CN105223362B - Campylobacter jejuni enzyme-linked immunologic detecting kit - Google Patents
Campylobacter jejuni enzyme-linked immunologic detecting kit Download PDFInfo
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- CN105223362B CN105223362B CN201510572362.2A CN201510572362A CN105223362B CN 105223362 B CN105223362 B CN 105223362B CN 201510572362 A CN201510572362 A CN 201510572362A CN 105223362 B CN105223362 B CN 105223362B
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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
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- G01N33/56922—Campylobacter
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- G—PHYSICS
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- 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
- G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
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Abstract
The invention discloses a kind of enzyme linked immunological kit for detecting campylobacter jejuni.Described test kit contains two strains can specifically be incorporated into the monoclonal antibody of campylobacter jejuni, one strain is monoclonal antibody 3G2D8H3G9 being made exclusively for capture antibodies, CGMCC No.8766, another strain is monoclonal antibody 4C9E1G7H3 as detection antibody, CGMCC No.8457.Testing confirmation in a large number, the test kit of the present invention can detect campylobacter jejuni special, efficiently, but and other 91 kinds of encountered pathogenic antibacterial no cross reactions, be the pathogenetic bacteria detection product that a kind of performance is fabulous.
Description
Technical field
The invention belongs to biotechnology and field of immunology, be specifically related to a kind of enzyme linked immunological examination detecting campylobacter jejuni
Agent box.
Background technology
Campylobacter jejuni (campylobacter jejuni enteritis) is the patients that certainly suffer from diarrhoea such as Butzler in 1973
Feces is separated, has the most recognized one of its Main Pathogenic Bacteria being mankind's diarrhoea.The sickness rate of campylobacter jejuni enteritis
Exceeding bacillary dysentery in developed country, in developing country, sickness rate 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 intestinal, mainly invade jejunum, ileum and
Colon, attacks intestinal mucosa, causes hyperemia and heamorrhagic lesions, observes that some bacterial strain can produce similar cholera intestinal 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
Aspergillosis is detection target, and technology required for protection relates to campylobacter jejuni and quickly detects product.
Summary of the invention
It is an object of the invention to provide a kind of test kit detecting campylobacter jejuni.
And then, the invention provides a kind of enzyme linked immunological kit for detecting campylobacter jejuni, described test kit
Contain:
A () solid phase carrier, described solid phase carrier is coated with the anti-campylobacter jejuni monoclonal anti as capture antibodies
Body 3G2D8H3G9, described anti-campylobacter jejuni monoclonal antibody 3G2D8H3G9 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 4C9E1G7H3 by mouse hybridoma cell system 4C9E1G7H3,
CGMCC No.8457 produces.
In a preference, described solid phase carrier is enzyme reaction plate.
In another preference, described detection antibody is with detectable label.
In another preference, described detectable label is horseradish peroxidase.
In another preference, described test kit is possibly together with positive control and negative control.
In another preference, described positive control is the campylobacter jejuni bacterium solution of inactivation, and described negative control is
The brucella broth of sterilizing.
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 immunogen, and immunity Balb/c mice, isolated and purified obtain
Two strains anti-campylobacter jejuni monoclonal antibody, i.e. 3G2D8H3G9, preserving number CGMCC No.8766 and 4C9E1G7H3, preserving number
CGMCC No.8457, the antibody titer of above-mentioned two strain monoclonal antibodies can reach 1:100000, and it can specificity, efficiently
Be combined with campylobacter jejuni.It is coated enzyme reaction plate as capture antibodies using said monoclonal antibody 3G2D8H3G9, uses Radix Cochleariae officinalis mistake
Said monoclonal antibody 4C9E1G7H3 of oxide enzyme labelling, as detection antibody, makes enzyme-linked immunologic detecting kit.Result
Display, above-mentioned campylobacter jejuni detection kit detection sensitivity reaches 105Cfu/ml, has good excellent of repeatability, accuracy
Point, between hole, error is less than 5%, and in plate, CV is less than 3%.It increases Liszt, hog cholera sramana, mouse typhus sramana, enteritis sand with single
Door, Fu Shi will he, Song's Nei Shi will he, Boydii will he, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestinal pestis
Bacterium etc. amount to 91 kinds of equal no cross reactions of antibacterial.
On this basis, the present inventor and then according to DASELISA immunization, through repeatedly testing, finally obtain
A kind of can quickly, the enzyme linked immunological kit of efficient detection food-borne campylobacter jejuni.
Antibody
The present invention includes two strains anti-campylobacter jejuni monoclonal antibody.The two strains anti-campylobacter jejuni monoclonal anti of the present invention
Body can utilize mouse hybridoma cell system 3G2D8H3G9 (CGMCC No.8766) and mouse hybridoma cell system 4C9E1G7H3
(CGMCC No.8457) secretes generation respectively.
The present invention includes the corresponding aminoacid with anti-campylobacter jejuni monoclonal antibody 3G2D8H3G9 and 4C9E1G7H3
The monoclonal antibody of sequence, and there is other protein of these chains or protein conjugate and fusion expressed product.Specifically
Ground, the present invention includes having the light chain containing hypervariable region (complementary determining region, CDR) and any protein of heavy chain or protein molecule
Thing and fusion expressed product (i.e. immune conjugate and fusion expressed product), if the light chain of this hypervariable region and the present invention and heavy chain
Hypervariable region is identical or at least 90% homology, preferably at least 95% homology.As it is known by the man skilled in the art, immunity is even
Connection thing and fusion expressed product include: medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or
Treat conjugate that is that molecule is combined and that formed with anti-campylobacter jejuni monoclonal antibody or its fragment.Present invention additionally comprises with anti-
Cell surface marker thing that campylobacter jejuni 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 or its fragment.The sequence of these DNA moleculars can be used
Routine techniques, utilizes mouse hybridoma cell system 3G2D8H3G9 (CGMCC No.8766) and 4C9E1G7H3 (CGMCC
No.8457) 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 synthetic 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 chemosynthesis completely
Thing) DNA sequence.Then this DNA sequence 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 chemosynthesis.
The invention still further relates to comprise above-mentioned suitable DNA sequence 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 cell, such as bacterial cell；Or the eukaryotic cell such as low, such as yeast cells；Or it is high
Deng eukaryotic cell, 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 eukaryote, 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, supersound 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 the knot of these methods
Close.
Anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 of the present invention and 3G2D8H3G9, its titer height (can reach
1:100000), it is possible to detect campylobacter jejuni specifically, efficiently, Liszt, hog cholera sramana, mouse typhus sand are increased with single
Door, enteritis sramana, Fu Shi will he, Song's Nei Shi will he, Boydii will he, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, little
Intestinal yersinias etc. amount to 91 kinds of antibacterials (strain) all no cross reactions, and this is the maximum innovative point of the present invention.Above-mentioned monoclonal
Antibody 4C9E1G7H3 can use horseradish peroxidase, alkaline phosphatase, nanogold particle labelling, in specific manner as inspection
Survey antibody uses.Said monoclonal antibody 3G2D8H3G9, in various detections are used, can make as capture antibodies in specific manner
With.
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 capture antibodies capture jejunum
After aspergillosis, then being produced by mouse hybridoma cell system 4C9E1G7H3 (CGMCC No.8457) with detectable label labelling
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, feces, food etc..Preferably, described sample is food.
As used herein, described " capture antibodies ", " 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 capture antibodies 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 capture antibodies 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 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 " specificity " 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 capture antibodies is fixed on carrier, and then capture antibodies is 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 based on a strain
For the test kit that monoclonal antibody, a strain multi-resistance are developed, two strain monoclonal antibodies the kit measurement effect developed is the most excellent, accurately
Advantage is had more in degree, specificity and stability.
Specifically, the enzyme linked immunological kit of the present invention contains:
A () enzyme reaction plate, described enzyme reaction plate is coated with the anti-campylobacter jejuni Dan Ke as capture antibodies
Grand antibody 3G2D8H3G9, described anti-campylobacter jejuni monoclonal antibody 3G2D8H3G9 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 4C9E1G7H3 is by mouse hybridoma cell system 4C9E1G7H3
(CGMCC No.8457) produces.
As the optimal way 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 capture antibodies used at the test kit determining the present invention and/or detection
After antibody, this area can be used to be conventionally used for and detect the various labels that antibodies carries out detecting.The present invention is to institute
The label used has no particular limits, as long as with described detection antibodies, and can 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 specificity anti-detection antibody
Anti antibody 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), glucoseoxidase, 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 optimal way 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
Labelling HRP on antibody, reaction directly add substrate colour developing after terminating 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 substance.
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 test kit of the present invention when detection more convenient, described test 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 mixture, stop buffer, enrichment liquid, diluent.
Additionally, operation instructions also can be comprised in described test 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 test kit of the present invention uses DASELISA immunization.When on enzyme reaction plate pre-coated have anti-
Campylobacter jejuni monoclonal antibody (capture antibodies), after adding sample solution or standard substance, adds with detectable labelling
Another strain anti-campylobacter jejuni monoclonal antibody (detection antibody) of thing such as horseradish peroxidase, exists in sample or standard substance
Campylobacter jejuni will capture antibodies coated with on enzyme reaction plate combine, after detection antibody to be added, can be formed " anti-
Isoantigen enzyme labelled antibody " complex, 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 by microplate reader.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 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, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestinal yersinia etc. amount to
91 kinds of antibacterial (strain) all no cross reactions, this is the maximum innovative point of the present invention.
In a word, the pre-treatment of sample is required low by the test kit of the present invention, and easy and simple to handle, detectable limit is 105Cfu/ml,
Specificity and good stability, and all there is no cross reaction with most of food-borne pathogens.
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: lab guide (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and
Number is calculated by weight.
Unless otherwise defined, the meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words
Justice is identical.Additionally, any method similar or impartial to described content and material all can be applicable in the present invention.Described in literary composition
Preferable implementation only present a demonstration with material and be used.
Embodiment 1. anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 and the preparation of 3G2D8H3G9
One, immunogen and the preparation of positive criteria product
Campylobacter jejuni (CICC No.22937) is seeded on brucella broth, 37 DEG C, 150r/min shaken cultivation 17h, meter
Number, adds 0.3% formalin room temperature and inactivates 1 day.Concentration is extremely to adjust campylobacter jejuni (CICC No.22937) with normal saline
5×109Cfu/ml is as immunogen；Adjusting concentration with normal saline is 108Cfu/ml is as positive control standard substance, Bu Shi meat
Soup is negative control standard substance.
Two, the preparation of monoclonal antibody
1, laboratory animal: select 38 week old, about body weight 20g, female Balb/c mice be laboratory animal.
2, immunization method: every mouse peritoneal injection 0.2ml immunogen, at interval of 2 weeks with same dosage booster injection one
Secondary.
3, blood sampling: take a blood sample from tail vein after 3 booster immunizations, uses indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum
Titer.Treat that titer no longer rises, lumbar injection same amount immunogen, 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 euzymelinked immunosorbent assay (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 amplification culture, it is injected in the Balb/c mouse peritoneal through paraffin oil pretreatment, 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 bacterium of cfu/ml bacterial concentration join in ELISA Plate, and each pathogenic bacterium 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 Plate, every hole 100 μ l.4 DEG C of overnight incubation, close according to conventional ELISA method
Washing.
(2) campylobacter jejuni positive control standard substance and negative control standard substance.
(3) monoclonal antibody 4C9E1G7H3 of the anti-campylobacter jejuni of horseradish peroxidase-labeled.
(4) enzyme labelled antibody diluent: 0.01M PBS, pH 7.6.
(5) 10 × concentration washing liquid: the 0.1M phosphate buffer containing 0.5% tween 20 and 0.2% sodium azide, pH
7.4, during use, concentration washing liquid 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 capture antibodies coated elisa plate
With being coated buffer, 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: 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 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 dehydrated alcohol precipitation enzyme of use 6ml pre-cooling, 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 test kit
(1) detection method
1, sample pre-treatments
Take measuring samples 25g (ml) and add 225ml brucella broth homogenizing 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 test kit
30min is placed under the micropore of taking-up requirement and all reagent room 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 liquids in each micropore, rock the several seconds gently, quickly turn over
Turn by liquid in micropore to the greatest extent, to one fold clean absorbent 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 by microplate reader.
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 microplate reader, 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, test kit repeatability and stability test
Precision test in plate: take 6 micropores in 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 Plate, 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 ratio of its meansigma methods.
Reperformance test result in table 4. plate
Reperformance test result between table 5. plate
2, test kit cross reaction test
Remove to detect other 91 kinds of food-borne pathogens and conventional food Carried bacteria, checking test kit detection sky with test kit
The specificity of intestinal Campylobacter spp, sees whether that the pathogenic bacterium with other have cross reaction and false positive to occur.
Table 6. test kit specificity is tested
Note: "+" represent that testing result is positive；" " represents that testing result is negative.
3, test kit storage life experiment
Test kit preservation condition is 28 DEG C, after 12 months, and the testing result of test kit and the test kit of new lot
Result is consistent.Occur in view of having improper preservation condition during transport and use, by test kit at 37 DEG C of bars preserved
Placing 8 days under part, be accelerated degradation, result shows that the indices of test kit complies fully with requirement.Therefore test 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 parahaemolyticus 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 teachings 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 (6)
1. the enzyme linked immunological kit being used for detecting campylobacter jejuni, it is characterised in that described test kit contains:
A () solid phase carrier, described solid phase carrier is coated with the anti-campylobacter jejuni monoclonal antibody as capture antibodies
3G2D8H3G9, described anti-campylobacter jejuni monoclonal antibody 3G2D8H3G9 by mouse hybridoma cell system 3G2D8H3G9,
CGMCC No.8766 produces；
(b) container a, equipped with anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 as detection antibody in described container a,
Described anti-campylobacter jejuni monoclonal antibody 4C9E1G7H3 is by mouse hybridoma cell system 4C9E1G7H3, CGMCC
No.8457 produces.
2. test kit as claimed in claim 1, it is characterised in that described solid phase carrier is enzyme reaction plate.
3. test kit as claimed in claim 1, it is characterised in that described detection antibody is with detectable label.
4. test kit as claimed in claim 3, it is characterised in that described detectable label is horseradish peroxidase
Enzyme.
5. test kit as claimed in claim 1, it is characterised in that described test kit is right possibly together with positive control and feminine gender
According to.
6. test kit as claimed in claim 5, it is characterised in that described positive control is the campylobacter jejuni bacterium of inactivation
Liquid, described negative control is the brucella broth of sterilizing.
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| EP2579901B1 (en) * | 2010-06-09 | 2019-08-07 | The Board of Trustees of The University of Arkansas | Vaccine and methods to reduce campylobacter infection |
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