consumption.Other consideration comprise probe be easy to synthesize, be easy to power operation, can automatization, convenience etc.
The mode of this marker bonding probes changes with the essence of this marker.For radioactively labelled substance, can use multiple technologies.Commonly used is with α-
32P-dNTP carries out nick translation, or carries out the terminal phosphate hydrolysis with alkaline phosphatase, then use γ-
32P-NTP and T4 polynucleotide kinase carry out radioactivity
32The P mark.Perhaps, can synthesizing ribonucleotide, wherein one or more elements of Cun Zaiing are replaced by radio isotope, for example, replace hydrogen with tritium.
The favourable enzyme of thing of serving as a mark comprises lytic enzyme, particularly esterase and Glycosylase or oxydo-reductase, particularly peroxidase.Fluorescent chemicals comprises fluorescein and derivative, Luo Dan amine and derivative thereof, dansyl, umbelliferone etc.The chemoluminescence agent comprises luciferin and 2,3-dihydro phthalazine diketone class, for example, luminol,3-aminophthalic acid cyclic hydrazide.
The general probe at dna sample that invests the water-insoluble porous support that uses of this hybridization carries out.This dna sample by sex change so that single-chain nucleic acid be fixed.For cracking, chemical cracking is easy to use, uses dilute alkaline soln usually, for example, and 0.1 to 1M sodium hydroxide.This alkali can also be used to making the DNA sex change.Other sex change reagent includes but not limited to organic solvent as alcohol, acid amides, amine, urase, phenol and sulfoxide, or some mineral ion such as thiocyanate ion and perchlorate, can also realize by elevated temperature.
Also can be by the affinity purification antibody test blood helicobacter pylori of immunization method with anti-helicobacter pylori, this method includes but not limited to immunoblotting and immunoassays (as basic sandwich immunoassay calibrating, the calibrating of three sandwich immunoassay and immune chromatograph).
Immunoblotting need be through polyacrylamide gel electrophoresis with the blood sample fractional separation, albumen after will separating then/peptide band is transferred on the nitrocellulose filter, more isolating albumen/peptide band and anti-helicobacter pylori one-level antibody is done in order to form antigen-antibody complex.Then, add secondary antibody with the anti-one-level antibody of detection reagent mark to show this detection reaction.
Basis and the calibrating of three sandwich immunoassay all need at least three key elements: the antigenic first kind of antibody of anti-helicobacter pylori, antigenic second kind of antibody of anti-helicobacter pylori and test sample (that is blood sample).
First and second kinds of antibody (being that anti-helicobacter pylori is antigenic) are polyclonal antibody preferably.This antibody can pass through to rabbit or other animal such as goat or the acquisition of cow injection helicobacter pylorus mycetocyte, and preferably these cells break by supersound process, or break by other cell rupture means, so that expose a plurality of antigen sites.
In a word, by first injection, then booster shots are so that this reaction maximization prepares antibody.In order to prepare these antibody, thus with antigen and assistant agent for example freund's adjuvant mix with immune animal and strengthen this immune response.The amount of institute's injections of antigens must be suitable for producing the detectable antibody of capacity.Multiple injection can be carried out so that production of antibodies the best with the cycle of rule.Infusion protocol depends on used animal.Give the animal bloodletting, earlier by detecting this blood production of antibodies amount.Detect production of antibodies with tentative bloodletting and enzyme immunoassay (EIA).By chromatogram, preferred affinity column chromatography is with antibody purification.
In the sandwich calibrating in basis, if first kind of antibodies solid carrier, second kind of antibody must be used the detection reagent mark, and it can be any marker in the known immunodetection.For example, and enzyme labelling thing (as alkaline phosphatase or horseradish peroxidase etc.), fluorescent agent, luminescence reagent or radioactively labelled substance (as fluorescent yellow, Luo Mingdan, europium, luminol,3-aminophthalic acid cyclic hydrazide or acridine pigment and radio isotope such as I
125Deng), perhaps colloidal solid (as gold and selenium etc.) is the marker that can be used for this immunoassays.In these markers, the most frequently used is enzyme labelling thing such as horseradish peroxidase (HRP) or alkaline phosphatase.Specifically, by HRP and diaminobenzidine, tetramethyl benzidine, 4-chloro-1-naphthol reaction, or, in colorimetric estimation, can detect the HRP marker by other similar chemical reaction.This colorimetric estimation reaction product can detect in board-like reader, scanner, photodensitometer or estimate.By comparative sample reading and the reading that contains the antigenic standard substance of known quantity, can measure the amount of helicobacter pylori.Preferred several standard substance is used for the concentration of the marker of " containing lid " tested sample.
Radio isotope as
125I iodine or beta emitter as
14C carbon is another kind of marker commonly used, and it can detect by gamma counter or scintillometer.Fluorescent marker also can be used for traget antibody, so that measure by fluorometric analysis.
Solid carrier can be any solid carrier that becomes known for immunoassays.It can be as the porous plate of using among the ELISA, its can be in board-like reader reading of data.Perhaps, it can be or to be dissolved in a kind of carrier that the sample in the stationary phase carries out stratographic analysis to liquid.The example of this carrier comprises the medium of film such as nitrocellulose or column chromatography.
Embodiment
Following non-limiting examples is used for illustrating the detection to Heliobacter pylori antigen in the blood.
Embodiment 1
Preparation Heliobacter pylori antigen and nucleic acid
Under the room temperature helicobacter pylori bacterial classification of ATCC bacterial strain 43504,43571,43629 and 49053 being preserved thing thaws respectively and dilutes in 5ml brucella meat soup.Bacterial suspension 0.2ml after will diluting immediately coats on the trypticase soybean blood agar dish, wherein is added with 5% sheep blood.These agar disks are cultivated under little aerobic condition and are allowed bacterial growth.After the cultivation, bacterium colony is scraped and uses the PBS washed twice from dish.Then, the precipitation after the washing is suspended among the PBS.
In order from each helicobacter pylorus bacteria strain, to collect antigen, the helicobacter pylori cell transfer carried out 10 minutes ultrasonication to ice-cold container and with Microson XL200 supersonic cell disruptor.Then, will keep the immunogen of supernatant liquor through the bacterial suspension of ultrasonication centrifugal 30 minutes at 2-8 ℃ with 25000 * g as the preparation helicobacter pylori antibody.
In order from the helicobacter pylorus bacteria strain, to collect nucleic acid, can be with the 1%SDS that is present in 100mM Tris-HCl (pH8.8) with helicobacter pylori lysis.Then, can this lysate be extracted once, use isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) to extract subsequently twice with isopyknic phenol/chloroform.Behind chloroform-phenol extraction, can precipitate chromosomal DNA under the Virahol room temperature with 0.6 volume.With 13000 * g centrifugal 15 minutes, with this nucleic acid precipitation of 70% washing with alcohol, and it is dissolved in 10mM Tris-HCl and 1mM EDTA, among the pH8.0.
Embodiment 2
With the helicobacter pylori in the pcr amplification technology for detection blood
Can the DNA digestion of helicobacter pylorus bacteria strain will be derived from restriction enzyme.Suitable restriction enzyme includes but not limited to HindIII, EcoRI, BamHI, ClaI and XbaI.Can in Tris-acetate-edta buffer liquid, walk electrophoresis in gained fragment on the sepharose.Extract test kit with a kind of sepharose and from this sepharose, extract dna fragmentation available from BoehringerMannheim (Germany).Because the helicobacter pylorus bacteria strain is highly multifarious at gene level, so relatively the dna fragmentation of each helicobacter pylorus bacteria strain is favourable to find the consensus sequence fragment.
Can on two chains, determine the segmental dna sequence dna of consensus sequence with double-stranded DNA template and dideoxy chain termination method (as Sanger etc., Proc.Natl.Acad.Sci.USA (1977), 71:1342-1346 is described).Based on the segmental conserved sequence of this consensus sequence, can be by the scheme that following manufacturers provides dna synthesizer synthetic oligonucleotide primer thing.The primer normal length that is used for PCR is about 20bp and preferred 15-25bp.Identical and have essentially identical Nucleotide when forming when two primer lengths, can obtain to increase preferably.The preferred only PCR primer of antigenic therewith nucleic acid specificity hybridization is because the existence of helicobacter pylori specificity nucleotide sequence has been indicated in the existence of amplification.
With the conventional amount used of ordinary method and experiment, according to the method that obtains typical nucleic acid, those skilled in the art can obtain the antigen fragment of dna encoding.
Pcr amplification contain the plasma dna extract be template, helicobacter pylori primer, Dynazyme damping fluid (can be available from Finnzymes, Espoo, Finland), carry out in the reaction mixture of the mixture of all four kinds of deoxynucleotides and Dynazyme.The extraction of plasma dna is identical with the mode of extracting helicobacter pylori nucleic acid, and different is in the end to have increased the step that allows this extract pass through Microcon 100 strainers.This final step is in order to remove residual abiotic inhibitory substance and polysaccharide compound, to have found that they are strong PCR inhibitor, and DNA can't be precipitated.
Can this reaction be covered and (Norwalk, CT are heated to 94 ℃ and kept 10 minutes in USA) at the Perkin-ElmerDNA thermal cycler before beginning PCR circulation with mineral oil.5 loop parameters of beginning can for: 94 ℃ of sex change 2 minutes, extended 1 minute 42 ℃ of annealing 1 minute and at 72 ℃.Can carry out 30 round-robin PCR with following parameters subsequently:, extended 1 minute 59 ℃ of annealing 1 minute and at 72 ℃ 94 ℃ of sex change 2 minutes.Circulation in the end, extension can be carried out 10 minutes.Can under 4 ℃, stop the PCR reaction, and on sepharose, the PCR product be analyzed.Can estimate the PCR product size and with the consensus sequence fragment in helicobacter pylori, found relatively.
Its size equals the segmental size of consensus sequence and is regarded as positive findings.Can use by Southern blot hybridization method
32The dna probe of P CTP mark (consensus sequence fragment amplification thus) confirms that this PCR product is the helicobacter pylori sequence.
Perhaps, can use restriction enzyme such as AluI, HinFI and HaeIII digest this PCR product and consensus sequence fragment, and analyze the digestion pattern of this digest by agarose gel electrophoresis.Identical collection of illustrative plates indicates this PCR product to derive from the helicobacter pylori sequence.
Embodiment 3
With the helicobacter pylori DNA in the LCR augmentation detection blood
Ligase chain reaction (LCR) calibrating needs two oligonucleotide and dna ligase of two covers.First the cover oligonucleotide (being Oligo A and Oligo B) be each other successive and with the double-helical chain complementation of target DNA.Therefore the second cover oligonucleotide (being Oligo C and Oligo D) is a complementary with first cover, and has occupied the contiguous site on second chain of target DNA.All four oligonucleotide probes can according to the design of the conserved sequence of helicobacter pylorus bacteria strain and AppliedBiosystems (Foster City, CA) synthetic and in the oligonucleotide synthesizer by the PAGE purifying.Oligo A and Oligo D can by under 37 ℃ adenosine 5 ' (γ-
32P) under the existence of triphosphoric acid and polynucleotide kinase, in 50mM Tris-HCl (pH7.5), 7mM magnesium chloride and 1mM dithiothreitol (DTT), hatched 30 minutes, carry out radio-labeled at its 5 ' end.Then, make this polynucleotide kinase inactivation by heating down at 70 ℃.The radiolabeled oligonucleotide probe A and the D of equivalent, and oligonucleotide probe B and C can join in the Eppendorf pipe, adds the dna profiling that extracts from serum sample simultaneously.Each pipe contains a kind of reaction buffer, this damping fluid by 50mM two-Tris pH6.5, the 10mM magnesium chloride, 10mM ammonium chloride, 10mM Repone K, 1mM dithiothreitol (DTT) and 1mM NAD form.Then, can in each pipe, add an amount of mineral oil, and these pipes are heated to 100 ℃ and kept 3 minutes, be cooled to 85 ℃ and kept 1 minute subsequently, and place down, add dna ligase simultaneously at 55 ℃.Preferred dna ligase is the Pfu dna ligase, and it derives from Pyrococcus furiosus.These reaction tubess can be placed then the DNA thermal cycler (RoboCycler, Stratagene) in and in 85 ℃ and 50 ℃ circulation 20,30 or 40 times, each temperature 1 minute.Then, stop dyestuff with 95% methane amide a five equilibrium of each reaction is made 1: 1 dilution of row.Sample after this dilution can be analyzed on acrylamide gel.
Embodiment 4
Preparation helicobacter pylori dna probe
Can cut away helicobacter pylorus bacteria strain dna fragmentation (general at least 25 bases, usually at least about 30 bases, and can up to about 10000 bases or more than, but usually be no more than about 5000 bases), and behind electrophoresis, from sepharose, extract.Can be connected to form construction of recombinant plasmid with this dna fragmentation digestion and with the host with restriction enzyme.For example, can with ClaI with the digestion of this dna fragmentation and connect the Pev-Vrf expressive host that enters ClaI digestion (Crowl etc., Gene (1985), 38:31-38).Then, the plasmid of this reorganization can be transferred in the host cell, it can be prokaryotic cell prokaryocyte such as E.coli RRI, or eukaryotic cell such as NIH 3T3 cell or HeLa cell.The plasmid of this reorganization can be bred by duplicating in this host cell.Can be according to So etc., Infect.Immun. (1978), the method for 21:405-411 is separated this recombinant plasmid.By from then on discharge the dna fragmentation of helicobacter pylori in the plasmid with the digestion of identical restriction enzyme.Can confirm the helicobacter pylori dna fragmentation of this release by sepharose or polyacrylamide gel electrophoresis.Then, can use radio isotope (as
32P,
3H,
14C etc.) or the dna fragmentation after fluorescent substance (as using digoxin base and DNA probe labeled with biotin and combined with fluorescent detection method) this propagation of mark and as dna probe.
Embodiment 5
Dot blot preparation with the helicobacter pylori dna probe
In water, boil or autoclave sterilization can be sterilized nitrocellulose filter.Place the surface of agar also to use the serum drop of processed its DNA of release an one bactericidal film.For example, can use dilute alkaline aqueous solution (for example 0.1 to 1M sodium hydroxide) with this serum sample cracking.This alkali can also be used to making the DNA sex change.Other sex change operation includes but not limited to elevated temperature, with an organic solvent (as alcohol, acid amides, amine, urea, phenol and sulfoxide) or certain mineral ion (as thiocyanate ion and perchlorate).
After this sample sex change, in aqueous buffer solution (it is about 6 to 8 that pH is generally, and is generally 7), can wash this filter membrane.After cracking, sex change and washing, can this sample DNA drop filter membrane is dry at elevated temperatures, be generally about 50 to 70 ℃, so that sample DNA is fixed on this filter membrane.
Then, under the intensification of gentleness, this filter membrane and the hybridization solution that does not contain probe are hatched time enough to incite somebody to action thoroughly wetting this filter membrane.Can use multiple hybridization solution, wherein contain about 20% to 60%, the inertia polar organic solvent of preferred 3%0 volume percent.A kind of common hybridization solution use about 50% methane amide, about 0.5 to 1M sodium-chlor, about 0.05 to 0.1N Sodium Citrate, about 0.05 to 0.2% sodium lauryl sulphate (SDS) and a spot of EDTA, phenanthrene can (about 300-500kdal), polyvinylpyrrolidone (about 250-500kdal) and serum albumin.The DNA (for example, tire ox thymus gland or salmon seminal fluid) that in this hybridization solution, can also contain 0.5 to 5mg/ml the ultrasonic sex change of having an appointment, and the glycine of about 0.5 to 2% weight/volume (wt/vol) of existing of selectivity.Can also contain other additive, for example about T 500 of 100 to 1000kdal and about hybridization solution of 8 to 15wt%.
The amount of labeled DNA probe alters a great deal, and whether this depends on the characteristic of marker and can combine with filter membrane effectively, and the stringent condition that should hybridize.In a word, should use stoichiometry basically excessive probes to improve the speed that combines of this probe and fixed sample DNA.
Under the room temperature,, can detect the duplex that this filter membrane exists now according to the essence of marker with after containing second solution with sodium-chlor, Sodium Citrate and the SDS of the similar concentration of hybridization solution and cleaning this filter membrane.When this marker when being radioactive, that this filter membrane is dry and make the x-ray film exposure with it.If this marker is a fluorescent substance, then can directly use fluorescence microscope.
This probe does not need with the sequence of its hybridization complementary fully; Wherein can exist 30% or more base mismatch right.Influence condition that the DNA crossbred forms and be know and by Crosa etc., J.Bact. (1973), 115 (3): 904-911 describes in detail.
Embodiment 6
The rabbit polyclonal antibody of preparation anti-helicobacter pylori
New Zealand white rabbit with being present in the Heliobacter pylori antigen of 0.5 in the complete Freund's adjuvant to 1.0mg, is carried out immunity by intramuscular injection earlier, carries out booster immunization every 4 weeks with the 0.5-1.0mg same antigen that is present in the incomplete Freund's adjuvant again.Blood sample collection is analyzed behind the 3rd booster immunization.In case this antibody titers has arrived about 10
6Desired level, just begin bloodletting.
The blood of individuality is diluted to 1 * 10 earlier in PBS
6Then rabbit anteserum after the dilution of 100 μ l is joined the Heliobacter pylori antigen bag by in the hole.After hatching 1 hour under the room temperature, with PBS with this plate washing 4 times and add 100 μ l goat anti-rabbit igg-HRP binding substancess.This plate was at room temperature hatched 30 minutes again.After PBS washing 4 times, in each hole, add 100 μ l tetramethyl benzidine (TMB) substrates with dyeing, and detect colour intensity in each hole with the micropore reader at 450/650nm.OD450/650 must be greater than 1.0 preparations that just can be used for antibody.
Embodiment 7
Preparation antibody-HRP enzyme conjugates
Select enzyme horseradish peroxidase (HRP) to come and the helicobacter pylori antibody coupling.The preparation of this antibody-HRP enzyme conjugates is the Nakane method (Nakane etc. according to improvement, 1978:Inimmunoflorescence and related staining techniques, editors such as Knapp, p215-220, Elsivier/North-Holland Biomedical Press, Amsterdam).
In brief, with sodium periodate HRP is carried out oxide treatment earlier.This oxidation produces aldehyde radical on the Kohlenhydrate side chain.HRP after this antibody and the oxidation mixes under alkaline pH again, allows amino and the reaction of the aldehyde radical on the HRP on this antibody, forms Schieff alkali, and will be reduced to covalent linkage between antibody and the HRP.Then, by carry out gel-filtration antibody purification-HRP binding substances with the Sephacryl-S300 post.
This HRP can with the rabbit antibody of one-level antibody such as anti-helicobacter pylori, or combine with the goat antibody of secondary antibody such as anti-rabbit igg.
Embodiment 8
Gel electrophoresis and immunoblotting assay
Serum sample can be analyzed by SDS-PAGE.Behind the electrophoresis, can be by silver-colored dyeing process (Oakley etc., Anal.Biochem. (1980), 105:361-363) immobilized gel and the protein isolate of improvement.
Perhaps, protein can be transferred on the nitrocellulose paper by the 1amp electroblotting in 1 hour again.After the binding site sealing that will not occupy with encapsulant such as polysorbas20 and skimmed milk, in this nitrocellulose paper, add the described helicobacter pylori antibody of embodiment 6 (on seeing) of capacity.Then, this nitrocellulose paper at room temperature can be hatched 1 hour.The alkaline phosphatase enzyme conjugates that can in this nitrocellulose paper, add at last, the secondary antibody (as goat anti-rabbit igg) that resists the animal species immunoglobulin (Ig) that produces helicobacter pylori antibody.This reaction can detect by adding 5-bromo-4-chloro-3-isobutyl-phosphoric acid (BCIP)/nitroblue tetrazolium(NBT) (NBT).
Embodiment 9
The serum Heliobacter pylori antigen of preparation free form
The serum Heliobacter pylori antigen can free form and the form of immunocomplex exist.The free form of helicobacter pylori can be by immunoassays or any can detection method easily detection.Form the antigen of immunocomplex for helicobacter pylori antibody in those and the serum, their backs of can only dissociating in immunocomplex are from then on detected.
The serum Heliobacter pylori antigen can free form and the form of immunocomplex exist, and the free form of helicobacter pylori can easily detect by ELISA of the present invention, but immunocomplex can not easily detect by identical method, and this fact is shown in table 1.
Table 1 is the research of serum helicobacter pylori admixture.Tested sample does not contain Heliobacter pylori antigen.Although there is not helicobacter pylori in the serum, but may contain helicobacter pylori antibody (i.e. " antibody positive sample ") in the serum sample, this may be that it is opposite with the sample that does not contain any helicobacter pylori antibody (i.e. " negative antibody sample ") because original infection causes.In the admixture group, with the Heliobacter pylori antigen (1 * 10 of known quantity
5Bacterium/ml),, join " negative antibody " and reach in " antibody positive " serum sample no matter be whole bacterial cells or cell pyrolysis liquid.In " non-admixture " group, do not add Heliobacter pylori antigen.
The result of table 1 shows in " negative antibody " sample, by OD
450/650The increase of reading has reflected the adding of Heliobacter pylori antigen.On the contrary, adding Heliobacter pylori antigen in " antibody positive " sample can be with OD
450/650Reading be increased to " negative antibody " sample in identical level.A possible explanation is that Heliobacter pylori antigen and the serum helicobacter pylori antibody that adds formed immunocomplex, and it disturbs ELISA to detect.
For the immunocomplex with the serum Heliobacter pylori antigen dissociates, can be with dissociating reagent or under the condition of dissociating of sample, handle this serum sample.Four kinds of main reagent that dissociate are arranged, and it is very effective to these immunocomplexs that dissociate.The first kind of reagent that dissociates is the high level salt solution of sodium-chlor or Repone K.The concentration of preferred high level salt solution is 1M at least.Preferred salt is Repone K.The second kind of reagent that dissociates is the solution that contains washing composition such as SDS, polysorbas20, octyl glucoside, deoxycholate salt or Triton X-100.These washing composition can separately or be united use.The preferred concentration of this washing composition is greater than 1%.The third reagent that dissociates is the solution that contains chaotropic agent example hydrochloric acid guanidine, urea and potassium sulfocyanate (KSCN).The concentration of these chaotropic agents can be greater than 2M.The 4th kind of reagent that dissociates is to contain the solution of organic solvent as 10% dioxan and 40% ethylene glycol.
Perhaps, this serum sample can be handled under the dissociated condition of sample, and this condition can be high or low pH, perhaps the temperature of Sheng Gaoing.Preferred high pH is 9 or higher.Preferred low pH is 3 or lower.Preferred elevated temperature is at least 50 ℃.
The research of table 1. serum Heliobacter pylori antigen admixture
| 1×10 5Bacterium/ml | Admixture not | |
| Sample | OD 450/650 | OD 450/650 |
| Damping fluid | 3.866 | 0.077 |
| Ab negative sample 1 | 3.973 | 0.096 |
| Ab positive sample 1 | 0.336 | 0.120 |
| Ab positive sample 2 | 0.390 | 0.082 |
Embodiment 10
The affinity purification of rabbit helicobacter pylori antibody
A. prepare affinity column
1 to the 5 helicobacter pylorus mycetocyte paste that restrains is suspended in 12.5ml PBS damping fluid, and (0.1M sodium phosphate, 0.15M sodium-chlor pH7.2), wherein contain octyl glucoside, and at room temperature stir 30 minutes.Then with this suspension in ice bath with the output rating ultrasonication of maximum 10 minutes, the ultrasonication of per minute has 1 minute the timed interval.After the supersound process, removed insolubles in centrifugal 30 minutes with 25000g.Keep supernatant liquor and protein concentration is adjusted to 1-3mg/ml with PBS.To in PBS, join in this supernatant liquor with the gel filled ratio of the every ml of 1-10mg albumen by the aminolink coupling gel (by the Pierce preparation) of pre-equilibration, then every milliliter of gel filled middle 5M sodium borohydride that is present in the 10-40 μ l in the water that adds.Again with this reaction slurry in 4 ℃ of following overnight incubation (at least 6 hours), mix gently simultaneously.After hatching, this gel slurry is poured in the chromatographic column of suitable size and discharged excessive liquid.Then, with this post of coupling buffer wash-out of two column volumes, use the 1M Tris HCl of two column volumes subsequently, the pH7.4 wash-out.With the gel resuspending behind the wash-out in the 1M of 1 column volume Tris HCl, pH7.4.Behind the resuspending, the 5M sodium borohydride of every milliliter of gel filled adding 10-40 μ l.Hatched gained suspension under the room temperature 1 hour, and leniently stirred simultaneously.Drain then water in the post washs to remove any unconjugated antigen in the post in a large number with PBS.
B. by the affinity column antibody purification
For purified rabbit antibody, in isopyknic rabbit anteserum, slowly add isopyknic saturated ammonium sulphate solution, with the precipitation helicobacter pylori antibody.Stir under the room temperature after 30 minutes, centrifugal 30 minutes collecting precipitations dissolve in PBS again and dialyse with 100 times of excessive PBS.After the dialysis, by 0.2 μ m strainer this solution is filtered, and application of sample is to this affinity column.Behind the application of sample, arrive baseline up to elutriant with this post of PBS thorough washing.Anti-helicobacter pylori specific antibody in this post 3MKSCN aqueous solution wash-out.Collection contains the fraction of immunoglobulin (Ig) and with PBS dialysis, minimumly changes twice dialyzate to remove excessive KSCN.Then, antibody purified is concentrated into about 1.0-2.0mg/ml and preservation under-20 ℃.
Embodiment 11
Serum antigen ELISA test
With standard method serum is separated from whole blood.Collect the free serum sample according to embodiment 1.Between 20 μ g/ml and 1.0 μ g/ml, the antibody of affinity purification is made serial dilution in phosphoric acid buffer.The 0.1ml equal portions of each diluent are joined among the Costar Strip Plate, cover also overnight incubation at room temperature.This plate once and was at room temperature sealed 4 hours with the 1%BSA that is present among the PBS with the PBS washing.After removing BSA solution, in the micropore batten of this antibody sandwich, add the serum sample of 0.1ml free form, cover also and hatched 2 hours under the room temperature.Then, with PBS/ tween washings this plate is washed 5 times.Then, in each micropore, add 0.1ml and horseradish peroxidase bonded rabbit helicobacter pylori antibody, cover and at room temperature hatched 1 hour.Again this plate washing was also at room temperature hatched 10 minutes with the 0.1ml tetramethyl benzidine for 5 times with PBS/ tween washings.Measure change in color at 450nm.Stop this reaction with 0.1ml 1N sulfuric acid.Elect the diluent that obtains maximum optical density and minimum background as best diluent.
Table 2 and 3 has been represented two tests carrying out with different patients serum's samples in the different time.These test cards understand that serum ELISA detects the result of helicobacter pylori.First test (table 2) comprises 5 objects.Second test (table 3) comprises 3 objects.The result is with OD
450/650Expression.The existence of helicobacter pylori and quantitatively can under the 450nm wavelength, detect (wavelength that 650nm represents to detect background [i.e. this plate]).OD
450/650Represented at OD
450Reading deduct at OD
650Reading.OD
450/650≤ 0.1 sample is represented negative findings (that is, not having helicobacter pylori infection).
Table 2. serum ELISA test (test 1)
| OD 450/650 | The result | |
| Pos sample #1 | 0.712 | Positive |
| Pos sample #2 | 0.487 | Positive |
| Pos sample #3 | 0.187 | Positive |
| Neg sample #4 | 0.076 | Negative |
| Neg sample #5 | 0.048 | Negative |
Table 3. serum ELISA test (test 2)
| OD 450/650 | The result | |
| Pos sample #6 | 0.311 | Positive |
| Pos sample #7 | 3.846 | Positive |
| Neg sample #8 | 0.083 | Negative |
| Damping fluid | 0.097 | Negative |
Embodiment 12
The comparative studies of helicobacter pylori antibody in immunoassays with affinity purification and DEAE purifying
Table 4 has shown in ELISA with the comparative studies between the helicobacter pylori antibody of the helicobacter pylori antibody of affinity column purifying and DEAE column purification.
Be described in embodiment 5 (on seeing) by affinity column purifying helicobacter pylori antibody, DEAE (DEAE-cellulose) post is described below:
With balance DEAE post under 0.0175M potassiumphosphate (pH6.5) room temperature.The supernatant liquor that will contain helicobacter pylori antibody places on this post.Collect and flow out fraction.Detect protein concentration (OD
280) and collect all greater than 0.200 fraction.
In phosphoric acid buffer, make serial dilution with affinity purification between 20 μ g/ml and 1.0 μ g/ml with the DEAE antibody purified.The 0.1ml equal portions of each diluent are joined among the Costar EIA StripPlate, cover also overnight incubation at room temperature.This plate once and was at room temperature sealed 4 hours with the 1%BSA that is present among the PBS with the PBS washing.Some positives and negative sample are carried out dilution in 1: 5 in sample diluent (PBS-BSA).Each sample (0.1ml) is joined in the hole (in contrast) of the hole of DEAE antibody purification bag quilt or affinity purification antibody sandwich, and the DEAE that has generally acknowledged earlier with 0.1ml simultaneously and/or the rabbit anti-helicobacter pylori horseradish peroxidase binding substances of affinity purification are hatched (seeing Table 4).After hatching 60 minutes under the room temperature, the antibody of unconjugated sample and enzyme labelling is removed in the thorough washing of sample.Add under tetramethyl benzidine substrate and the room temperature and hatched 10 minutes.Stop this color change reactions and at OD with 0.1ml 1N sulfuric acid
450/650The numerical value that reads each hole spectrophotometric analysis is to determine the reactivity of each sample.
Table 4 clearlys show that with regard to low background reading and height (sensitiveer) positive sample reading, the antibody of affinity purification and the enzyme conjugates of affinity purification have produced best result.The antibody of affinity purification be used for the both sides of plate and enzyme conjugates as reference standard (detecting 1).Other all detected results and reference standard are relatively.When comparing with reference standard, because the OD of background (being the higher OD of negative sample) that raises and positive sample reduces (detecting 3), DEAE plate and DEAE binding substances have shown false positive results.Replace the DEAE plate with affine plate, positive signal raises, but higher background still exists, and this has caused false positive results (detecting 2).By reducing DEAE binding substances concentration to original 50%, background reduces but positive signal also reduces, and has caused false negative result (detecting 4) like this.
The comparison of table 4.DEAE and affinity purification antibody
| Detect 1 | Detect 2 | Detect 3 | Detect 4 | |
| Plate | Affine | Affine | DEAE | DEAE |
| Enzyme conjugates | Affine | DEAE | DEAE | DEAE (half intensity) |
| Negative control | 0.009 | 0.233 | 0.212 | 0.085 |
| Positive control | 1.320 | 1.366 | 1.111 | 0.879 |
| Positive sample 1 | 2.364 | 2.813 | 1.259 | 0.766 |
| Negative sample 2 | 0.013 | 0.154 | 0.114 | 0.049 |
| Negative sample 3 | 0.024 | 0.323 | 0.245 | 0.112 |
| Positive sample 4 | 0.354 | 0.398 | 0.158 | 0.084 |
Embodiment 13
Immune chromatograph detects
Immunochromatographitest test device has the outer plastic box that has two forms: " application of sample form " and " observation window as a result ".This " application of sample form " to wherein adding moving phase, wherein also contains " label pad " or contains the storage storehouse of second kind of antibody, and this antibody is with detection reagent such as immunity-Jin mark.With this pad place add sampling point and " observation window as a result " than between the lower boundary." observation window as a result " on stationary phase, it contains the check line, and it is located with one-level antibody, and control line, and its antibody with anti-second kind of antibody is located.This check line is adding between sampling point and the control line.
In order to detect, 4-6 bled joins the sample area of this box clearly.This sample flow through contain purifying, with the red color of immunity-Jin or the label pad of other marker link coupled helicobacter pylori antibody.The sample that contains the antibody of mark now will move this check bar at first by the check line, pass through control line then.So contain Heliobacter pylori antigen in the sample, this antibody then will combine this antigen and red immunity-Jin coupling with the helicobacter pylori antibody on being fixed on nitrocellulose filter, forms a line.When helicobacter pylori antibody-antigen-antibody complex is hunted down, in form as a result, can see a red check line (film-antibody: antigen: antibody-red immunity-Jin).This control line goat anti-rabbit antibodies drop.When this sample flow when (if a kind of monoclonal antibody is used for the coupling coloured particle, this control line goat anti-mouse drop), helicobacter pylori-immunity-Jin-antibody is caught by the goat antirabbit line, to guarantee correctly to carry out this test.
Liquid sample wherein may contain other moving phase, will move to higher part (chromatographic effect) from lower part, and by the top absorption pad, all excessive liquid will be arranged the top to this box.If in this sample, do not contain antigen, have only control line to see in the later stage of test.If there is antigen, will see the check line.
Through detailed description of the present invention and with reference to preferred embodiment, do not deviating under the preceding topic of the scope of the invention for a person skilled in the art, improved form of the present invention and version are conspicuous, scope of the present invention limits in the claims.
Claims (14)
1, detects the method for Heliobacter pylori antigen in the blood, use the antigenic antibody of anti-helicobacter pylori, utilize immunological method to detect the Heliobacter pylori antigen that exists with the antigen-antibody complex form in the blood sample, and the antigenic antibody of described anti-helicobacter pylori is the antibody of affinity purification.
2, the method for Heliobacter pylori antigen in the described detection blood of claim 1, wherein said immunological detection method is the immunoassays method.
3, the method for Heliobacter pylori antigen in the described detection blood of claim 2, wherein said immunoassays comprise that basic sandwich detection, three sandwich detections or immune chromatograph detect.
4, the method for Heliobacter pylori antigen in the described detection blood of claim 1, wherein said blood sample is a serum.
5, the method for Heliobacter pylori antigen in the described detection blood of claim 1, wherein said detection method is the immunoblotting detection method.
6, the method for Heliobacter pylori antigen in claim 2 or the 4 described detection blood wherein, implements to dissociate processing earlier to blood sample.
7, the method for Heliobacter pylori antigen in the described detection blood of claim 6, wherein, described dissociate to handle comprise and adopt the reagent or under condition, handle of dissociating at sample solution.
8, the method for Heliobacter pylori antigen in the described detection blood of claim 7, the wherein said reagent that dissociates comprises that volumetric molar concentration is at least sodium-chlor or the Klorvess Liquid of 1M.
9, the method for Heliobacter pylori antigen in the described detection blood of claim 7, the wherein said reagent that dissociates comprises washing composition, it comprises and is selected from following at least a material: SDS, polysorbas20, octyl glucoside, deoxycholate salt and Triton X-100.
10, the method for Heliobacter pylori antigen in the described detection blood of claim 7, the wherein said reagent that dissociates comprises organic solvent, it comprises and is selected from following at least a material: dioxan and ethylene glycol.
11, the method for Heliobacter pylori antigen in the described detection blood of claim 7, the wherein said reagent that dissociates comprises a kind of chaotropic agent, it is selected from Guanidinium hydrochloride, urea or potassium sulfocyanate.
12, the method for Heliobacter pylori antigen in the described detection blood of claim 7, the wherein said reagent that dissociates comprises at least a enzyme, this enzyme is selected from proteolytic enzyme and lipase.
13, the method for Heliobacter pylori antigen in the described detection blood of claim 7, wherein, sample solution comprises from condition that pH with sample is adjusted to and is not less than 9 or be not more than 3.
14, the method for Heliobacter pylori antigen in the described detection blood of claim 7, wherein, sample solution comprises that from condition temperature regulation with described sample is to being not less than 50 ℃.
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|---|---|---|---|---|
| CN103834741A (en) * | 2014-03-19 | 2014-06-04 | 四川万可泰生物技术有限责任公司 | Noninvasive helicobacter pylori gene detection kit and preparation and detection method thereof |
| CN103834741B (en) * | 2014-03-19 | 2016-05-18 | 四川万可泰生物技术有限责任公司 | A kind of without wound helicobacter pylori gene detecting kit and preparation and determination methods method thereof |
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| Publication number | Publication date |
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| CN1330153A (en) | 2002-01-09 |
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