CN105223363B - The method of detection campylobacter jejuni and monoclonal antibody thereof - Google Patents
The method of detection campylobacter jejuni and monoclonal antibody thereof Download PDFInfo
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- CN105223363B CN105223363B CN201510573715.0A CN201510573715A CN105223363B CN 105223363 B CN105223363 B CN 105223363B CN 201510573715 A CN201510573715 A CN 201510573715A CN 105223363 B CN105223363 B CN 105223363B
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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
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- 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
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
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Abstract
The invention belongs to microorganism detection field.Specifically, the present invention relates to a kind of detect campylobacter jejuni method, for two strains of the method by the monoclonal antibody of the anti-campylobacter jejuni produced with monoclonal antibody preparation process, this monoclonal antibody can specifically be combined with campylobacter jejuni.It is produced by mouse hybridoma cell system 3G2D8H3G9, CGMCC No.8766 or 4C9E1G7H3, CGMCC No.8457.Present invention also offers the purposes of the monoclonal antibody of anti-campylobacter jejuni.
Description
Technical field
The invention belongs to microorganism detection field.Specifically, the present invention relates to a kind of anti-campylobacter jejuni monoclonal resist
Body, produce the hybridoma of this monoclonal antibody and the purposes of the monoclonal antibody of anti-campylobacter jejuni.
Background technology
Campylobacter jejuni (campylobacter jejuni enteritis) is the patients that certainly suffer from diarrhoea such as Butzler in 1973
Ight soil is separated, has the most recognized one of its Main Pathogenic Bacteria being mankind's diarrhoea.The incidence of disease of campylobacter jejuni enteritis
Exceeding bacillary dysentery in developed country, in developing country, the incidence of disease is nearly identical to bacillary dysentery.This bacterium is as important
Food-borne pathogens, breed rapidly under containing micro amount of oxygen environment along with food enters after enteron aisle, mainly invade jejunum, ileum and
Colon, attacks intestinal mucosa, causes hyperemia and heamorrhagic lesions, observes that some bacterial strain can produce similar cholera intestines poison in recent years
Element, in causing enteric cavity, liquid secretion increases.
The at present detection of campylobacter jejuni depends on the biochemical identification of GB defined, its shortcoming be complex operation,
The detection cycle is longer, it is impossible to adapt to substantial amounts of sample examination.Immunology detection be occur in recent years the most quickly, accurately, stable
Quick detection means, but there is no at present both at home and abroad can operate with detection practice quickly detect product, this patent is curved with jejunum
Aspergillus is detection target, and technology required for protection relates to campylobacter jejuni and quickly detects product.
Summary of the invention
A kind of method that it is an object of the invention to provide vitro detection vibrio parahemolyticus.
It is a further object to provide two strains for said method by with a monoclonal antibody preparation process
Produce may specifically bind in the monoclonal antibody of vibrio parahemolyticus difference antigen site.
It is a further object to provide the hybridoma producing said monoclonal antibody.
A first aspect of the present invention there is provided a kind of method of vitro detection campylobacter jejuni, and described method is used for non-disease
Sick diagnostic purpose, comprises the following steps:
A testing sample is loaded onto and is coated with the solid phase carrier catching antibody, so that the jejunum in testing sample is curved by ()
Aspergillus seizure antibody on solid phase carrier is combined, and forms the solid phase with " campylobacter jejuni-seizure antibody " binary complex
Carrier；Described seizure antibody is monoclonal antibody 3G2D8H3G9 being specifically incorporated into campylobacter jejuni, described Dan Ke
Grand antibody 3G2D8H3G9 is produced by mouse hybridoma cell system 3G2D8H3G9, CGMCC No.8766；
B detection antibody is loaded onto the solid phase carrier that (a) obtains by (), thus formed with " detection antibody-jejunum campylobacter
Bacterium-seizure antibody " solid phase carrier of ternary complex；Described detection antibody is the list being specifically incorporated into campylobacter jejuni
Clonal antibody 4C9E1G7H3, described monoclonal antibody 4C9E1G7H3 is by mouse hybridoma cell system 4C9E1G7H3, CGMCC
No.8457 produces, and described monoclonal antibody 4C9E1G7H3 carries a detectable；With
Detectable in (c) detection ternary complex, so that it is determined that the existence of testing sample jejuni
Whether and the amount that exists.
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 specifically to be incorporated into campylobacter jejuni
Monoclonal antibody, it is produced by mouse hybridoma cell system 3G2D8H3G9, CGMCC No.8766.
A third aspect of the present invention there is provided a kind of hybridoma producing monoclonal antibody, and it is Mouse Hybridoma Cells
Clone 3G2D8H3G9, CGMCC No.8766.
A fourth aspect of the present invention there is provided the purposes of above-mentioned immunoglobulin (Ig), and it is used for detecting campylobacter jejuni,
Described purposes is used for non-diseases diagnostic purpose.
A fifth aspect of the present invention there is provided a kind of immunoglobulin (Ig), and it is specifically to be incorporated into campylobacter jejuni
Monoclonal antibody, it is produced by mouse hybridoma cell system 4C9E1G7H3, CGMCC No.8457.
A sixth aspect of the present invention there is provided a kind of hybridoma producing monoclonal antibody, and it is Mouse Hybridoma Cells
Clone 4C9E1G7H3, CGMCC No.8457.
Present invention also offers the purposes of above-mentioned immunoglobulin (Ig), it is used for detecting campylobacter jejuni, and described purposes is used
In non-diseases diagnostic purpose.
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By hereafter and claim
Description, the feature of the present invention, purpose and advantage will become apparent from.
Accompanying drawing explanation
The SDS-PAGE of the monoclonal antibody of Fig. 1 present invention
Lane1:4C9E1G7H3, Lane3:3G2D8H3G9.
Detailed description of the invention
The research of the present inventor shows, using campylobacter jejuni as immunogene, and immunity Balb/c mouse, isolated and purified obtain
Anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 and 3G2D8H3G9, its antibody titer can reach 1:100000, and it can be special
The opposite sex, is combined with campylobacter jejuni efficiently, with single increasing Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi
91 kinds of bacteriums (strain) such as will he, Song's Nei Shi will he, Boydii will he, large intestine O157:H7, small intestine Yersinia ruckeri are all anti-without intersecting
Should.
The invention provides anti-campylobacter jejuni monoclonal antibody.The anti-campylobacter jejuni monoclonal antibody of the present invention is permissible
Mouse hybridoma cell system 4C9E1G7H3 (CGMCC No.8457) or 3G2D8H3G9 (CGMCC No.8766) secretion is utilized to produce
Raw.The present invention includes having the corresponding amino acid sequence of anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 and 3G2D8H3G9
Monoclonal antibody, and there is other protein of these chains or protein conjugate and fusion expressed product.Specifically, this
Bright including has the light chain containing hypervariable region (complementary determining region, CDR) and any protein of heavy chain or protein conjugate and melts
Close expression product (i.e. immune conjugate and fusion expressed product), if the light chain of this hypervariable region and the present invention and the hypermutation of heavy chain
District is identical or at least 90% homology, preferably at least 95% homology.As it is known by the man skilled in the art, immune conjugate and
Fusion expressed product includes: medicine, toxin, cell factor (cytokine), radionuclide, enzyme and other diagnosis or treatment point
That sub and anti-campylobacter jejuni monoclonal antibody or its fragment is combined and the conjugate that formed.Present invention additionally comprises curved with anti-jejunum
Cell surface marker thing that aspergillus monoclonal antibody or its fragment combine or antigen.
For anti-campylobacter jejuni monoclonal antibody heavy and the sequence of light chain of the present invention, can measure by conventional method.
The hypervariable region of anti-campylobacter jejuni monoclonal antibody V chain or complementary determining region (complementarity determining
Region, CDR) particularly interesting, because they at least partly relate to conjugated antigen.Therefore, the present invention includes those
There is light chain immunoglobulin and the molecule of weight chain variable chain of band CDR, if its CDR and anti-campylobacter jejuni monoclonal antibody
CDR has the homology of more than 90% (preferably more than 95%).The present invention not only includes complete monoclonal antibody, also includes
There is immunocompetent antibody fragment, such 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 be used
Routine techniques, utilizes hybridoma cell line 4C9E1G7H3 (CGMCC No.8457) or 3G2D8H3G9 (CGMCC No.8766) to obtain
?.Additionally, also the coded sequence of light chain and heavy chain can be merged, form single-chain antibody.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then proceeds to cell, then by conventional method relevant sequence of isolated from the host cell after propagation.
Additionally, can also be used with the method for Prof. Du Yucang to synthesize relevant sequence, when especially fragment length is shorter.Generally, logical
Synthesize multiple small fragment after first, be attached the most again obtaining the fragment that sequence is the longest.
At present, it is already possible to obtain code book invention albumen (or its fragment, or it derives by chemical synthesis completely
Thing) DNA sequence dna.Then this DNA sequence dna can be introduced various existing DNA molecular (or such as carrier) that in this area, oneself knows and
In cell.Additionally, sudden change is introduced in protein sequence of the present invention also by chemical synthesis.
The invention still further relates to comprise above-mentioned suitable DNA sequence dna and suitable promoter or control the carrier of sequence.This
A little carriers may be used for converting suitable host cell, allows it to marking protein.
Host cell can be prokaryotic, such as bacterial cell；Or the eukaryotic such as low, such as yeast cells；Or it is high
Deng eukaryotic, such 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 is former
When core biology is such as Enterohemorrhagic E.coli, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaC12Method
Processing, step used is generally well-known in the art.Another kind of method is to use MgC12.Also can electricity consumption wear if it is required, convert
The method in hole is carried out.When host is eucaryote, following DNA transfection method can be used: calcium phosphate precipitation, conventional mechanical
Method such as microinjection, electroporation, liposome packaging etc..
The transformant obtained can be cultivated by conventional method, expresses the polypeptide of the coded by said gene of the present invention.According to used
Host cell, culture medium used in cultivation is selected from various conventional medium.Under conditions of being suitable to host cell growth
Cultivate.When after host cell growth to suitable cell density, with suitable method (such as temperature transition or chemical induction)
The promoter that induction selects, is further cultured for a period of time by cell.
Recombinant polypeptide in the above methods can intracellular or on cell membrane express or be secreted into extracellular.As
Fruit needs, and can utilize its physics, chemical being separated and the albumen of purification of Recombinant with other characteristic by various separation methods.This
A little methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, uses
Protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, ultrasonically treated, ultracentrifugation, sieve chromatography (gel filtration),
Adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various LC technology and the knot of these methods
Close.
The anti-campylobacter jejuni monoclonal antibody of the present invention, its titer is high (can reach 1:100000), it is possible to specific
Ground, detect campylobacter jejuni efficiently, congratulate with single Liszt of increasing, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will,
91 kinds of bacteriums (strain) such as Song's Nei Shi will he, Boydii will he, large intestine O157:H7, small intestine Yersinia ruckeri all no cross reactions, this
Maximum innovative point for the present invention.Said monoclonal antibody 4C9E1G7H3 can use horseradish peroxidase, alkaline phosphate ester
Enzyme, nanogold particle mark, and use as detection antibody in specific manner.Said monoclonal antibody 3G2D8H3G9 is in various detections
In utilization, can use as seizure antibody in specific manner.
Detection kit
The present inventor is through studying widely and testing, it has unexpectedly been found that, when using mouse hybridoma cell system
Anti-campylobacter jejuni monoclonal antibody produced by 3G2D8H3G9 (CGMCC No.8766) is curved as catching antibody capture jejunum
After aspergillus, then being produced by mouse hybridoma cell system 4C9E1G7H3 (CGMCC No.8457) with detectable label mark
Raw anti-campylobacter jejuni monoclonal antibody, as detection antibody, can extremely efficient be incorporated into campylobacter jejuni, thus logical
Cross double-antibody method and detect campylobacter jejuni in high sensitivity.
As used herein, described " sample " refers to the materials such as food, human and animal excreta, vomitus, includes but not limited to: blood
Clearly, the enrichment liquid of blood plasma, ight soil, food etc..Preferably, described sample is food.
As used herein, described " seizure antibody ", " coated antibody ", " first antibody " was used interchangeably with " one resists ",
May specifically bind the monoclonal antibody in campylobacter jejuni all referring to described, it is by mouse hybridoma cell system
3G2D8H3G9, CGMCC No.8766 produces.
Described seizure antibody can be coated on solid phase carrier.The present invention solid phase carrier to being used is the most particularly
Limit, as long as it can be with seizure antibody phase coupling (connection).Such as, described solid phase carrier is enzyme reaction plate.
As used herein, described " detection antibody ", " SA ", " enzyme labelled antibody " was used interchangeably with " two resist ",
All referring to specifically binding to another strain monoclonal antibody of campylobacter jejuni, it is by mouse hybridoma cell system
4C9E1G7H3, CGMCC No.8457 produces.
As used herein, described " specifically " refers to that antibody can only be incorporated into campylobacter jejuni；More particularly, those are referred to
Can be combined with campylobacter jejuni but nonrecognition and be incorporated into the antibody of other non related antigen molecule.
The present inventor and then the principle according to double-antibody method, be prepared for one and can be used for detecting sample jejuni
Enzyme linked immunological kit.The way of double-antibody method routine is that seizure antibody is fixed on carrier, then catches antibody with anti-
Former reaction, after washing, (described detection antibody carries detectable, or can be able to detect with carrying with detection antibody response again
The material of label combines), finally carry out chemiluminescence or enzyme connection chromogenic reaction detection signal.Further, relative to monoclonal antibody body
For competition law, the mensuration effect of double antibody sandwich method is the most excellent, thus only needs little sample size when measuring.So using
No matter double antibody sandwich method has more advantage in sensitivity, accuracy, the degree of accuracy, specific and stability.
Specifically, the enzyme linked immunological kit of the present invention contains:
A () enzyme reaction plate, described enzyme reaction plate is coated with as the anti-campylobacter jejuni Dan Ke catching antibody
Grand antibody 3G2D8H3G9, described anti-campylobacter jejuni monoclonal antibody is by mouse hybridoma cell system 3G2D8H3G9, CGMCC
No.8766 produces；
B () container a, equipped with the anti-campylobacter jejuni monoclonal antibody as detection antibody in described container a
4C9E1G7H3, described anti-campylobacter jejuni monoclonal antibody is by mouse hybridoma cell system 4C9E1G7H3, CGMCC
No.8457 produces.
As the preferred embodiment of the present invention, described detection antibody is with detectable label.
As used herein, described " detectable label " refers to for determining detected sample jejuni
The mark of the amount of presence or absence and existence.The seizure antibody used at the kit determining the present invention and/or detection
After antibody, this area can be used to be conventionally used for and detect antibody and be combined the various labels carrying out detecting.The present invention is to institute
The label used has no particular limits, as long as can be combined with described detection antibody, and after appropriate processing can
The presence or absence of instruction detected sample jejuni and the label of amount are all available exactly.Described
Label can directly be arranged on detection antibody；Or, described label also may be placed at specific anti-detection antibody
Antiantibody on, those skilled in the art can select suitable label according to the kind of the antibody used and characteristic.Such as, institute
The label stated can be selected from: horseradish peroxidase (HRP), alkaline phosphatase vinegar enzyme (AP), glucose oxidase, β-D-gala
Glycosidase, urase, catalase or glucoamylase.
When using some enzyme marker as implied above, in addition it is also necessary to use some substrates combined with corresponding enzyme, from
And existence situation or the amount of label can be reported by modes such as colour developings.As used herein, described " with label
Corresponding substrate " refer to be labeled thing institute catalyzed coloration, for showing that detection antibody occurs combination with campylobacter jejuni
Identify signal.Described substrate is such as: for the adjacent benzene two limb (OPD) of horseradish peroxidase, tetramethyl biphenyl limb (TMB),
ABTS；P-nitrophenyl phosphoric acid vinegar (p-nitro phenyl phosphate, p-NPP) for alkaline phosphatase etc..This
Field personnel can select suitable substrate according to the kind of the label used and characteristic.
As the preferred embodiment of the present invention, described detection antibody is joined directly together with label and connects.It is highly preferred that it is described
Label is HRP.Compared with reacting with streptavidin HRP again with after detecting antibody, reaction with biotin labeling, directly in detection
On antibody mark HRP, reaction terminate after directly add substrate colour developing the most simple and convenient.
In order to obtain quantitative result, it is also possible to arrange during detection and know the mark of multiple campylobacter jejunis of concentration containing oneself
Quasi-product.The method that can use routine for the method to set up of standard items.
In order to eliminate false positive and false negative, it is possible to arrange Quality Control (comparison) during detection.Excellent as the present invention
The campylobacter jejuni bacterium solution selecting mode, described positive control to be inactivation, described negative control is the brucella broth of sterilizing
(Brucella broth)。
Additionally, in order to make the kit of the present invention when detection more convenient, described kit preferably also comprises it
Its some auxiliary reagent, described auxiliary reagent is more conventional use of reagent, the characteristic of these reagent in ELISA kit
And their compound method is all well-known to those skilled in the art.Described reagent is such as (but not limited to): developer,
Cleaning solution, stop buffer, enrichment liquid, dilution.
Additionally, operation instructions also can be comprised in described kit, for the user of the reagent that explanation wherein loads
Method.
The Cleaning Principle of the enzyme linked immunological kit of the present invention and having the beneficial effect that:
The kit of the present invention uses DASELISA immunization.When on enzyme reaction plate pre-coated have anti-
Campylobacter jejuni monoclonal antibody (seizure antibody), after adding sample solution or standard items, adds with detectable mark
Another strain anti-campylobacter jejuni monoclonal antibody (detection antibody) of thing such as horseradish peroxidase, exists in sample or standard items
Campylobacter jejuni will combine by seizure antibody coated with on enzyme reaction plate, after detection antibody to be added, can be formed " anti-
Isoantigen enzyme labelled antibody " compound, yellow substance can be formed after TMB (tetramethyl benzidine) develops the color, thus can sentence
The presence or absence of disconnected sample jejuni and the amount of existence.
Under 450nm, absorbance is measured with ELIASA.Negative control≤0.1, positive control >=0.3, experimental result has
Effect, otherwise result be judged to invalid；During detection OD value >=0.2, hole, it is determined that for the positive；Detection hole OD value is between 0.1-0.2
Time, it is determined that for the weak positive；Detection OD value≤0.1, hole is judged to feminine gender.
Test shows, the campylobacter jejuni enzyme linked immunological kit of the present invention, has higher sensitivity and the degree of accuracy.Between plate
Error is less than 5%, and in plate, CV is less than 3%.Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is increased with single
He, Song's Nei Shi will he, Boydii will he, Escherichia coli O 157: H7, small intestine Yersinia ruckeri etc. amount to 91 kinds of pathogenetic bacterias all without handing over
Fork reaction, this is the maximum innovative point of the present invention.
In a word, the pre-treatment of sample is required low by the kit of the present invention, and easy and simple to handle, detectable limit is 105Cfu/ml,
Specific and good stability, and all there is no cross reaction with most of food-borne pathogens.
Detection method
A kind of method that present invention also offers kit vitro detection campylobacter jejuni utilizing the present invention, including following
Step:
A testing sample is loaded onto and is coated with the solid phase carrier catching antibody, so that the jejunum in testing sample is curved by ()
Aspergillus seizure antibody on solid phase carrier is combined, and forms the solid phase with " campylobacter jejuni-seizure antibody " binary complex
Carrier；Described seizure antibody is monoclonal antibody 3G2D8H3G9 being specifically incorporated into campylobacter jejuni, described Dan Ke
Grand antibody 3G2D8H3G9 is produced by mouse hybridoma cell system 3G2D8H3G9, CGMCC No.8766；
B detection antibody is loaded onto the solid phase carrier that (a) obtains by (), thus formed with " detection antibody-jejunum campylobacter
Bacterium-seizure antibody " solid phase carrier of ternary complex；Described detection antibody is the list being specifically incorporated into campylobacter jejuni
Clonal antibody 4C9E1G7H3, described monoclonal antibody 4C9E1G7H3 is by mouse hybridoma cell system 4C9E1G7H3, CGMCC
No.8457 produces, and described monoclonal antibody 4C9E1G7H3 carries a detectable；With
Detectable in (c) detection ternary complex, so that it is determined that the depositing of detected sample jejuni
Whether and the amount that exists.
As a kind of preferred embodiment of the present invention, quantitatively the method for detection campylobacter jejuni is specific as follows:
() antigen-antibody reaction: the seizure antibody of the present invention is coated on porous plate, afterwards in the micropore of porous plate
It is separately added into the standard items of variable concentrations, quality-control product (optional), or testing sample；
() integrated enzyme reaction: detection antibody (being provided with label) solution is added each hole, vibrates, hatch, wash；
() chromogenic reaction: every hole adds the substrate corresponding to label, developer, hatches, and every hole adds reaction terminating
Liquid, terminates to react:
() ELIASA mensuration OD value:
() result calculates:
A) making calibration curve: with campylobacter jejuni standard concentration as abscissa, it is ordinate that standard items measure OD value,
Make calibration curve；
B) quality-control product concentration (optional) is passed judgment on: according to the OD value of quality-control product, from calibration curve, read corresponding concentration value；
When quality-control product mensuration concentration value is in given range, this time measures effectively；
C) testing sample concentration is calculated: when calibration curve and quality-control product are all determined effective, according to the OD of sample to be tested
Value calculates the jejunum campylobacter bacteria concentration of testing sample from calibration curve.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1. anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 and the preparation of 3G2D8H3G9
One, immunogene and the preparation of positive criteria product
Campylobacter jejuni (CICC No.22937) is seeded on brucella broth, 37 DEG C, 150r/min vibration anaerobic condition training
Support 17h, counting, add 0.3% formalin room temperature and inactivate 1 day.Campylobacter jejuni (CICC is adjusted with physiological saline
No.22937) concentration is to 5 × 109Cfu/ml is as immunogene；Adjusting concentration with physiological saline is 108Cfu/ml is right as the positive
According to standard items, brucella broth is negative control standard items.
Two, the preparation of monoclonal antibody
1, animal used as test: select 38 week old, about body weight 20g, female Balb/c mouse be animal used as test.
2, immunization method: every mouse peritoneal injection 0.2ml immunogene, at interval of 2 weeks with same dosage booster shots one
Secondary.
3, blood sampling: take a blood sample from tail vein after 3 booster immunizations, uses indirect non-competing ELISA to measure antiserum
Titer.Treat that titer no longer rises, lumbar injection same amount immunogene, conventionally carry out cell fusion after 3 days.
4, cell merges: takes immune mouse spleen cell and melts with SP2/0 myeloma cell's routine under 50%PEG effect
Close, be inoculated in 96 well culture plates respectively, be placed in 37 DEG C, 5%CO2Incubator is cultivated.
5, filtering hybridoma: use indirect non-competing ELISA, screens the hybridoma in strong positive hole, will
It is transferred to 24 well culture plates.
6, clone cultivates and prepared by antibody: carry out colonized culture with limiting dilution assay.When cell grows to be paved with at the bottom of hole
When 1/10, then detect with same method, strong positive hole is cloned, 3-4 time the most repeatedly, until positive rate reaches 100% again.
By hybridoma expand cultivate, be injected in through paraffin oil pretreatment Balb/c mouse peritoneal, every 2 × 106Individual hybridoma
Cell, 7～10 days mouse web portion protuberances, living body puncture extraction ascites.
Three, monoclonal antibody-purified and identify
After ascites first slightly carries with caprylic acid-ammonium, then purify with Protein G Sepharose affinity chromatography；Pure
Antibody after change measures titer (being shown in Table 1) with indirect elisa method after doubling dilution；Using SDS-PAGE purity assay again, 5% amasss
Layer glue, 10% separation gel, 120V voltage, bottom electrophoresis to glue, gel Coomassie Brilliant Blue dyes, and after decolouring, gel imaging divides
Analysis system observed result (see Fig. 1).
1. liang of strain antibody titer measurement result (OD of table450)
Embodiment 2. monoclonal antibody 4C9E1G7H3 and the CHARACTERISTICS IDENTIFICATION of 3G2D8H3G9
One, monoclonal antibody subgroup identification
1, antigen coated: being coated mountain sheep anti mouse two anti-igg+A+M with 0.01M PBS, every hole 50 μ l, 4 DEG C are coated overnight, secondary
Day discards liquid in hole, washes plate 3 times.
2, close: every hole adds 1%BSA 200 μ l, closes overnight for 4 DEG C.Pat dry plank next day and do not wash plate.
3, monoclonal antibody hybridoma cell supernatant, 8 micropores of each sample, every hole 50 μ l are added.37 DEG C, hatch 1h.
4, after washing plate 4 times, it is separately added into rabbit anti-mouse igg 1, IgG2a, IgG2b, IgG3, IgA, IgM, the κ of specific bond,
λ, hatches 1h for 37 DEG C.
5, after washing plate 4 times, every hole adds the horseradish peroxidase-labeled anti-rabbit two anti-igg (H+L) diluted, and 37
DEG C, hatch 30min.
6, after washing plate 4 times, 100 μ l substrate nitrite ions are added, 37 DEG C, lucifuge colour developing 10min.Read under 450nm wavelength
Result.
As shown in table 2, H3 belongs to IgG2a subclass, and G9 belongs to IgG1 subclass.
2. liang of strain monoclonal antibody subgroup identification results of table
Two, monoclonal antibody cross reaction test
1, antigen coated: by 96 kind 108The pathogenic bacteria of cfu/ml bacterial concentration join in ELISA Plate, and each pathogenic bacteria add
3 each holes, every hole 100 μ l, 4 DEG C, it is coated overnight.
2, closing: after washing plate 3 times, every hole adds 3%BSA 200 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.Add the mountain sheep anti mouse two diluted and resist in all micropores, every hole 100 μ l, incubate for 37 degrees Celsius
Educate 1h.
5, plate is washed 4 times.Addition substrate nitrite ion, 37 DEG C, lucifuge colour developing 15min.Result is read under 450nm wavelength.
3. liang of strain monoclonal antibody cross reaction testing results of table
Embodiment 3. detects the composition of the enzyme linked immunological kit of campylobacter jejuni, prepares and apply
One, enzyme linked immunological kit is made up of following substances
(1) ELISA Plate of pre-coated antibody: with the dilution of 0.02M acetate buffer solution (pH 2.0) solution, anti-campylobacter jejuni
Monoclonal antibody 3G2D8H3G9 is coated 96 hole ELISA Plates, every hole 100 μ l.4 DEG C of overnight incubation, close according to conventional ELISA method
Washing.
(2) campylobacter jejuni positive control standard items and negative control standard items.
(3) monoclonal antibody 4C9E1G7H3 of the anti-campylobacter jejuni of horseradish peroxidase-labeled.
(4) enzyme labelled antibody dilution: 0.01M PBS, pH 7.6.
(5) 10 × concentration washing lotion: the 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% Sodium azide, pH
7.4, during use, concentration washing lotion 10 times is diluted.
(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 campylobacter jejuni monoclonal antibody 3G2D8H3G9 as catching antibody coated elisa plate
With being coated buffer solution, campylobacter jejuni monoclonal antibody 3G2D8H3G9 being diluted to 5 μ g/ml, every hole adds 100 μ
L, 4 DEG C overnight, inclines next day and is coated liquid, with the wash liquid diluted 3 times, pats dry, and then adds 220 μ l in every hole and closes
Liquid, 37 DEG C of incubation 2h, liquid in hole of inclining, seal with aluminium foil bag after drying and preserve.
It is coated buffer solution: 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 monoclonal antibody 4C9E1G7H3 of horseradish peroxidase-labeled
Campylobacter jejuni monoclonal antibody 4C9E1G7H3 and horseradish peroxidase (HRP) are carried out coupling, the side of employing
Method is the Over-voltage protection of improvement, and method is as follows:
A, 5mg horseradish peroxidase (HRP) is dissolved in 0.5ml 0.2M acetate buffer solution (pH 5.6).
B, the 0.06M NaIO of the existing preparation of addition4Solution 0.5ml, 4 DEG C of oxidation 20min.
C, the addition 0.4M ethylene glycol solution 0.5ml containing 22%NaCl, room temperature stands 30min.
D, the absolute ethyl alcohol precipitation enzyme of use 6ml precooling, 1500rpm is centrifuged 10min.
E, remove supernatant, precipitation is dissolved in the 0.01M PBS (pH 7.4) of 2.5ml.
F, addition 10mg monoclonal antibody 4C9E1G7H3, and immediately with 0.5M carbonic acid buffer (pH 9.6) regulation pH extremely
9.0.4 DEG C stand overnight.
G, addition 10mg/ml sodium borohydride 50 μ l, stand 2h by 4 DEG C.
H, use 0.01M PBS (pH 7.4) 4 DEG C of dialysed overnight.
I, purification storage.
Three, the application of kit
(1) detection method
1, sample pre-treatments
Take measuring samples 25g (ml) and add 225ml brucella broth homogeneous mixing, 36 DEG C ± 1 DEG C cultivation under homogenizer
16h.Cultured sample heats in 100 DEG C of water-baths 10min, and 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.Take 200 μ l measuring samples and be added to micropore
In, 37 DEG C, hatch 30min.Remove liquid in hole, add 200 μ l washing lotions in each micropore, rock the several seconds gently, quickly turn over
Turn by liquid in micropore to the greatest extent, to one fold clean blotting paper clap several under, repeat operation and wash plate altogether 3 times.Add 100 μ l enzymes
Mark monoclonal antibody 4C9E1G7H3 working solution, hatches 60min by 37 DEG C.Remove in hole liquid and wash plate 4 times.Will colour developing A liquid and
Colour developing B liquid mixed in equal amounts is 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 with ELIASA.
Result judges: negative control≤0.1, positive control >=0.3, and experimental result is effective, otherwise result be judged to invalid；
During detection OD value >=0.2, hole, it is determined that for the positive；When detection hole OD value is between 0.1-0.2, it is determined that for the weak positive；Detection hole
OD value≤0.1 is judged to feminine gender.
If without ELIASA, can with the naked eye judge: Positive control wells has macroscopic yellow, negative control hole without color,
Experimental result is effective, is otherwise judged to that experimental result is invalid；It is positive findings when detecting hole and having obvious macroscopic yellow；
When having faint yellow, it is determined that for weak positive findings；It is negative findings when being visible by naked eyes yellow.
(2) detection of enzyme linked immunological kit effect
1, kit repeatability and stability test
Precision test in plate: take 6 micropores on same ELISA Plate, enter with the milk polluted by campylobacter jejuni
Row test, experiment is repeated 4 times.
Precision test between plate: take with a batch of 4 pieces of ELISA Plates, survey with the milk polluted by campylobacter jejuni
Examination, experiment is repeated 3 times.
The computational methods of the coefficient of variation: the standard deviation of the coefficient of variation (CV)=measurement result and the percentage of its mean value.
Reperformance test result in table 4. plate
Reperformance test result between table 5. plate
2, kit cross reaction test
Remove to detect other 91 kinds of food-borne pathogens and conventional food Carried bacteria, checking kit detection sky with kit
Intestines Campylobacter spp specific, sees whether that the pathogenic bacteria with other have cross reaction and false positive to occur.
Table 6. kit is specifically tested
Note: "+" represent that testing result is positive；" " represents that testing result is negative.
3, kit storage life experiment
Kit preservation condition is 28 DEG C, after 12 months, and the testing result of kit and the kit of new lot
Result is consistent.Occur in view of having improper preservation condition during transport and use, by kit at 37 DEG C of bars preserved
Placing 8 days under part, be accelerated degradation, result shows that the indices of kit complies fully with requirement.Therefore kit
At least can be able to preserve more than 12 months at 28 DEG C.
The preservation of biomaterial
Produce the hybridoma cell strain 3G2D8H3G9 of campylobacter jejuni monoclonal antibody through above-mentioned qualification in 2014
It was preserved in (north, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on January 9
Occasion West Road 1 institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is " anti-jejunum campylobacter
Bacterium hybridoma ", preserving number is CGMCC No.8766.
Produce the hybridoma cell strain 4C9E1G7H3 of vibrio parahemolyticus monoclonal antibody through above-mentioned qualification in 2013
On November 15, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and (Beijing is exposed to the sun
North Star West Road, district 1 institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is " anti-jejunum
Campylobacter spp hybridoma ", preserving number is CGMCC No.8457.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
Claims (9)
1. a method for vitro detection campylobacter jejuni, described method is used for non-diseases diagnostic purpose, comprises the following steps:
A testing sample is loaded onto and is coated with the solid phase carrier catching antibody by (), so that the campylobacter jejuni in testing sample
Seizure antibody on solid phase carrier is combined, and forms the solid phase carrier with " campylobacter jejuni-seizure antibody " binary complex；
Described seizure antibody is monoclonal antibody 3G2D8H3G9 being specifically incorporated into campylobacter jejuni, and described monoclonal resists
Body 3G2D8H3G9 is produced by mouse hybridoma cell system 3G2D8H3G9, CGMCC No.8766；
B detection antibody is loaded onto the solid phase carrier that (a) obtains by (), thus formed with " detection antibody-campylobacter jejuni-catch
Catch antibody " solid phase carrier of ternary complex；Described detection antibody is the monoclonal being specifically incorporated into campylobacter jejuni
Antibody 4C9E1G7H3, described monoclonal antibody 4C9E1G7H3 is by mouse hybridoma cell system 4C9E1G7H3, CGMCC
No.8457 produces, and described monoclonal antibody 4C9E1G7H3 carries a detectable；With
Detectable in (c) detection ternary complex, so that it is determined that the presence or absence of testing sample jejuni
And the amount existed.
2. the method for claim 1, it is characterised in that described solid phase carrier is enzyme reaction plate.
3. the method for claim 1, it is characterised in that described detectable is horseradish peroxidase.
4. the monoclonal antibody being specifically incorporated into campylobacter jejuni, it is characterised in that it is by mouse hybridoma cell
Being 3G2D8H3G9, CGMCC No.8766 is produced.
5. the hybridoma producing monoclonal antibody, it is characterised in that it is mouse hybridoma cell system
3G2D8H3G9, CGMCC No.8766.
6. the purposes of a monoclonal antibody as claimed in claim 4, it is characterised in that it is used for detecting jejunum campylobacter
Bacterium, described purposes is used for non-diseases diagnostic purpose.
7. the monoclonal antibody being specifically incorporated into campylobacter jejuni, it is characterised in that it is by mouse hybridoma cell
Being 4C9E1G7H3, CGMCC No.8457 is produced.
8. the hybridoma producing monoclonal antibody, it is characterised in that it is mouse hybridoma cell system
4C9E1G7H3, CGMCC No.8457.
9. the purposes of a monoclonal antibody as claimed in claim 7, it is characterised in that it is used for detecting jejunum campylobacter
Bacterium, described purposes is used for non-diseases diagnostic purpose.
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