CN102659942A - Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit - Google Patents
Vibrio parahaemolyticus flagellin monoclonal antibody and antigen capture ELISA (enzyme-linked immunosorbent assay) kit Download PDFInfo
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Abstract
The invention discloses a vibrio parahaemolyticus flagellin monoclonal antibody and an antigen capture ELISA (enzyme-linked immunosorbent assay) kit. The vibrio parahaemolyticus flagellin monoclonal antibody is produced by secreting of a hybridoma cell strain with the preservation number of CGMCC (China General Microbiological Culture Collection) No.6061. The vibrio parahaemolyticus flagellin monoclonal antibody can be used for detecting vibrio parahaemolyticus. The invention also discloses a vibrio parahaemolyticus flagellin capture ELISA (enzyme-linked immunosorbent assay) kit.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of Vibrio parahemolyticus flagellin monoclonal antibody, hybridoma cell strain and antigen capture ELISA test kit.
Background technology
(Vibrio parahemolyticus VP) is a kind of halophilism bacterium to Vibrio parahemolyticus.Vibrio parahaemolytisus poisoning is that feed contains due to the food of this bacterium, mainly from sea-food, like inkfish, ocean fish, extra large shrimp, sea crab, jellyfish, and contains the higher pickling food of salinity, like salted vegetables, butcher's meat etc.This bacterium survival ability is strong, on rag and chopping block, can survive more than 1 month, can survive in the seawater 47 days.Be cardinal symptom with Acute onset, stomachache, vomiting, diarrhoea and watery stool clinically.This disease is many to betide the coastland in summer and autumn, often causes collective's morbidity.Owing to the seafood air transport, the inland city case also increases gradually in recent years.
The method that detects at present pathogenic microorganism in the food is main with traditional method mainly, i.e. isolation and identification method, and this method required time is long, needs 5-7 days generally speaking, reaches 10-15 days sometimes, is difficult to satisfy the needs of Rapid identification; The round pcr that grew up in recent years, be a kind of fast, sensitive, technology that specificity is good, but at present should technology still depend on traditional method before increase the bacterium step, often contain the PCR suppressor factor in the enrichment liquid, thereby influence the amplification of PCR; With antibody is that basic immunology detection has become hazardous and noxious substances and the indispensable important technical of harmful organism detection in the food.Developed multiple specificity immunology detection technique at present, like radioimmunoassay (RIA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), time resolved fluoro-immunoassay (TrFIA), chemiluminescence immune assay (CIA), (BLIA)bioluminescence immunoassay (BIA), immunoprecipitation, immuno agglutination reaction, ELISA detection kit, immunity colloidal gold test paper strip, immune latex detection reagent etc.Wherein ELISA detection kit, immunity colloidal gold test paper strip etc. are multiple is the immunology detection technology on basis with antibody;, practical characteristics easy, quick, sensitive, accurate with it is the important composition in the border inspection and quarantine safety detection technical system always, become harmful organism and hazardous and noxious substances and detected indispensable important technical.The ELISA method detects fast, as screening method have fast, sensitive characteristics, be the screening method that receives extensive welcome, but employed test kit costs an arm and a leg all from abroad, and needs its special instrument of outfit.Thereby research and development have the antibody of food-borne pathogenic mikrobe of independent intellectual property right, are the exploitation ELISA detection method that has independent intellectual property right, Radioactive colloidal gold detection method, based on the basis of immunoreation for the immune nanometer magnetic bead enriching method on basis.
Flagellum is the important virulence factor of Vibrio parahemolyticus, has specific flagellar antigen (H antigen), one of foundation of Chang Zuowei serological identification.The flagellum of bacterium has very important physicochemical property, and its immunogenicity has great importance in biology and mechanism of causing a disease research.For example; The antigen that flagellin is considered to a kind of protectiveness is used for the vaccine research of pathogenic bacterium; There is result of study to show; Flagellin can be used as the inductor that the natural immunity is replied, and induces the natural immunity of generation to reply the acquired immunity that can help to set up to exogenous antigen and replys, thereby demonstrate the characteristic of flagellin as immunological adjuvant.
In Vibrio parahemolyticus MONOCLONAL ANTIBODIES SPECIFIC FOR process before; The VP thalline that use deactivation as antigen more; And most of albumen of thalline total albumen that is Pseudomonas, specificity between not having kind, the monoclonal antibody that causes gathering in the crops is difficult to avoid cross reaction between the kind in the vibrios Pseudomonas.Specific flagellin was as antigen between this research was selected for use and had kind, and in the positive colony screening process, got rid of cross reaction, the good VP monoclonal antibody of preparation specificity, and be applied to prepare Vibrio parahemolyticus ELISA detection kit.
Summary of the invention
First technical problem to be solved by this invention provides a kind of Vibrio parahemolyticus flagellin monoclonal antibody.This monoclonal antibody can be used to detect Vibrio parahemolyticus.
Second technical problem to be solved by this invention provides a kind of Vibrio parahemolyticus flagellin and catches the ELISA test kit.
For solving the problems of the technologies described above, technical scheme provided by the present invention is following:
Vibrio parahemolyticus flagellin monoclonal antibody provided by the present invention is to be the mouse hybridoma cell strain secretion generation of CGMCC No.6061 by deposit number.This mouse hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 26th, 2012 and (is called for short CGMCC; The address is No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), deposit number is CGMCC No.6061 for its deposit number.
Deposit number is that the mouse hybridoma cell strain of CGMCC No.6061 also belongs to protection scope of the present invention.
The present invention also provides a kind of Vibrio parahemolyticus flagellin to catch the ELISA test kit, and this test kit comprises the solid phase carrier that encapsulates Vibrio parahemolyticus flagellin polyclonal antibody and the monoclonal antibody of the present invention of enzyme labelling.Said enzyme is preferably HRPO.Further, for the ease of detecting, test kit of the present invention also comprises positive control and negative control, and the required enzyme linked immunosorbent detection reagent of ELISA reaction.Wherein, said positive control is the Vibrio parahemolyticus flagellin, and said negative control is normal mouse serum or its diluent of not immune Vibrio parahemolyticus flagellin.Said enzyme linked immunosorbent detection reagent is conventional enzyme linked immunosorbent detection reagent, includes but not limited to substrate reactions liquid, washings and the reaction terminating liquid of enzyme.
The present invention has the following advantages and effect
At first, the present invention comprises the non-Vibrio parahemolyticus reference culture of 30 strain Vibrio parahemolyticus and 28 strains through the specificity experiment, demonstrates good specificity and stability.Secondly, the susceptibility experimental result shows that the detection sensitivity of test kit of the present invention is 10
3/ hole apparently higher than the mikrobe traditional detection method, and has advantages such as quick, efficient.
Description of drawings
Fig. 1. the SDS-PAGE figure of Vibrio parahemolyticus flagellin; Swimming lane 1: Vibrio parahemolyticus flagellin; Swimming lane 2: albumen Marker;
Fig. 2. Vibrio parahemolyticus ATCC17802 thalline and flagellum Electronic Speculum;
Fig. 3. Vibrio parahemolyticus ATCC17802 flagellin Electronic Speculum;
The SDS-PAGE figure of Fig. 4 .mAb; Swimming lane 1: albumen Marker; Swimming lane 2:mAb.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
1. the extraction of yeast culture and flagellin
Vibrio parahemolyticus (Vibrio parahemolyticus) (ATCC 17802, and preserve in the laboratory) is inoculated in T1N1 substratum (available from Beijing Luqiao Technology Co., Ltd., article No. CM168), 36 ℃ of activation 24h; Picking list colony inoculation is in the TSA substratum that contains 2%NaCl (available from Beijing Luqiao Technology Co., Ltd., article No. CM417) growth 18 ~ 24h; With aseptic cotton carrier well-grown bacterium colony is uniformly coated on TSA (2%NaCl) substratum, cultivates 18 ± 2h for 36 ℃.Inferior daily aseptic cotton carrier is scraped in the 0.15M NaCl solution that hypothallus is suspended in precooling, ice bath 15 ~ 30min, and 1000rpm homogenate 45sec places 10min on ice immediately.The centrifugal 10min of 10000rpm under 4 ℃ of conditions of homogenate; Get the centrifugal 2h of 16000rpm under 4 ℃ of conditions of supernatant, abandon supernatant, deposition is resuspended in TET (10mM Tris, 2mM EDTA, pH8.0, the 1%Triton X-100) damping fluid.Centrifugal process repeats 3 times.Operating process keeps carrying out under the low temperature.Leave and take deposition at last, (pH7.8) among the buffer, 4 ℃ of preservations are subsequent use for 0.1M Tris, 0.1mM EDTA to be dissolved in a small amount of Tris-EDTA.
2.SDS-PAGE and Electronic Speculum is identified
The flagellin that extracts carries out the SDS-PAGE electrophoresis.5% spacer gel, 10% separation gel, 120V voltage, electrophoresis to glue bottom.Gel dyes with the Xylene Brilliant Cyanine G method, decolouring back gel imaging analysis systematic observation result.See Fig. 1.
Vibrio parahemolyticus thalline and single living flagellum Electronic Speculum result are as shown in Figure 2.With twice thalline of aseptic deionized water centrifuge washing, suspend, get one on copper mesh, the phospho-wolframic acid negative staining is dried, transmission electron microscope observing.The flagellin Electronic Speculum adopts same procedure, sees Fig. 3.
3. the immunity of animal
(1) immunity of BALB/c mouse
With the flagellin is antigen, and 5 of the BALB/c mouses in immune 6 ~ 8 ages in week are established 2 and do not done negative control as immune mouse.Initial immunity antigen add equivalent Fu Shi Freund's complete adjuvant fully emulsified after, subcutaneous injection immune mouse, 40 μ g/ mouse.Whenever immune at a distance from two weeks later on same dose antigen and the fully emulsified back of freund 's incomplete adjuvant.The 5th immunity tail vein blood after 3 ~ 5 days detects antiserum titre.
(2) immunity of new zealand purebred rabbit
With the female new zealand rabbit 0.5mg/ of Vibrio parahemolyticus flagellin immunity only.Whenever once, be total to immunity 5 times at a distance from two all immunity.Five exempt from the back carried out heart in 3-5 days and measures blood greatly, put 37 ℃ 1 hour, put refrigerator then and spend the night for 4 ℃, get the serum purifying every other day and get Vibrio parahemolyticus flagellin polyclonal antibody ,-20 ℃ are frozen subsequent use.
4. antiserum titre
Antiserum titre adopts indirect ELISA method: with PBS dilution VP antigen concentration to 1 μ g/mL, add 96 orifice plates, and 100 μ L/ holes, 4 ℃ are spent the night.Wash plate 3 times with PBST.2% bovine serum albumin is dissolved in the PBS solution, 200 μ L/ holes, 37 ℃ of sealing 1h.Wash plate with PBST.5 immune mouse serums add in the corresponding aperture with the PBS gradient dilution, 100 μ L/ holes, and blank is a PBS solution, negative control is washed plate for 37 ℃ of immune serums not after encapsulating 45min.The sheep anti mouse of HRP mark adds with 1:2500 multiple dilution, and plate is washed after encapsulating 40min for 37 ℃ in 100 μ L/ holes.Every hole adds freshly prepared substrate solution 100 μ L, and every hole adds the 2M vitriol oil 50 μ L behind 37 ℃ of effect 15min, joins detector with enzyme and measures OD450nm value, reading and observations.Tire height and satisfy the mouse of P/N value＞2.0 of selection, one week the back booster immunization, get mouse boosting cell in 3 ~ 5 days and carry out cytogamy.
5.P2/0 myeloma cell's recovery and cultivation
In advance frozen myeloma cell (SP2/0) is recovered, after myeloma cell frozen in-80 ℃ of deep freezers is taken out fast, place 38 ℃ of water-baths slightly to rock and make its rapid thawing; Notice that the frozen mouth of pipe can not run into water, in order to avoid pollute, then with cell transfer to containing 6 ~ 10ml RPMI-1640 perfect medium (RPMI-1640 substratum that contains 10% calf serum; Calf serum is available from Hyclone; Article No. SH30541.03) in the Tissue Culture Flask, puts into 37 ℃, 5%CO
2Cultivated in the incubator 4 ~ 6 hours, and when treating the whole adherent growth of cell, in time changed liquid, whenever went down to posterity once at a distance from 2 ~ 3 days later, and the adjustment cell makes it be in the righttest stand density, when cell reaches certain activity, counting is prepared to merge.Preceding 1 ~ 2 day pair cell of cytogamy carries out 1 to 4 and goes down to posterity, and using every bottle of cell concn of fresh culture adjustment is 1 ~ 2 * 10
5/ ml, the general cell that can obtain logarithmic phase in 1 ~ 2 day.
6. the preparation of feeder cell
(1) draws neck to put to death BALB/c mouse, after the tap water flushing, be soaked in 10min in 75% alcohol fully, move in the plate of Bechtop, make its belly up.
(2) mention the mouse skin of chest abdomen with tweezers, cut off an osculum, skin is torn a bigger mouth, use new tweezers then again instead and mention mouse peritoneum with two tweezers with scissors; Cut off, find the thymus gland of mouse, use tweezers, little scissors carefully takes out thymus gland; Be placed in the disposable plate, incidental fat, reticular tissue etc. on the thymus gland are peelled off in carefulness, add RPMI-1640 substratum (available from Hyclone, article No. SH30809.01) 5ml; Grind thymus gland, sieve, thymus cell suspension is added in the centrifuge tube; The centrifugal 10min of 1000rpm abandons supernatant, twice of centrifuge washing.
(3) gently that cell is resuspended and mixed even with 5ml HAT nutrient solution, counting, adding HAT nutrient solution to cell concn is 1 ~ 2 * 10
5/ ml.
(4) cell suspension is splashed in the 96 porocyte culture plates, 37 ℃, 5%CO are put in 100 μ l/ holes (two)
2Cultivate in the incubator.
7. the preparation of immune spleen cell suspension
(1) booster immunization selected the higher BALB/c mouse of serum titer after 3 ~ 5 days, extractd eyeball, bloodletting, and collection and separation of serum are as the negative control of antibody test.
(2) disconnected neck is put to death, and is soaked in 10min in 75% alcohol after the tap water flushing, takes out mouse and is placed in the plate of aseptic Bechtop, makes its belly up.
(3) mention the mouse skin of chest abdomen with tweezers, cut off an osculum, with two tweezers skin is torn a bigger mouth again with scissors; Again use new tweezers then instead and mention mouse peritoneum; Cut off, find the spleen of mouse, carefully spleen is taken out; Be placed in the disposable plate careful fat and the reticular tissue removed.
(4) with after the flushing of RPMI-1640 washing lotion, add new RPMI-1640 washing lotion, grind with the syringe nook closing member; Sieve then, splenocyte as far as possible all is expressed in the solution through mesh, splenocyte suspension is moved in the centrifuge tube; The centrifugal 10min of 1000rpm abandons supernatant, twice of centrifuge washing.
(5) gently that splenocyte is resuspended with 10ml HAT nutrient solution, counting, subsequent use.
The preparation of 8SP2/0 myeloma cell's suspension
(1) gets 4 bottles of cultured myeloma cells of 100ml culturing bottle (merge and change liquid previous day, cell should be in logarithmic phase during fusion), be collected in the 50ml centrifuge tube.
(2) 1000rpm is centrifugal 5 ~ 10 minutes, abandons supernatant.
(3) add 30ml RPMI-1640 washing lotion in the deposition, resuspended gently, mixing, centrifuge washing is once again with method.
(4) gently that splenocyte is resuspended and mixed even with 10ml HAT nutrient solution, counting, subsequent use.
9 cytogamy
(1) blow down cultured myeloma cell gently, it is transferred in the 50mL centrifuge tube, the centrifugal 7min of 1000r/min abandons supernatant, and 10mL substratum washing lotion suspends, counting.
The suspension that (2) will contain 1 * 108 splenocyte with contain 2 * 10
7Individual myeloma cell's suspension is mixed in the centrifuge tube of a 50ml, and the supplemented medium washing lotion is to 30ml, fully mixing.
(3) 1000rpm is centrifugal 7 minutes, and supernatant discarded cleans twice, removes supernatant as far as possible.
(4) flick centrifuge tube with have gentle hands at the bottom of, make cell mass loose evenly in the pasty state.Put 37 ℃ of water-baths, preheating 5 ~ 10min is to reach fusion temperature.
(5) 50%PEG (MW4000), the RPMI-1640 washing lotion from 4 ℃ of refrigerators, taking out to have prepared are placed in 37 ℃ of water-baths, and temperature is subsequent use in advance.
(5) centrifuge tube is put in the beaker that contains 37 ~ 40 ℃ of water; Rotate centrifuge tube, draw 50% PEG 1ml, dropwise, slowly add centrifuge tube along tube wall with the 1ml suction pipe; Time was controlled in 60 seconds; And then draw cell suspension with time of 30 seconds, and left standstill 30 seconds, in 30 seconds, cell slowly is blown in the centrifuge tube again.
(6) add the good RPMI-1640 washing lotion of temperature in advance, make the PEG dilution and lose and shortly melt effect, concrete grammar: add 2ml in preceding two minutes, added 3ml on the 3rd minute, in 3min, add 20ml at last.
(7) the centrifugal fused cell of room temperature, the centrifugal 7min of 800rpm abandons supernatant.
(8) add HAT nutrient solution (50 *,, article No. H0262), pressure-vaccum, resuspended sedimentation cell gently available from Sigma.
(9) adding of fused cell suspension is contained in 96 well culture plates of feeder cell, 37 ℃, 5%CO are put with culture plate then in 100 μ l/ holes (two)
2Incubator in cultivate.
The screening of 10 positive colonies and cloning are cultivated
Merge back beginning in the 3rd day, observe each porocyte growing state every day,, handle with sodium azide immediately if pollution is arranged.Merge back 6d, 9d changes liquid with HAT nutrient solution half amount, after HAT selects nutrient solution to keep two weeks of cultivation, use instead HT (50 *; Available from Sigma, article No. H0137) nutrient solution half amount is changed liquid, later on according to the cell growing state; 1 ~ 2d changes a not good liquor, uses complete culture solution instead after two weeks.Treat that hybridoma covers with (about about 10d) of hole floorage at 1/10 o'clock, draw the hole supernatant that the clone occurs and be used for specific detection.Adopt the indirect elisa method among the 1.2.3.Cross reaction: the flagellin with vibrio cholerae (VC), Vibrio vulnificus (VV) and vibrio alginolyticus (VA) is an antigen, encapsulates 96 orifice plates, measures the cross reaction situation of VP antiserum(antisera) and 3 kinds of vibrios.Join detector with enzyme and measure OD450, it is positive to satisfy P/N value＞2.0.VP antigen coated microplate detected result is positive, and other bacterial antigens encapsulate hybridoma that the plate detected result is negative to carry out cloning with limiting dilution assay and cultivates.When cell cover with the hole floorage 1/10 the time use again with quadrat method and detect, clone in the strong positive hole again.3 ~ 4 times so repeatedly, reach 100% up to the positive colony rate.
The preparation of 11 ascites is identified with antibody
Positive colony hybridoma to filtering out at last carries out enlarged culturing; And be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 26th, 2012 and (be called for short CGMCC; The address is No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city), deposit number is CGMCC No.6061.Induce method in the conventional ascites body and prepare odd contradictive hydroperitoneum.The CHARACTERISTICS IDENTIFICATION of mAb: the detection of (1) Ig class and subclass: class and subclass with Ig in the mouse mAb subclass detection kit mensuration Hybridoma Cell Culture supernatant, carry out on the operation steps by specification.(2) Western blot detects: adopt Western blot experimental technique commonly used, measure the specificity of ascites under 1: 2000 extent of dilution.(3) antibody purification and titration: ascites obtains antibody after with Protein G Sepharose affinity chromatography purifying, and the antibody behind the purifying is measured with indirect elisa method behind doubling dilution and tired.Antibody is through the SDS-PAGE purity assay.
Antibody obtains two bands clearly through the SDS-PAGE electrophoresis behind the purifying: heavy chain molecule amount 50KDa, and light chain molecular weight 25KDa, shown in Figure 4.The antibody ELISA result's (seeing table 1) that tires can find out, extent of dilution is situation under to still have very strong positive reaction at 1: 2187000.
The ELISA of table 1.mAb tires.
1. the Vibrio parahemolyticus flagellin is caught ELISA test kit composition
The solid phase carrier that has encapsulated Vibrio parahemolyticus flagellin polyclonal antibody is an enzyme plate, the Vibrio parahemolyticus flagellin monoclonal antibody of horseradish peroxidase-labeled, substrate reactions liquid, the positive and negative control, washings and the reaction terminating liquid of enzyme.
(1) how anti-enzyme plate encapsulates
With the PBS damping fluid Vibrio parahemolyticus flagellin polyclonal antibody (preparing like embodiment 1) being diluted to concentration is 10 μ g/mL, encapsulates the efficient desmoenzyme target of 96 hole EIA, 100 μ L/ holes, and 4 ℃ are spent the night.Take out the back and wash plate 3 times, dry with PBST.2% BSA is dissolved in the PBS solution as confining liquid, and 100 μ L/ holes add enzyme plate, 37 ℃ of sealing 1h.Take out the back and wash plate 3 times with PBST, each 3min dries, and dry final vacuum sealing is preserved.
Wherein, PBS buffered soln: Na
2HPO
412H
2O 13.76g, NaH
2PO
42H
2O 1.794g, NaCl 9g add deionized water to 1000ml; The PBST washings: contain the PBS aqueous solution of 0.005%Tween 20, PH is 7.4.
(2) mark of anti-Vibrio parahemolyticus flagellin monoclonal antibody
To resist Vibrio parahemolyticus flagellin hybridoma cell strain CGMCC No.6061 excretory monoclonal antibody and horseradish peroxidase (HRP) to adopt the periodates oxidation style to carry out coupling.
(3) substrate
Adopt 3,3,5,5-TMB TMB (SIGMA) is as the substrate of horseradish peroxidase.Sodium acetate soln (pH=4.3) 4mL, the H of adding 30%
2O
25mL, TMB 1mL, 10mL substrate reactions liquid (existing with join at present).
(4) positive and negative control
Positive control:, can extract 50 μ g/mL according to embodiment 1 described method by the Vibrio parahemolyticus flagellin.
Negative control: 100 times of diluents of the normal mouse serum of not immune Vibrio parahemolyticus flagellin.
(5) washings
Be the PBST washings
(6) reaction terminating liquid
2M H with the tri-distilled water preparation
2SO
4Solution.
Embodiment 3 Vibrio parahemolyticus flagellins are caught the method for use of ELISA test kit
1. the processing of sample to be checked
Get sample enrichment liquid 1mL to be checked, abandon supernatant behind the centrifugal 2min of 10000rpm, deposition adds the resuspended deposition of a small amount of 200 μ LPBS with after the PBS damping fluid washed twice.Boiling water bath 5min.
2. add contrast and sample to be checked
Get sample 100 μ L to be checked and add corresponding enzyme mark hole, a side that adds model is knocked in positive control and negative control 100 μ L/ holes, with at the bottom of guaranteeing the even coverage hole of sample, 37 ℃ hatch 1h after PBST wash plate 3 times, drying.
3. adding monoclonal antibody linked with peroxidase
Add monoclonal antibody linked with peroxidase working fluid (PBS damping fluid 1:1000 dilution enzyme mark monoclonal antibody) 100 μ L/ holes, 37 ℃ hatch 1h after PBST wash plate 3 times, dry.
4. add the substrate colour developing
Add freshly prepared tmb substrate 100 μ L/ holes, room temperature lucifuge 10 ~ 15min.
5. adding reaction terminating liquid
Add 2M H
2SO
4Solution 50 μ L/ holes, termination reaction.
6. survey the OD450nm value
Enzyme plate places ELIASA to measure the OD450nm value.
7. the result judges
As stated above, 100 times of diluents of 20 parts of negative normal mouse serum purified products are detected.According to formula threshold value=negative OD MV+3 * standard deviation, calculating negative threshold value is 0.117.Sample OD value to be checked is judged to the positive greater than 0.117, otherwise is judged to feminine gender.
Embodiment 4 Vibrio parahemolyticus flagellins are caught the specificity and the sensitivity testing of ELISA test kit
(1) specific assay
Catch the specificity of ELISA test kit in order to verify Vibrio parahemolyticus flagellin of the present invention; Form with method according to embodiment 2 and embodiment 3 described test kits 1 strain Vibrio parahemolyticus ATCC17802 and 30 strain Vibrio parahemolyticus (oneself separates the laboratory) and the non-Vibrio parahemolyticus reference culture of 28 strains are detected, see table 2.The result shows; Test kit of the present invention to Vibrio parahemolyticus ATCC17802 and 30 strain Vibrio parahemolyticus OD450nm values between 0.601 ~ 2.143; The non-Vibrio parahemolyticus reference culture of 28 strains OD450nm value between 0.065 ~ 0.091, the specificity that demonstration can be good.
Table 2
| Strain name | The OD450nm value |
| Vibrio parahemolyticus (laboratory is from separating 30 strains) | 0.601~2.143 |
| Vibrio parahemolyticus ATCC 17802 | 2.175 |
| Vibrio cholerae 75 | 0.089 |
| Vibrio alginolyticus ATCC 1833 | 0.091 |
| Vibrio vulnificus ATCC 1758 | 0.084 |
| Ge Shi listeria spp ATCC 700545 | 0.065 |
| Xi Er listeria spp ATCC 35967 | 0.071 |
| Listeria innocua ATCC 33090 | 0.080 |
| Mo Shi listeria spp ATCC 2540 | 0.075 |
| Singly increase listeria spp ATCC 15313 | 0.078 |
| Klebsiella pneumonia CGMCC 1.1736 | 0.089 |
| Enterobacter cloacae CGMCC 1.57 | 0.090 |
| Bacillus cereus ATCC 11778 | 0.067 |
| Streptococcus aureus ATCC 25923 | 0.069 |
| Staphylococcus epidermidis ATCC 12228 | 0.075 |
| Salmonella paratyphi A CMCC 50001 | 0.084 |
| Intestinal bacteria ATCC 25912 | 0.079 |
| Rhodococcus equi ATCC 6936 | 0.069 |
| Salmonella enteritidis CMCC 50041 | 0.073 |
| Shigella flexneri CMCC 51571 | 0.088 |
| -Escherichia coli O 157 | 0.071 |
| Salmonella typhimurium CMCC 50115 | 0.084 |
| Enterobacter sakazakii ATCC 29544 | 0.078 |
| Citrobacter CMCC 48017 | 0.090 |
| The third type Hemolytic streptococcus CMCC 32206 | 0.067 |
| Beta hemolytic streptococcus CMCC 32210 | 0.074 |
| Pseudomonas aeruginosa ATCC 15442 | 0.079 |
| Yersinia entero-colitica CMCC 52212 | 0.080 |
| Shigella dysenteriae CMCC 51252 | 0.068 |
| Enterococcus faecalis CGMCC 1.2135 | 0.072 |
| Negative control | 0.087 |
(2) sensitivity testing
With Vibrio parahemolyticus ATCC17802 inoculation in the APW substratum, after 36 ℃ of shaking tables are cultivated 1h, 10 times of gradient dilutions of saline water, plate count simultaneously, obtaining cell concentration is 10
8~ 10
2The thalline solution of CFU/mL is formed according to embodiment 2 and embodiment 3 described test kits then and method detects, and sees table 3.The result shows that the detection sensitivity of test kit of the present invention is 10
3/ hole.
Table 3
Obviously, the above embodiment of the present invention only be for clearly the present invention is described and is done for example, and be not to be qualification to embodiment of the present invention.For the those of ordinary skill in affiliated field, on the basis of above-mentioned explanation, can also make other multi-form variation or change.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (9)
1. Vibrio parahemolyticus flagellin monoclonal antibody is to be the hybridoma cell strain secretion generation of CGMCC No. 6061 by deposit number.
2. secretion produces the hybridoma cell strain of Vibrio parahemolyticus flagellin monoclonal antibody, and its deposit number is CGMCC No. 6061.
3. a Vibrio parahemolyticus flagellin is caught the ELISA test kit, it is characterized in that, comprises the solid phase carrier that encapsulates Vibrio parahemolyticus flagellin polyclonal antibody and the described monoclonal antibody of claim 1 of enzyme labelling.
4. test kit according to claim 3 is characterized in that, said enzyme is a HRPO.
5. according to claim 3 or 4 described test kits, it is characterized in that said test kit also comprises positive control and negative control.
6. test kit according to claim 5 is characterized in that, said positive control is the Vibrio parahemolyticus flagellin, and said negative control is normal mouse serum or its diluent of not immune Vibrio parahemolyticus flagellin.
7. the application of the described monoclonal antibody of claim 1 in the test kit of preparation detection Vibrio parahemolyticus.
8. the application of the described monoclonal antibody of claim 1 in detecting Vibrio parahemolyticus.
9. the application of the described test kit of claim 3 in detecting Vibrio parahemolyticus.
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Cited By (11)
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| CN102993301A (en) * | 2012-12-11 | 2013-03-27 | 北京出入境检验检疫局检验检疫技术中心 | Vibrio cholerae flagellin monoclonal antibody and antigen capture ELISA kit |
| CN103160602A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | Real-time fluorescent PCR (polymerase chain reaction) detection kit of vibrio parahaemolyticus and detection method thereof |
| CN103160603A (en) * | 2013-04-08 | 2013-06-19 | 北京出入境检验检疫局检验检疫技术中心 | LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof |
| CN103484434A (en) * | 2013-06-28 | 2014-01-01 | 朱艮苗 | Hybridoma cell strain and applications thereof |
| CN104316690A (en) * | 2014-11-03 | 2015-01-28 | 上海理工大学 | Rapid vibrio parahaemolyticus immune colloidal gold test strip |
| CN104360066A (en) * | 2014-11-03 | 2015-02-18 | 上海理工大学 | Method for detecting bibrio parahemolyticus and monoclonal antibodies thereof |
| 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 |
| CN105158470A (en) * | 2015-06-10 | 2015-12-16 | 北京农学院 | ELISA kit for detecting Vibrio parahemolyticus in aquatic product |
| CN105424927A (en) * | 2015-11-09 | 2016-03-23 | 山东省海洋生物研究院 | Method for detecting vibrio parahaemolyticus |
| CN110540966A (en) * | 2018-12-20 | 2019-12-06 | 湖北云璐生物工程有限公司 | Human haemophilus influenzae surface protein monoclonal antibody and antigen capture ELISA kit |
| CN113234688A (en) * | 2021-05-19 | 2021-08-10 | 上海理工大学 | Vibrio parahaemolyticus broad-spectrum colloidal gold immunochromatographic test strip, monoclonal antibody and hybridoma cell strain thereof |
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| CN102993301A (en) * | 2012-12-11 | 2013-03-27 | 北京出入境检验检疫局检验检疫技术中心 | Vibrio cholerae flagellin monoclonal antibody and antigen capture ELISA kit |
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| CN104316690A (en) * | 2014-11-03 | 2015-01-28 | 上海理工大学 | Rapid vibrio parahaemolyticus immune colloidal gold test strip |
| CN104360066A (en) * | 2014-11-03 | 2015-02-18 | 上海理工大学 | Method for detecting bibrio parahemolyticus and monoclonal antibodies thereof |
| 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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| CN110540966B (en) * | 2018-12-20 | 2023-04-28 | 湖北云璐生物工程有限公司 | Human haemophilus influenzae surface protein monoclonal antibody and antigen capture ELISA kit |
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