CN103777017B - Detect method and the monoclonal antibody thereof of enterohemorrhagic Escherichia coli O 157: H7 - Google Patents

Detect method and the monoclonal antibody thereof of enterohemorrhagic Escherichia coli O 157: H7 Download PDF

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CN103777017B
CN103777017B CN201410028114.7A CN201410028114A CN103777017B CN 103777017 B CN103777017 B CN 103777017B CN 201410028114 A CN201410028114 A CN 201410028114A CN 103777017 B CN103777017 B CN 103777017B
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escherichia coli
monoclonal antibody
antibody
enterohemorrhagic escherichia
cgmcc
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CN103777017A (en
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刘箐
李浩林
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University of Shanghai for Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/265—Enterobacter (G)

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Abstract

The invention belongs to microorganism detection field.Specifically, the present invention relates to a kind of method of enterohemorrhagic Escherichia coli O 157: H7 of detection, for two strains of the method by the antihaemorrhagics Escherichia coli O 157 produced with monoclonal antibody preparation process: the monoclonal antibody of H7, this monoclonal antibody can specifically with enterohemorrhagic Escherichia coli O 157: H7 is combined.It is by mouse hybridoma cell system GS2-D3, and CGMCC No.6610 or 3A8F11B10E7, CGMCC No.8458 produced.Present invention also offers antihaemorrhagics Escherichia coli O 157: the purposes of the monoclonal antibody of H7.

Description

Detect method and the monoclonal antibody thereof of enterohemorrhagic Escherichia coli O 157: H7
Technical field
The invention belongs to microorganism detection field.Specifically, the present invention relates to and a kind of detect the method for enterohemorrhagic Escherichia coli O 157: H7, the two strain antihaemorrhagics Escherichia coli O 157s for the method: H7 monoclonal antibody and uses thereof and produce the hybridoma of this monoclonal antibody.
Background technology
Enterohemorrhagic Escherichia coli O 157: H7(Escherichia coli O157:H7) be the colibacillary predominant serotypes of enteron aisle, by beef and the propagation such as goods, milk and goods thereof, chicken, veterinary antibiotics, beverage, water thereof.Enterohemorrhagic Escherichia coli O 157: H7 can cause enteritis, the typical clinical symptom of infected patient is bloody stool, stomachache, low-heat or do not generate heat, about there is the patient of 10% can develop into hemolytic uremia and thrombotic thrombocytopenic purpura, and damage can be caused to the enteron aisle of infected patient, liver,spleen,kidney, lymph, brain, respiratory system and nervous system.Old man and children are people at highest risk, and easily hemolytic uremia occurs, mortality ratio is up to more than 30%.The World Health Organization (WHO) is by enterohemorrhagic Escherichia coli O 157: H7 enteritis is classified as emerging infectious disease.Enterohemorrhagic Escherichia coli O 157: H7 infects has become a global hygienic issues.
The detection of current enterohemorrhagic Escherichia coli O 157: H7 depends on the biochemical identification of GB defined, and its shortcoming is that complex operation, sense cycle are longer, cannot adapt to a large amount of sample examinations.Molecular biology and immunology detection are quick, accurate, the most stable quick detection meanss occurred in recent years; but the whole dependence on import of testing product; China there is no at present and has complete independent intellectual property right; and the quick testing product detecting practice can be applied to; this patent is with enterohemorrhagic Escherichia coli O 157: H7 is for detecting target, and technology required for protection relates to enterohemorrhagic Escherichia coli O 157: the quick testing product of H7.
Summary of the invention
The object of this invention is to provide a kind of vitro detection enterohemorrhagic Escherichia coli O 157: the method for H7.
Two strains that another object of the present invention is to provide for said method can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically by what produce with monoclonal antibody preparation process: the monoclonal antibody of the different antigen site of H7.
Another object of the present invention is to provide the hybridoma producing said monoclonal antibody.
A first aspect of the present invention there is provided a kind of vitro detection enterohemorrhagic Escherichia coli O 157: the method for H7, comprises the following steps:
A testing sample application of sample is caught the solid phase carrier of antibody in being coated with by (), thus making the enterohemorrhagic Escherichia coli O 157 in testing sample: the seizure antibody of H7 on solid phase carrier is combined, and forms the solid phase carrier with " enterohemorrhagic Escherichia coli O 157: H7-catches antibody " binary complex; Described seizure antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody 3A8F11B10E7 of H7, and described monoclonal antibody 3A8F11B10E7 is produced by mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458;
B () will detect the solid phase carrier that antibody application of sample obtains in (a), thus form the solid phase carrier with " detect antibody-enterohemorrhagic Escherichia coli O 157: H7-and catch antibody " ternary complex; Described detection antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody GS2-D3 of H7, described monoclonal antibody GS2-D3 is by mouse hybridoma cell system GS2-D3, CGMCC No.6610 produces, and described monoclonal antibody GS2-D3 carries a detectable; With
C () detects the detectable in ternary complex, thus determine enterohemorrhagic Escherichia coli O 157 in testing sample: whether and the amount existed the existence of H7.
In a preference, described solid phase carrier is enzyme reaction plate.
In another preference, described detectable is horseradish peroxidase.
A second aspect of the present invention there is provided a kind of immunoglobulin (Ig), and it is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody of H7, and it is by mouse hybridoma cell system 3A8F11B10E7, and CGMCC No.8458 produced.
In a preference, above-mentioned immunoglobulin (Ig) is with enterohemorrhagic Escherichia coli O 157: H7, ATCC No.43895 immunity Balb/c mouse obtains.
A third aspect of the present invention there is provided a kind of hybridoma producing said monoclonal antibody, and it is mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458.
In a preference, the monoclonal antibody that above-mentioned hybridoma produces can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically: H7.
A fourth aspect of the present invention there is provided the purposes of above-mentioned immunoglobulin (Ig), and it is used to detect enterohemorrhagic Escherichia coli O 157: H7.
A fifth aspect of the present invention there is provided a kind of immunoglobulin (Ig), and it is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody of H7, and it is by mouse hybridoma cell system GS2-D3, and CGMCC No.6610 produced.
In a preference, above-mentioned immunoglobulin (Ig) is with enterohemorrhagic Escherichia coli O 157: H7, ATCC No.43895 immunity Balb/c mouse obtains.
A sixth aspect of the present invention there is provided a kind of hybridoma producing said monoclonal antibody, and it is mouse hybridoma cell system GS2-D3, CGMCC No.6610.
In a preference, the monoclonal antibody that above-mentioned hybridoma produces can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically: H7.
Present invention also offers the purposes of above-mentioned immunoglobulin (Ig), it is used to detect enterohemorrhagic Escherichia coli O 157: H7.
The details of various aspects of the present invention is able to detailed description by chapters and sections subsequently.By hereafter and the description of claim, feature of the present invention, object and advantage will be more obvious.
Accompanying drawing explanation
The SDS-PAGE electrophoretogram of Fig. 1 monoclonal antibody of the present invention
Lane1 is GS2-D3, Lane2 is 3A8F11B10E7
Embodiment
Monoclonal antibody
The research of the present inventor shows, using enterohemorrhagic Escherichia coli O 157: H7 is as immunogene, immunity Balb/c mouse, separation and purification obtains two strain antihaemorrhagics Escherichia coli O 157s: H7 monoclonal antibody GS2-D3 and 3A8F11B10E7, its antibody titer can reach 1:100000, it can specificity, efficiently with enterohemorrhagic Escherichia coli O 157: H7 is combined, with Listeria monocytogenes, salmonella, Shigella, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, staphylococcus aureus, streptococcus, bacillus cereus, other Escherichia coli etc. amount to 76 kinds of equal no cross reactions of pathogenetic bacteria.
The invention provides antihaemorrhagics Escherichia coli O 157: H7 monoclonal antibody.Antihaemorrhagics Escherichia coli O 157 of the present invention: H7 monoclonal antibody can utilize mouse hybridoma cell system GS2-D3(CGMCC No.6610) or 3A8F11B10E7(CGMCC No.8458) secretion generation.The present invention includes and there is antihaemorrhagics Escherichia coli O 157: the monoclonal antibody of the corresponding amino acid sequence of H7 monoclonal antibody GS2-D3 and 3A8F11B10E7, and there is other protein of these chains or protein conjugate and fusion expressed product.Particularly, the present invention includes and have containing hypervariable region (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (i.e. immune conjugate and fusion expressed product), as long as this hypervariable region is identical with the hypervariable region of heavy chain with light chain of the present invention or at least 90% homology, preferably at least 95% homology.As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: medicine, toxin, cell factor (cytokine), radioactive nuclide, enzyme and other diagnosis or treatment molecule and antihaemorrhagics Escherichia coli O 157: that H7 monoclonal antibody or its fragment are combined and the conjugate that formed.The present invention also comprises and antihaemorrhagics Escherichia coli O 157: the cell surface marker thing that H7 monoclonal antibody or its fragment are combined or antigen.
For antihaemorrhagics Escherichia coli O 157 of the present invention: H7 monoclonal antibody heavy and sequence of light chain, can measure by conventional method.Antihaemorrhagics Escherichia coli O 157: the hypervariable region of H7 monoclonal antibody V chain or complementary determining region (complementaritydetermining region, CDR) interesting especially because relate to conjugated antigen at least partly in them.Therefore, the present invention includes those and there is the band light chain immunoglobulin of CDR and the molecule of weight chain variable chain, as long as its CDR and antihaemorrhagics Escherichia coli O 157: H7 monoclonal antibody CDR has the homology of more than 90% (preferably more than 95%).The present invention not only comprises complete monoclonal antibody, also comprises and has immunocompetent antibody fragment, as Fab or (Fab ') 2fragment; Heavy chain of antibody; Light chain of antibody.
Present invention also offers the DNA molecular of above-mentioned immunoglobulin (Ig) or its fragment.The sequence of these DNA moleculars can use routine techniques, utilizes hybridoma cell line GS2-D3(CGMCC No.6610) or 3A8F11B10E7(CGMCC No.8458) obtain.In addition, also the coded sequence of light chain and heavy chain can be merged, form single-chain antibody.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by conventional method and obtains relevant sequence.
In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for Prof. Du Yucang.Usually, by first synthesizing multiple small fragment, and then carry out connect can obtain the very long fragment of sequence.
At present, the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in the various existing DNA molecular (or as carrier) that in this area, oneself knows and cell.In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.
The invention still further relates to the carrier comprising above-mentioned suitable DNA sequence dna and suitable promoter or control sequence.These carriers may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic, as bacterial cell; Or the eukaryotic such as low, as yeast cells; Or higher eucaryotic cells, as mammalian cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotes as Enterohemorrhagic E.coli time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaC1 2method process, step used is well-known in this area.Another kind method uses MgC1 2.If needed, transform and also can be undertaken by the method for electroporation.When host is eucaryote, following DNA transfection method can be used: calcium phosphate precipitation, conventional mechanical methods as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by conventional method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, nutrient culture media used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promoter selected with the induction of suitable method (as temperature transition or chemical induction), cultivates a period of time again by cell.
Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cell membrane.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, ultrasonic process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Antihaemorrhagics Escherichia coli O 157 of the present invention: H7 monoclonal antibody GS2-D3 and 3A8F11B10E7, its height of tiring (can 1:100000 be reached), enterohemorrhagic Escherichia coli O 157 can be detected specifically, efficiently: H7, amount to 76 kinds of equal no cross reactions of pathogenetic bacteria with Listeria monocytogenes, salmonella, Shigella, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, staphylococcus aureus, streptococcus, bacillus cereus, other Escherichia coli etc., this is maximum innovative point of the present invention.Said monoclonal antibody GS2-D3 can use horseradish peroxidase, alkaline phosphatase, nanogold particle to mark, and uses in specific manner as detecting antibody.Said monoclonal antibody 3A8F11B10E7, in various detection is used, can use as catching antibody in specific manner.
Detection kit
The present inventor is through to study widely and test, be surprised to find that, when adopting mouse hybridoma cell system 3A8F11B10E7(CGMCC No.8458) the antihaemorrhagics Escherichia coli O 157 that produces: H7 monoclonal antibody is as seizure antibody capture enterohemorrhagic Escherichia coli O 157: after H7, again with detectable label mark by mouse hybridoma cell system GS2-D3(CGMCCNo.6610) the antihaemorrhagics Escherichia coli O 157 that produces: H7 monoclonal antibody is as detection antibody, extremely effectively can be incorporated into enterohemorrhagic Escherichia coli O 157: H7, thus detect enterohemorrhagic Escherichia coli O 157 in high sensitivity by double-antibody method: H7.
As used herein, described " sample " refers to the materials such as food, human and animal excreta, vomitus, includes but not limited to: the enrichment liquid of serum, blood plasma, ight soil, food etc.Preferably, described sample is food.
As used herein, described " seizure antibody ", " coated antibody ", " first antibody " and " primary antibodie " is used interchangeably, all refer to and described can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody of H7, it is by mouse hybridoma cell system 3A8F11B10E7(CGMCC No.8458) produce.
Described seizure antibody can be coated on solid phase carrier.The present invention has no particular limits adopted solid phase carrier, if its can with seizure antibody phase coupling (connection).Such as, described solid phase carrier is enzyme reaction plate.
As used herein, described " detection antibody ", " second antibody ", " enzyme labelled antibody " was used interchangeably with " two resist ", all refer to can specific binding in enterohemorrhagic Escherichia coli O 157: another strain monoclonal antibody of H7, it is produced by mouse hybridoma cell system GS2-D3, CGMCC No.6610.
As used herein, described " specificity " refers to that antibody can only be incorporated into enterohemorrhagic Escherichia coli O 157: H7; More particularly, refer to that those to be combined with enterohemorrhagic Escherichia coli O 157: H7 but nonrecognition and be incorporated into the antibody of other non related antigen molecule.
The present inventor and then according to double antibodies sandwich ratio juris, has prepared the enzyme linked immunological kit that one can be used for detecting enterohemorrhagic Escherichia coli O 157: H7 in sample.The way of double-antibody method routine is that seizure antibody is fixed on carrier, then antibody and antigen-reactive is caught, after washing, (described detection antibody carries detectable with detection antibody response again, or can be combined with the material carrying detectable), finally carry out chemiluminescence or enzyme connection chromogenic reaction detection signal.Further, relative to the competition law of monoclonal antibody body, the mensuration effect of double antibody sandwich method is more excellent, only needs little sample size when thus measuring.So adopt double antibody sandwich method no matter to have more advantage in sensitivity, degree of accuracy, accuracy, specificity and stability.
Specifically, enzyme linked immunological kit of the present invention contains:
(a) enzyme reaction plate, described enzyme reaction plate is coated with the antihaemorrhagics Escherichia coli O 157 as catching antibody: H7 monoclonal antibody 3A8F11B10E7, described antihaemorrhagics Escherichia coli O 157: H7 monoclonal antibody is produced by mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458;
(b) container a, antihaemorrhagics Escherichia coli O 157 as detecting antibody is housed: H7 monoclonal antibody GS2-D3 in described container a, described antihaemorrhagics Escherichia coli O 157: H7 monoclonal antibody is produced by mouse hybridoma cell system GS2-D3, CGMCC No.6610.
As optimal way of the present invention, described detection antibody is with detectable label.
As used herein, described " detectable label " refers to for determining enterohemorrhagic Escherichia coli O 157 in detected sample: whether and the mark of the amount existed the existence of H7.Determining seizure antibody that kit of the present invention adopts and/or after detecting antibody, this area can adopted conventional for being combined with detection antibody the various labels carrying out detecting.The present invention has no particular limits adopted label, as long as can be combined with described detection antibody, and enterohemorrhagic Escherichia coli O 157 in detected sample can be indicated exactly after appropriate processing: the existence of H7 whether and the label of amount be all available.Described label directly can be arranged at and detect on antibody; Or described label also can be arranged on the antiantibody of the anti-detection antibody of specificity, those skilled in the art according to the kind of adopted antibody and characteristic, can select suitable label.Such as, described label can be selected from: horseradish peroxidase (HRP), alkaline phosphatase vinegar enzyme (AP), glucose oxidase, beta-D-galactosidase, urase, hydrogen peroxidase or glucoamylase.
When adopting some enzyme marker as implied above, also need the substrate adopting some to be combined with corresponding enzyme, thus report label by modes such as colour developings there is situation or amount.As used herein, described " substrate corresponding with label " refers to and can be labeled thing institute catalyzed coloration, for showing the identification signal detecting antibody and enterohemorrhagic Escherichia coli O 157: H7 and occur to combine.Described substrate is such as: for adjacent benzene two limb (OPD), tetramethyl biphenyl limb (TMB), the ABTS of horseradish peroxidase; For the p-nitrophenyl phosphoric acid vinegar (p-nitro phenyl phosphate, p-NPP) etc. of alkaline phosphatase.Those skilled in the art according to the kind of adopted label and characteristic, can select suitable substrate.
As optimal way of the present invention, described detection antibody is directly connected with label.More preferably, described label is HRP.With detect antibody with biotin labeling, react after compare with streptavidin HRP reacting phase again, directly detection antibody on mark HRP, reaction terminate after directly add substrate colour developing more simple and convenient.
In order to obtain quantitative result, the multiple enterohemorrhagic Escherichia coli O 157s knowing concentration containing oneself can also be set in testing process: the standard items of H7.Method to set up for standard items can adopt conventional method.
In order to eliminate false positive and false negative, also Quality Control (contrast) can be set in testing process.As optimal way of the present invention, described positive control is the enterohemorrhagic Escherichia coli O 157 of deactivation: H7 bacterium liquid, and described negative control is the EC meat soup of sterilizing.
In addition, in order to make kit of the present invention more convenient when detecting, preferably some other auxiliary reagent is also comprised in described kit, described auxiliary reagent is conventional some reagent used in ELISA kit, and the characteristic of these reagent and their compound method are all well-known to those skilled in the art.Described reagent is (but being not limited to) such as: developer, cleansing solution, stop buffer, enrichment liquid, dilution.
In addition, in described kit, also operation instructions can be comprised, for illustration of the using method of the reagent wherein loaded.
Cleaning Principle and the beneficial effect of enzyme linked immunological kit of the present invention are as follows:
What kit of the present invention adopted is DASELISA immunization.Antihaemorrhagics Escherichia coli O 157 is had: H7 monoclonal antibody (seizure antibody) when pre-coated on enzyme reaction plate, after adding sample solution or standard items, add with detectable label another strain antihaemorrhagics Escherichia coli O 157 as horseradish peroxidase again: H7 monoclonal antibody (detection antibody), the enterohemorrhagic Escherichia coli O 157 existed in sample or standard items: H7 will combine with seizure antibody enzyme reaction plate wrapping quilt, after detection antibody to be added, " antibody-antigen-enzyme labelled antibody " compound can be formed, through TMB(tetramethyl benzidine) colour developing after can form yellow substance, thus can in judgement sample enterohemorrhagic Escherichia coli O 157: H7 existence whether and the amount existed.
Under 450nm, absorbance is measured by microplate reader.Negative control≤0.1, positive control >=0.3, experimental result is effective, otherwise result be judged to be invalid; During detect aperture OD value >=0.2, be judged to be the positive; When detect aperture OD value is between 0.1-0.2, be judged to be the weak positive; Detect aperture OD value≤0.1 is judged to be feminine gender.
Test shows, enterohemorrhagic Escherichia coli O 157 of the present invention: H7 enzyme linked immunological kit, has higher sensitivity and accuracy.Between plate, error is less than 5%, and in plate, CV is less than 3%.Amount to 76 kinds of equal no cross reactions of pathogenetic bacteria with Listeria monocytogenes, salmonella, Shigella, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, staphylococcus aureus, streptococcus, bacillus cereus, other Escherichia coli etc., this is maximum innovative point of the present invention.
In a word, kit of the present invention requires low, easy and simple to handle to the pre-treatment of sample, and detection limit is 10 5cfu/ml, specificity and good stability, and all there is no cross reaction with most of food-borne pathogens.
Detection method
Present invention also offers one utilizes kit box of the present invention to detect enterohemorrhagic Escherichia coli O 157 outward: the method for H7, comprises the following steps:
A testing sample application of sample is caught the solid phase carrier of antibody in being coated with by (), thus making the enterohemorrhagic Escherichia coli O 157 in testing sample: the seizure antibody of H7 on solid phase carrier is combined, and forms the solid phase carrier with " enterohemorrhagic Escherichia coli O 157: H7-catches antibody " binary complex; Described seizure antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody 3A8F11B10E7 of H7, and described monoclonal antibody 3A8F11B10E7 is produced by mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458;
B () will detect the solid phase carrier that antibody application of sample obtains in (a), thus form the solid phase carrier with " detect antibody-enterohemorrhagic Escherichia coli O 157: H7-and catch antibody " ternary complex; Described detection antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody GS2-D3 of H7, described monoclonal antibody GS2-D3 is by mouse hybridoma cell system GS2-D3, CGMCC No.6610 produces, and described monoclonal antibody GS2-D3 carries a detectable; With
C () detects the detectable in ternary complex, thus determine enterohemorrhagic Escherichia coli O 157 in detected sample: whether and the amount existed the existence of H7.
As a kind of optimal way of the present invention, quantitatively detect enterohemorrhagic Escherichia coli O 157: the method for H7 is specific as follows:
(I) antigen-antibody reaction: be coated on porous plate by seizure antibody of the present invention, adds the standard items of variable concentrations, quality-control product (optional) afterwards respectively in the micropore of porous plate, or testing sample;
(II) integrated enzyme reaction: antibody (being provided with label) solution will be detected and add each hole, and vibrate, hatch, wash;
(III) chromogenic reaction: every hole adds substrate, developer corresponding to label, and hatch, every hole adds reaction terminating liquid, terminates reaction:
(IV) microplate reader measures OD value:
(V) result calculates:
A) production standard curve: with enterohemorrhagic Escherichia coli O 157: H7 standard concentration is horizontal ordinate, it is ordinate that standard items measure OD value, makes typical curve;
B) quality-control product concentration (optional) is passed judgment on: according to the OD value of quality-control product, read corresponding concentration value from typical curve; When quality-control product mensuration concentration value is in given range, this time measures effectively;
C) calculate test serum sample concentration: when typical curve and quality-control product are all determined effective, calculate the enterohemorrhagic Escherichia coli O 157 of testing sample from typical curve according to the OD value of sample to be tested: H7 concentration.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise number percent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the meaning be familiar with identical.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1. antihaemorrhagics Escherichia coli O 157: the preparation of H7 monoclonal antibody GS2-D3 and 3A8F11B10E7
One, the preparation of immunogene and positive criteria product
Enterohemorrhagic Escherichia coli O 157: H7(ATCC43895) be seeded on sorbierite Mai Kangkai (SMAC) flat board, cultivate 24h for 37 DEG C, picking list bacterium colony increases bacterial context soup, 37 DEG C, 150r/min shaken cultivation 17h in improvement E.C ovobiocin, counting, adds 0.3% formalin room temperature deactivation 1 day.Enterohemorrhagic Escherichia coli O 157 is adjusted: H7(ATCC43895 with physiological saline) concentration to 5 × 10 9cfu/ml is as immunogene; Adjusting concentration with physiological saline is 10 8cfu/ml enterohemorrhagic Escherichia coli O 157: H7 bacterium liquid is as positive control standard items, and the EC meat soup of sterilizing is negative control standard items.
Two, the preparation of monoclonal antibody
1) animal used as test: select 38 week ages, about body weight 20g, female Balb/c mouse are animal used as test.
2) immunization method: every mouse peritoneal injection 0.2ml immunogene, at interval of 2 weeks with same dosage booster shots once.
3) take a blood sample: from tail vein blood sampling after 3 booster immunizations, adopt indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum titre.Wait to tire and no longer rise, lumbar injection same amount immunogene, conventionally carried out Fusion of Cells after 3 days.
4) Fusion of Cells: get that immune mouse spleen cell and SP2/0 myeloma cell are conventional under 50%PEG effect merges, is inoculated in 96 well culture plates respectively, is placed in 37 DEG C, 5%CO 2incubator is cultivated.
5) filtering hybridoma: adopt indirect non-competing euzymelinked immunosorbent assay (ELISA), the hybridoma in screening strong positive hole, transfers them to 24 well culture plates.
6) clone cultivates and antibody preparation: carry out colonized culture with limiting dilution assay.When Growth of Cells is to when to be paved with at the bottom of hole 1/10, then detect with same method, strong positive hole is cloned, 3-4 time so repeatedly, until positive rate reaches 100% again.Hybridoma is expanded and cultivates, be injected in through the pretreated Balb/c mouse peritoneal of paraffin oil, every 2 × 10 6individual hybridoma, 7 ~ 10 days mouse web portion protuberances, living body puncture extracts ascites.With caprylic acid-ammonium antibody purification from mouse ascites.
Three, the titration of monoclonal antibody
Enterohemorrhagic Escherichia coli O 157: H7 nutrient culture media is as follows: GB: GB4789.38-2012
EC meat soup (E.coli broth)
Composition: tryptone or trypticase 20.0g, No. 3 cholate or mixing cholate 1.5g, lactose 5.0g, dipotassium hydrogen phosphate (K 2hPO 4) 4.0g, potassium dihydrogen phosphate (KH 2pO 4) 1.5g, sodium chloride 5.0g, distilled water 1000.0mL;
Method for making: be dissolved in by mentioned component in distilled water, regulates pH6.9 ± 0.1,121 DEG C of autoclaving 15min.
The step that antibody titer measures is as follows:
(1) saturated culture of bacteria antigen is added 100 μ l together with nutrient culture media in the hole of correspondence, 4 DEG C spend the night (about wrapper sheet 36h).
(2) to turn liquid pat dry residual liquid, clean 3 times with 250 μ l cleansing solutions.
(3) 100 μ l1%BSA are added in each hole, 37 DEG C of closed 1h.
(4) to turn liquid pat dry residual liquid, clean 3 times with 250 μ l cleansing solutions.
(5) add 100 μ l serum in each hole, hatch 1h for 37 DEG C.
(6) to turn liquid pat dry residual liquid, add 250 μ lPBST cleansing solutions in each hole and wash 3 times.
(7) two anti-(sigma) that in each hole, the HRP of 50 μ l marks, incubated at room 1h.
(8) soak 5min with cleansing solution, turned letter liquid also pats dry residual liquid, adds 250 μ lPBST cleansing solutions and wash 3 times in each hole.
(9) add 100 μ l substrates in each hole, colour developing 30min, add stop buffer 100 μ l also immediately at OD 450reading.
Table 1. liang strain antibody titer measurement result (OD450)
Four, the purity analysis of monoclonal antibody
With the purity of SDS-PAGE analysis list clonal antibody, 5% spacer gel, 10% separation gel, 120V voltage, bottom electrophoresis to glue, gel Coomassie Brilliant Blue dyes, with Labworks image acquisition and analysis software observations (Fig. 1) after decolouring.
As shown in Figure 1, swimming lane 1 and swimming lane 2 are respectively GS2-D3 and 3A8F11B10E7.The heavy chain molecule amount of GS2-D3 is 48kDa, and light chain molecule amount is 26kDa; The heavy chain molecule amount of 3A8F11B10E7 is 48kDa, and light chain molecule amount is 28kDa.The CHARACTERISTICS IDENTIFICATION of embodiment 2. monoclonal antibody GS2-D3 and 3A8F11B10E7
One, monoclonal antibody subgroup identification
1, antigen coated: with 0.01M PBS bag by goat against murine two anti-igg+A+M, every hole 50 μ l, 4 DEG C of bags are spent the night, and discard liquid in hole next day, wash plate 3 times.
2, close: every hole adds 1%BSA200 μ l, close for 4 DEG C and spend the night.Next day pats dry plank and does not wash plate.
3, monoclonal antibody hybridoma cell supernatant is added, each sample 8 micropores, every hole 50 μ l.37 DEG C, hatch 1h.
4, after washing plate 4 times, the rabbit anti-mouse igg 1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, 37 DEG C adding specific bond respectively hatches 1h.
5, after washing plate 4 times, every hole adds horseradish peroxidase-labeled anti-rabbit two anti-igg (H+L) of having diluted, and 37 DEG C, hatches 30min.
6, after washing plate 4 times, 100 μ l substrate nitrite ions are added, 37 DEG C, lucifuge colour developing 10min.Result is read under 450nm wavelength.
Table 2. liang strain monoclonal antibody subgroup identification result
Two, monoclonal antibody cross reaction test
1, antigen coated: by 78 kind 10 8the pathogenic bacteria of cfu/ml bacterial concentration join in ELISA Plate, and each pathogenic bacteria add 3 each holes, and every hole 100 μ l, wraps and spent the night by 4 DEG C.
2, close: after washing plate 3 times, every hole adds 3%BSA200 μ l, hatches 2h for 37 DEG C.
3, plate is washed 3 times.Every hole adds the monoclonal antibody 100 μ l diluted, and hatches 1h for 37 degrees Celsius.
4, plate is washed 3 times.Adding the goat against murine two of having diluted resists in all micropores, and every hole 100 μ l, hatches 1h for 37 degrees Celsius.
5, plate is washed 4 times.Add substrate nitrite ion, 37 DEG C, lucifuge colour developing 15min.Result is read under 450nm wavelength.
Table 3. liang strain monoclonal antibody cross reaction testing result
Embodiment 3. detects enterohemorrhagic Escherichia coli O 157: the composition of the enzyme linked immunological kit of H7, preparation and application thereof
One, enzyme linked immunological kit is made up of following substances:
(1) ELISA Plate of pre-coated antibody: use 0.02M acetate buffer solution (pH2.0) solution dilution, antihaemorrhagics Escherichia coli O 157: H7 monoclonal antibody 3A8F11B10E7 wraps by 96 hole ELISA Plate, every hole 100 μ l.4 DEG C of overnight incubation, conveniently ELISA method closes washing.
(2) enterohemorrhagic Escherichia coli O 157: H7 positive control standard items and negative control standard items.
(3) the antihaemorrhagics Escherichia coli O 157 of horseradish peroxidase-labeled: the monoclonal antibody GS2-D3 of H7.
(4) enzyme labelled antibody dilution: 0.01M PBS, pH7.6.
(5) 10 × concentrated washing lotion: the 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% Sodium azide, pH7.4, dilutes concentrated washing lotion 10 times during use.
(6) nitrite ion A liquid, nitrite ion B liquid.Before using, A liquid is mixed with B liquid equal-volume.
(7) stop buffer: 2M sulfuric acid solution.
Two, the preparation of each component in enzyme linked immunological kit
(1) using Enterohemorrhagic E.coli monoclonal antibody 3A8F11B10E7 as seizure antibody coated elisa plate
Liquid is buffered by enterohemorrhagic Escherichia coli O 157: H7 monoclonal antibody 3A8F11B10E7 is diluted to 5 μ g/ml with bag, every hole adds 100 μ l, 4 DEG C are spent the night, incline next day coating buffer, with the wash liquid diluted 3 times, pats dry, then in every hole, add 220 μ l confining liquids, 37 DEG C of incubation 2h, liquid in hole of inclining, preserves with aluminium foil bag sealing after drying.
Bag is buffered liquid: 0.02M acetate buffer solution, regulates pH to 2.0 with 5M HCl.
Confining liquid: the 0.01M PBS containing 0.3% bovine serum albumin(BSA) and 10% sucrose.
(2) preparation of the monoclonal antibody GS2-D3 of horseradish peroxidase-labeled
By enterohemorrhagic Escherichia coli O 157: H7 monoclonal antibody GS2-D3 and horseradish peroxidase (HRP) carry out coupling, the method for employing is the Over-voltage protection of improvement, and method is as follows:
A, 5mg horseradish peroxidase (HRP) is dissolved in 0.5ml0.2M acetate buffer solution (pH5.6).
B, add the 0.06M NaIO of existing preparation 4solution 0.5ml, 4 DEG C of oxidation 20min.
C, add 0.4M ethylene glycol solution 0.5ml containing 22%NaCl, room temperature leaves standstill 30min.
The absolute ethyl alcohol precipitation enzyme of d, use 6ml precooling, the centrifugal 10min of 1500rpm.
E, remove supernatant, will precipitation be dissolved in the 0.01M PBS(pH7.4 of 2.5ml) in.
F, add 10mg monoclonal antibody GS2-D3, and use 0.5M carbonic acid buffer (pH9.6) to regulate pH to 9.0 immediately.4 DEG C of hold over night.
G, add 10mg/ml sodium borohydride 50 μ l, 4 DEG C, leave standstill 2h.
H, use 0.01M PBS(pH7.4) 4 DEG C of dialysed overnight.
I, purification storage.
Three, the application of kit
(1) detection method
1, sample pre-treatments
Get measuring samples 25g(ml) add 225ml improvement EC meat soup (mECn) homogeneous mixing under homogenizer, cultivate 16h for 36 DEG C ± 1 DEG C.Cultured sample is heated 10min in 100 DEG C of water-baths.It is stand-by that taking-up is cooled to room temperature.
2, detect with kit
30min is placed under the micropore of taking-up requirement and all reagent normal temperature.Getting 200 μ l measuring samples is added in micropore, 37 DEG C, hatches 30min.Remove liquid in hole, add 200 μ l washing lotions in each micropore, rock the several seconds gently, fast upset is by liquid in micropore to the greatest extent, to a folded clean thieving paper clap several under, repeat operation and wash plate altogether 3 times.Add 100 μ l monoclonal antibody linked with peroxidase GS2-D3 working fluids, 37 DEG C, hatch 60min.Remove liquid in hole and wash plate 4 times.Colour developing A liquid and colour developing B liquid mixed in equal amounts are made into substrate nitrite ion.Each micropore adds the substrate nitrite ion of 100 μ l, room temperature lucifuge colour developing 15-20min.Add stop buffer 100 μ l, under 450nm, measure absorbance by microplate reader.
Result judges: negative control≤0.1, positive control >=0.3, and experimental result is effective, otherwise result be judged to be invalid; During detect aperture OD value >=0.2, be judged to be the positive; When detect aperture OD value is between 0.1-0.2, be judged to be the weak positive; Detect aperture OD value≤0.1 is judged to be feminine gender.
If without microplate reader, can with the naked eye judge: Positive control wells has macroscopic yellow, negative control hole is without color, and experimental result is effective, otherwise is judged to be that experimental result is invalid; Be positive findings when detect aperture has obvious macroscopic yellow; When having faint yellow, be judged to be weak positive findings; Without during naked eyes visible yellow color being negative findings.
(2) detection of enzyme linked immunological kit effect
1, kit repeatability and stability test
Precision test in plate: get 6 micropores in same ELISA Plate, test with the milk polluted by large intestine O157:H7, experiment repetition 4 times.
Precision test between plate: 4 pieces of ELISA Plate of getting same batch, tests with the milk polluted by large intestine O157:H7, experiment repetition 3 times.
The computing method of the coefficient of variation: the coefficient of variation (the CV)=standard deviation of measurement result and the number percent of its mean value.
Reperformance test result in table 4. plate
Reperformance test result between table 5. plate
2, kit cross reaction test
Go to detect other 76 kinds of food-borne pathogens with kit, whether checking kit detects the specificity of enterohemorrhagic Escherichia coli O 157: H7, observe and have cross reaction and false positive to occur with other pathogenic bacteria.
Table 6. kit specificity is tested
Note: "+" represents that testing result is positive; "-" represents that testing result is negative.
3, kit storage life experiment
Kit preservation condition is 2-8 DEG C, and after 12 months, the testing result of kit is consistent with the kit results of new lot.Consider in transport and use procedure that having improper preservation condition occurs, placed 8 days under 37 DEG C of conditions of preserving by kit, carry out accelerated aging test, result shows that the indices of kit meets the requirements completely.Therefore kit at least can preserve more than 12 months at 2-8 DEG C.
The preservation of biomaterial
Produce the enterohemorrhagic Escherichia coli O 157 through above-mentioned qualification: the hybridoma cell strain GS2-D3 of H7 monoclonal antibody is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on September 24th, 2012, China, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is anti-Escherichia coli O 157 monoclonal hybridoma, and preserving number is CGMCC No.6610.
Produce the enterohemorrhagic Escherichia coli O 157 through above-mentioned qualification: the hybridoma cell strain 3A8F11B10E7 of H7 monoclonal antibody is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on November 15th, 2013, China, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is antihaemorrhagics Escherichia coli O 157: H7 hybridoma, and preserving number is CGMCC No.8458.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after having read above-mentioned instruction content of the present invention.

Claims (13)

1. a method of vitro detection enterohemorrhagic Escherichia coli O 157: H7, described method is used for non-diseases diagnostic purpose, comprises the following steps:
A testing sample application of sample is caught the solid phase carrier of antibody in being coated with by (), thus making the enterohemorrhagic Escherichia coli O 157 in testing sample: the seizure antibody of H7 on solid phase carrier is combined, and forms the solid phase carrier with " enterohemorrhagic Escherichia coli O 157: H7-catches antibody " binary complex; Described seizure antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody 3A8F11B10E7 of H7, and described monoclonal antibody 3A8F11B10E7 is produced by mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458;
B () will detect the solid phase carrier that antibody application of sample obtains in (a), thus form the solid phase carrier with " detect antibody-enterohemorrhagic Escherichia coli O 157: H7-and catch antibody " ternary complex; Described detection antibody is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody GS2-D3 of H7, described monoclonal antibody GS2-D3 is by mouse hybridoma cell system GS2-D3, CGMCC No.6610 produces, and described monoclonal antibody GS2-D3 carries a detectable; With
C () detects the detectable in ternary complex, thus determine enterohemorrhagic Escherichia coli O 157 in testing sample: whether and the amount existed the existence of H7.
2. the method for claim 1, is characterized in that, described solid phase carrier is enzyme reaction plate.
3. the method for claim 1, is characterized in that, described detectable is horseradish peroxidase.
4. an immunoglobulin (Ig), it is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody of H7, is characterized in that, it is by mouse hybridoma cell system 3A8F11B10E7, and CGMCC No.8458 produced.
5. immunoglobulin (Ig) as claimed in claim 4, it is characterized in that, it is with enterohemorrhagic Escherichia coli O 157: H7, ATCC No.43895 immunity Balb/c mouse obtains.
6. produce a hybridoma for monoclonal antibody, it is characterized in that, it is mouse hybridoma cell system 3A8F11B10E7, CGMCC No.8458.
7. hybridoma as claimed in claim 6, is characterized in that, the monoclonal antibody that it produces can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically: H7.
8. a purposes for immunoglobulin (Ig) as claimed in claim 4, is characterized in that, it is used to detect enterohemorrhagic Escherichia coli O 157: H7, and described purposes is used for non-diseases diagnostic purpose.
9. an immunoglobulin (Ig), it is incorporated into enterohemorrhagic Escherichia coli O 157 specifically: the monoclonal antibody of H7, is characterized in that, it is by mouse hybridoma cell system GS2-D3, and CGMCC No.6610 produced.
10. immunoglobulin (Ig) as claimed in claim 9, it is characterized in that, it is with enterohemorrhagic Escherichia coli O 157: H7, ATCC No.43895 immunity Balb/c mouse obtains.
11. 1 kinds of hybridomas producing monoclonal antibody, it is characterized in that, it is mouse hybridoma cell system GS2-D3, CGMCC No.6610.
12. hybridomas as claimed in claim 11, is characterized in that, the monoclonal antibody that it produces can be incorporated into enterohemorrhagic Escherichia coli O 157 specifically: H7.
The purposes of 13. 1 kinds of immunoglobulin (Ig)s as claimed in claim 9, is characterized in that, it is used to detect enterohemorrhagic Escherichia coli O 157: H7, and described purposes is used for non-diseases diagnostic purpose.
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