CN104360065A - Enzyme linked immunosorbent assay kit for detecting bibrio parahemolyticus - Google Patents

Enzyme linked immunosorbent assay kit for detecting bibrio parahemolyticus Download PDF

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CN104360065A
CN104360065A CN201410609238.4A CN201410609238A CN104360065A CN 104360065 A CN104360065 A CN 104360065A CN 201410609238 A CN201410609238 A CN 201410609238A CN 104360065 A CN104360065 A CN 104360065A
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kit
antibody
vibrio parahemolyticus
monoclonal antibody
3g9e9g3h7
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CN104360065B (en
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刘箐
刘武康
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University of Shanghai for Science and Technology
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    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting bibrio parahemolyticus. The kit contains two monoclonal antibodies which can be in specific binding with bibrio parahemolyticus. One monoclonal antibody is 3G9F7D5C9, CGMCC N0.9010 for specially catching antibodies, and the other monoclonal antibody is 3G9E9G3H7, CGMCC N0.8003 for detecting antibodies. As proved by substantive tests, the kit can efficiently and specially detect bibrio parahemolyticus and does not have cross reaction with other 77 types of common pathogenic bacteria, so that the kit is a product with excellent performance for detecting pathogenic bacteria.

Description

Vibrio parahemolyticus enzyme-linked immunologic detecting kit
Technical field
The invention belongs to biotechnology and field of immunology, be specifically related to a kind of enzyme linked immunological kit detecting vibrio parahemolyticus.
Background technology
Vibrio parahemolyticus (Bibrio parahemolyticus) is also called vibrio parahaemolytious, belongs to vibrio, is a kind of common pathogen.Vibrio parahemolyticus is a kind of Gram-negative bacteria of halophagia, and main habitat in the seawater.If eaten the seafood of round this fungi pollution, food poisoning can be caused; Clinically with Acute onset, stomachache, vomiting, diarrhoea and watery stool for cardinal symptom.
The detection of current vibrio parahemolyticus 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.Immunology detection is quick, accurate, the most stable quick detection means 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 detect target with vibrio parahemolyticus, and technology required for protection relates to the quick testing product of vibrio parahemolyticus.
Summary of the invention
The object of this invention is to provide a kind of kit detecting vibrio parahemolyticus.
And then the invention provides a kind of enzyme linked immunological kit for detecting vibrio parahemolyticus, described kit contains:
(a) solid phase carrier, described solid phase carrier is coated with the anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 as catching antibody, described anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 is produced by mouse hybridoma cell system 3G9F7D5C9, CGMCC No.9010;
(b) container a, anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 as detecting antibody is housed in described container a, described anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 is produced by mouse hybridoma cell system 3G9E9G3H7, CGMCC No.8003.
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 kit is also containing positive control and negative control.
In another preference, described positive control is the vibrio parahemolyticus bacterium liquid of deactivation, and described negative control is 3% sodium chloride basic peptone water of sterilizing.
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
Lane1：3G9F7D5C9，Lane2：3G9E9G3H7。
Embodiment
The research of the present inventor shows, using vibrio parahemolyticus as immunogene, immunity Balb/c mouse, separation and purification obtains the anti-vibrio parahemolyticus monoclonal antibody of two strains, i.e. 3G9F7D5C9, preserving number CGMCC No.9010 and 3G9E9G3H7, preserving number CGMCC No.8003, the antibody titer of above-mentioned two strain monoclonal antibodies can reach 1:100000, its can specificity, be combined with vibrio parahemolyticus efficiently.Using said monoclonal antibody 3G9F7D5C9 bag by enzyme reaction plate as seizure antibody, with the said monoclonal antibody 3G9E9G3H7 of horseradish peroxidase-labeled as detection antibody, make enzyme-linked immunologic detecting kit.Result shows, and above-mentioned vibrio parahemolyticus detection kit detection sensitivity reaches 10 5cfu/ml, have repeatability, advantage that accuracy is good, between hole, error is less than 5%, and in plate, CV is less than 3%.Its with singly increase Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, 77 kinds of equal no cross reactions of pathogenetic bacteria altogether such as bacillus cereus.
On this basis, the present inventor and then according to DASELISA immunization, through repeatedly testing, finally obtain a kind of can fast, the enzyme linked immunological kit of efficient detection food source property vibrio parahemolyticus.
Antibody
The present invention includes the anti-vibrio parahemolyticus monoclonal antibody of two strains.The anti-vibrio parahemolyticus monoclonal antibody of two strain of the present invention can utilize mouse hybridoma cell system 3G9F7D5C9 (CGMCC No.9010) and mouse hybridoma cell system 3G9E9G3H7 (CGMCC No.8003) to secrete generation respectively.
The present invention includes the monoclonal antibody of the corresponding amino acid sequence with anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 and 3G9E9G3H7, 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: that medicine, toxin, cell factor (cytokine), radioactive nuclide, enzyme and other diagnosis or treatment molecule are combined with anti-vibrio parahemolyticus monoclonal antibody or its fragment and the conjugate that formed.The present invention also comprises the cell surface marker thing or antigen that are combined with anti-vibrio parahemolyticus monoclonal antibody or its fragment.
For anti-vibrio parahemolyticus monoclonal antibody heavy of the present invention and sequence of light chain, can measure by conventional method.The hypervariable region of anti-vibrio parahemolyticus monoclonal antibody V chain or complementary determining region (complementarity determining 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 anti-vibrio parahemolyticus 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 mouse hybridoma cell system 3G9F7D5C9 (CGMCC No.9010) and 3G9E9G3H7 (CGMCC No.8003) to 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.
Anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 and 3G9F7D5C9 of the present invention, its height of tiring (can 1:100000 be reached), vibrio parahemolyticus can be detected specifically, efficiently, with singly increase Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc. amount to 77 kinds of equal no cross reactions of pathogenetic bacteria, this is maximum innovative point of the present invention.Said monoclonal antibody 3G9E9G3H7 can use horseradish peroxidase, alkaline phosphatase, nanogold particle to mark, and uses in specific manner as detecting antibody.Said monoclonal antibody 3G9F7D5C9, 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, after the anti-vibrio parahemolyticus monoclonal antibody adopting mouse hybridoma cell system 3G9F7D5C9 (CGMCC No.9010) to produce is as seizure antibody capture vibrio parahemolyticus, the anti-vibrio parahemolyticus monoclonal antibody produced by mouse hybridoma cell system 3G9E9G3H7 (CGMCC No.8003) marked with detectable label is again as detection antibody, extremely effectively can be incorporated into vibrio parahemolyticus, thus detect vibrio parahemolyticus in high sensitivity by double-antibody method.
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 the described monoclonal antibody that can be incorporated into vibrio parahemolyticus specifically, it is produced by mouse hybridoma cell system 3G9F7D5C9, CGMCC No.9010.
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 another strain monoclonal antibody of vibrio parahemolyticus, it is produced by mouse hybridoma cell system 3G9E9G3H7, CGMCC No.8003.
As used herein, described " specificity " refers to that antibody can only be incorporated into vibrio parahemolyticus; More particularly, refer to that those can be combined with vibrio parahemolyticus 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 a kind of enzyme linked immunological kit that can be used for detecting vibrio parahemolyticus 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 anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 as catching antibody, described anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 is produced by mouse hybridoma cell system 3G9F7D5C9, CGMCC No.9010;
(b) container a, anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 as detecting antibody is housed in described container a, described anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 is produced by mouse hybridoma cell system 3G9E9G3H7, CGMCC No.8003.
As optimal way of the present invention, described detection antibody is with detectable label.
As used herein, described " detectable label " refers to that whether and the mark of the amount existed existence for determining vibrio parahemolyticus in detected sample.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 can indicate exactly after appropriate processing the existence of vibrio parahemolyticus in detected sample 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 vibrio parahemolyticus 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 standard items knowing multiple vibrio parahemolyticus of concentration containing oneself can also be set in testing process.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 vibrio parahemolyticus bacterium liquid of deactivation, and described negative control is 3% sodium chloride basic peptone water (APW) 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.Anti-vibrio parahemolyticus monoclonal antibody (seizure antibody) is had when pre-coated on enzyme reaction plate, after adding sample solution or standard items, add with the anti-vibrio parahemolyticus monoclonal antibody of detectable label another strain as horseradish peroxidase (detection antibody) again, the vibrio parahemolyticus existed in sample or standard items 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, yellow substance can be formed after TMB (tetramethyl benzidine) colour developing, thus can in judgement sample vibrio parahemolyticus 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, vibrio parahemolyticus 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%.With singly increase Liszt, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc. amount to 77 kinds of equal no cross reactions of pathogenetic bacteria, 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.
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.
The preparation of the anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 and 3G9E9G3H7 of embodiment 1.
One, the preparation of immunogene and positive criteria product
Vibrio parahemolyticus (ATCC No.17802) is inoculated in 3% sodium chloride basic peptone water (APW), 37 DEG C, 150r/min shaken cultivation 17h, and counting, adds 0.3% formalin room temperature deactivation 1 day.Vibrio parahemolyticus (ATCCNo.17802) concentration to 5 × 10 are adjusted with physiological saline 9cfu/ml is as immunogene; Adjusting concentration with physiological saline is 10 8cfu/ml is as positive control standard items, and 3% sodium chloride basic peptone water (APW) 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
Vibrio parahemolyticus nutrient culture media is as follows: GB: GB/T 4789.7-2008
3% sodium chloride basic peptone water (APW)
Composition: peptone 10.0g, sodium chloride 30.0g, distilled water 1000.0mL
Method for making: mixed by mentioned component, regulates pH8.5 ± 0.2,121 DEG C of autoclaving 10min.
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 (OD 450)
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 3G9F7D5C9 and 3G9E9G3H7.The heavy chain molecule amount of 3G9F7D5C9 is 48kDa, and light chain molecule amount is 26kDa; The heavy chain molecule amount of 3G9E9G3H7 is 48kDa, and light chain molecule amount is 26kDa.
The CHARACTERISTICS IDENTIFICATION of embodiment 2. monoclonal antibody 3G9F7D5C9 and 3G9E9G3H7
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%BSA 200 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, the cross reaction test of monoclonal antibody 3G9F7D5C9 and 3G9E9G3H7
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 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%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.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 the composition of the enzyme linked immunological kit of vibrio parahemolyticus, preparation and application thereof
One, enzyme linked immunological kit is made up of following substances:
(1) ELISA Plate of pre-coated antibody: wrap by 96 hole ELISA Plate with 0.02M acetate buffer solution (pH 2.0) solution dilution, anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9, every hole 100 μ l.4 DEG C of overnight incubation, conveniently ELISA method closes washing.
(2) vibrio parahemolyticus positive control standard items and negative control standard items.
(3) the monoclonal antibody 3G9E9G3H7 of the anti-vibrio parahemolyticus of horseradish peroxidase-labeled.
(4) enzyme labelled antibody dilution: 0.01M PBS, pH 7.6.
(5) 10 × concentrated washing lotion: the 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% Sodium azide, pH 7.4, 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 vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 as seizure antibody coated elisa plate
Be buffered liquid with bag and vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 is diluted to 5 μ g/ml, 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 3G9E9G3H7 of horseradish peroxidase-labeled
Vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 and horseradish peroxidase (HRP) are carried out coupling, and the method for employing is the Over-voltage protection of improvement, and method is as follows:
A, 5mg horseradish peroxidase (HRP) is dissolved in 0.5ml 0.2M acetate buffer solution (pH 5.6).
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, precipitation is dissolved in the 0.01M PBS (pH 7.4) of 2.5ml.
F, add 10mg monoclonal antibody 3G9E9G3H7, and use 0.5M carbonic acid buffer (pH 9.6) 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 (pH 7.4) 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) and add 225ml3% sodium chloride basic peptone water (APW) 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 3G9E9G3H7 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 vibrio parahemolyticus, experiment repetition 4 times.
Precision test between plate: 4 pieces of ELISA Plate of getting same batch, tests with the milk polluted by vibrio parahemolyticus, 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 77 kinds of food-borne pathogens with kit, whether checking kit detects the specificity of vibrio parahemolyticus, 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 response characteristic qualification of embodiment 4. monoclonal antibody 3G9E9G3H7 and 3G9F7D5C9 and vibrio parahemolyticus flagellin
1, vibrio parahemolyticus flagellin extracts:
(1) vibrio parahemolyticus (Vibrio parahemolyticus) (ATCC 17802) is inoculated in 3% sodium chloride basic peptone water (APW), 37 DEG C, 150r/min shaken cultivation 17h, counting, adds 0.3% formalin room temperature deactivation 24h.
(2) secondary daily collection bacterium is centrifugal is resuspended in the 0.15M NaCl solution of precooling, and ice bath 15 ~ 30min, 1000rpm homogenate 45 seconds, is placed in 10min on ice immediately.
(3) the centrifugal 10min of 10000rpm under homogenate 4 DEG C of conditions; Get the centrifugal 2h of 16000rpm under supernatant 4 DEG C of conditions, abandon supernatant, precipitation is resuspended in TET (10mM Tris, 2mM EDTA, pH8.0,1%Triton X-100) damping fluid.Centrifugal process repeats 3 times.Operating process is carried out under keeping low temperature.Finally leave and take precipitation, be dissolved in a small amount of Tris-EDTA (0.1M Tris, 0.1mM EDTA, pH7.8) buffer, 4 DEG C save backup.
2, indirect ELISA measures the binding characteristic of 3G9E9G3H7 and 3G9F7D5C9 and flagellin
(1) flagellin bag quilt: joined by flagellin in ELISA Plate, 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%BSA 200 μ l, hatches 2h for 37 DEG C.
(3) plate is washed 3 times.Every hole adds the monoclonal antibody 3G9E9G3H7 and each 100 μ l of 3G9F7D5C9 that have diluted, 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.
The results are shown in Table 7.Research confirms: 3G9E9G3H7 and 3G9,F7D,5C9 two strain antibody and vibrio parahemolyticus flagellin no cross reaction.
The response characteristic of table 7 monoclonal antibody 3G9E9G3H7 and 3G9F7D5C9 and vibrio parahemolyticus flagellin
The preservation of biomaterial
Produce and be preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) through the hybridoma cell strain 3G9E9G3H7 of the vibrio parahemolyticus monoclonal antibody of above-mentioned qualification on July 16th, 2013, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is " producing the hybridoma cell strain of anti-vibrio parahemolyticus monoclonal antibody ", and preserving number is CGMCC No.8003.
Produce and be preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) through the hybridoma cell strain 3G9F7D5C9 of the vibrio parahemolyticus monoclonal antibody of above-mentioned qualification on April 10th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is " anti-vibrio parahemolyticus (Vi brio parahemolyticus) hybridoma ", and preserving number is CGMCC No.9010.
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 (6)

1. for detecting an enzyme linked immunological kit for vibrio parahemolyticus, it is characterized in that, described kit contains:
(a) solid phase carrier, described solid phase carrier is coated with the anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 as catching antibody, described anti-vibrio parahemolyticus monoclonal antibody 3G9F7D5C9 is produced by mouse hybridoma cell system 3G9F7D5C9, CGMCC No.9010;
(b) container a, anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 as detecting antibody is housed in described container a, described anti-vibrio parahemolyticus monoclonal antibody 3G9E9G3H7 is produced by mouse hybridoma cell system 3G9E9G3H7, CGMCC No.8003.
2. kit as claimed in claim 1, it is characterized in that, described solid phase carrier is enzyme reaction plate.
3. kit as claimed in claim 1, it is characterized in that, described detection antibody is with detectable label.
4. kit as claimed in claim 3, it is characterized in that, described detectable label is horseradish peroxidase.
5. kit as claimed in claim 1, is characterized in that, described kit is also containing positive control and negative control.
6. kit as claimed in claim 5, it is characterized in that, described positive control is the vibrio parahemolyticus bacterium liquid of deactivation, and described negative control is 3% sodium chloride basic peptone water of sterilizing.
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CN105158470A (en) * 2015-06-10 2015-12-16 北京农学院 ELISA kit for detecting Vibrio parahemolyticus in aquatic product
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CN113121682A (en) * 2019-12-30 2021-07-16 中国科学院上海营养与健康研究所 Monoclonal antibody for specifically recognizing vibrio parahaemolyticus O3: K6 and application thereof

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CN105158470A (en) * 2015-06-10 2015-12-16 北京农学院 ELISA kit for detecting Vibrio parahemolyticus in aquatic product
CN105004866A (en) * 2015-07-25 2015-10-28 江苏财经职业技术学院 Kit for rapidly detecting vibrio parahaemolyticus TDH toxin in food and application thereof
CN105004866B (en) * 2015-07-25 2016-08-17 江苏财经职业技术学院 Quickly detect test kit and the application thereof of vibrio parahaemolyticus TDH toxin in food
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CN106841607B (en) * 2017-04-19 2019-02-12 深圳市宝舜泰科技产业股份有限公司 Acute Hepatopancreatic necrosis syndrome dedicated test kit and preparation method thereof
CN110734491A (en) * 2019-11-21 2020-01-31 上海交通大学医学院 Kit for detecting vibrio parahaemolyticus and detection method
CN110734491B (en) * 2019-11-21 2022-03-25 上海交通大学医学院 Kit for detecting vibrio parahaemolyticus and detection method
CN113121682A (en) * 2019-12-30 2021-07-16 中国科学院上海营养与健康研究所 Monoclonal antibody for specifically recognizing vibrio parahaemolyticus O3: K6 and application thereof
CN113121682B (en) * 2019-12-30 2022-08-26 中国科学院上海营养与健康研究所 Monoclonal antibody for specifically recognizing vibrio parahaemolyticus O3: K6 and application thereof

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