CN103940995A - Listeria monocytogenes enzyme-linked immunosorbent assay kit - Google Patents
Listeria monocytogenes enzyme-linked immunosorbent assay kit Download PDFInfo
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- CN103940995A CN103940995A CN201410176192.1A CN201410176192A CN103940995A CN 103940995 A CN103940995 A CN 103940995A CN 201410176192 A CN201410176192 A CN 201410176192A CN 103940995 A CN103940995 A CN 103940995A
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- listeria monocytogenes
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
Abstract
The invention discloses a Listeria monocytogenes enzyme-linked immunosorbent assay kit. The kit contains two monoclonal antibodies which can be specifically bonded to the Listeria monocytogenes, wherein one monoclonal antibody is 1B4E7A6C9 specially used for capturing the antibody, and other monoclonal antibody is 6D4H7C8B6 used for detecting the antibody. The kit is proved to be capable of detecting the Listeria monocytogenes specifically in a high-efficiency manner and having no cross reaction with other 68 common pathogenic bacteria through substantive tests, and therefore, the kit has good performances on detecting the pathogenic bacteria.
Description
Technical field
The invention belongs to biotechnology and field of immunology, be specifically related to a kind of enzyme linked immunological kit that detects listeria monocytogenes.
Background technology
Listeria monocytogenes (Listeria monocytogenes) is that a kind of short and small Gram-positive is without brood cell's facultative Bacteroides nodosus, it is the strongest bacterium of pathogenicity in listeria, also be unique born of the same parents' endophyte that people is caused a disease, typical, can cause serious infectious diseases common to human beings and animals, as symptoms such as meningitis, septicemia, miscarriage and monocytosis.1988, the World Health Organization (WHO) (World Health Organization, WHO) delivered " food source property listeriosis is advised book ", instructs whole world various countries how to prevent Listeria pollution and poisoning.Since then, LM becomes new important food origin disease pathogen, and itself and E.coli O157, salmonella and staphylococcus aureus are listed as the large food-borne pathogens nineties four in 20th century by WHO.The main contaminated milk of this bacterium and dairy products, cheese product, meat products sausage, processing bird, raw meat, fish, shrimp, smoke fish, raw vegetables.
The detection of Listeria monocytogenes at present mainly 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 fast detecting means that occur 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 can apply to detect the fast detecting product of practice; this patent is taking Listeria monocytogenes as detecting target, and technology required for protection relates to listeria monocytogenes fast detecting product.
Summary of the invention
The object of this invention is to provide a kind of kit that detects listeria monocytogenes.
And then, the invention provides a kind of enzyme linked immunological kit for detection of listeria monocytogenes, described kit contains:
(a) solid phase carrier, on described solid phase carrier, be coated with as the anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 that catches antibody, described anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 is 1B4E7A6C9 by mouse hybridoma cell, and CGMCC No.8765 produces;
(b) container a, in described container a, be equipped with as the anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 that detects antibody, described anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 is 6D4H7C8B6 by mouse hybridoma cell, and CGMCC No.8002 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 kit also contains positive control and negative control.
In another preference, the listeria monocytogenes bacterium liquid that described positive control is deactivation, the Li Shi that described negative control is sterilizing increases bacterial context soup.
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By below and the description of claim, feature of the present invention, object and advantage will be more obvious.
Embodiment
The inventor's research shows, using listeria monocytogenes as immunogene, immunity Balb/c mouse, separation and purification obtains the anti-listeria monocytogenes monoclonal antibody of two strains, be 1B4E7A6C9, CGMCC No.8765 and 6D4H7C8B6, CGMCC No.8002, the antibody titer of above-mentioned two strain monoclonal antibodies can reach 1:100000, and it can specificity, be combined with listeria monocytogenes efficiently.Using the coated enzyme reaction plate of said monoclonal antibody 1B4E7A6C9 as catching antibody, as detecting antibody, make enzyme-linked immunologic detecting kit with the said monoclonal antibody 6D4H7C8B6 of horseradish peroxidase-labeled.Result demonstration, above-mentioned listeria monocytogenes detection kit detection sensitivity reaches 10
5cfu/ml, has advantages of that repeatability, accuracy are good, and between hole, error is less than 5%, and in plate, CV is less than 3%.Itself and enterohemorrhagic Escherichia coli O 157: H7, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will he, Song Nei Shi will he, Boydii will he, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc. amount to 68 kinds of equal no cross reactions of pathogenetic bacteria.
On this basis, the inventor and then according to DASELISA immunization, through test repeatedly, finally obtained a kind of can be fast, efficient detection eats the enzyme linked immunological kit of source property listeria monocytogenes.
Antibody
The present invention includes the anti-listeria monocytogenes monoclonal antibody of two strains.It is that 1B4E7A6C9 (CGMCC No.8765) and mouse hybridoma cell are that 6D4H7C8B6 (CGMCC No.8002) secretes respectively generation that the anti-listeria monocytogenes monoclonal antibody of two strain of the present invention can be utilized mouse hybridoma cell.
The present invention includes the monoclonal antibody of the corresponding amino acid sequence with anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 and 6D4H7C8B6, and there is other protein or protein conjugate and the fusion expressed product of these chains.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 (being immune conjugate and fusion expressed product), as long as the hypervariable region of this hypervariable region and light chain of the present invention and heavy chain is identical or at least 90% homology, preferably at least 95% homology.As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: medicine, toxin, cell factor (cytokine), radioactive nuclide, enzyme and other diagnosis or treatment molecule be combined with anti-listeria monocytogenes monoclonal antibody or its fragment and formation conjugate.The present invention also comprises cell surface marker thing or the antigen of being combined with anti-listeria monocytogenes monoclonal antibody or its fragment.
For anti-listeria monocytogenes monoclonal antibody heavy chain of the present invention and sequence of light chain, can measure by conventional method.(complementarity determining region, CDR) is interesting especially for the hypervariable region of anti-listeria monocytogenes monoclonal antibody V chain or complementary determining region, because relate at least partly conjugated antigen in them.Therefore, the present invention includes those and there is light chain immunoglobulin with CDR and the molecule of weight chain variable chain, as long as its CDR and anti-listeria monocytogenes monoclonal antibody CDR have 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.
The present invention also provides the DNA molecular of above-mentioned immunoglobulin (Ig) or its fragment.The sequence of these DNA moleculars can be used routine techniques, and utilizing mouse hybridoma cell is that 1B4E7A6C9 (CGMCC No.8765) and 6D4H7C8B6 (CGMCC No.8002) 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 can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated and obtains relevant sequence from the host cell propagation by conventional method.
In addition, also can synthesize relevant sequence by artificial synthetic method, especially fragment length more in short-term.Conventionally, by first synthetic multiple small fragments, and then connect and can obtain the fragment that sequence is very long.
At present, can be completely obtain the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.Then this DNA sequence dna can be introduced in the various existing DNA molecular (or as carrier) and cell that in this area, oneself knows.In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable DNA sequence dna and suitable promoter or control sequence.These carriers can be 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 is prokaryotes during as Enterohemorrhagic E.coli, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaC1
2method processing, step used is well-known in this area.Another kind method is to use MgC1
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eucaryote, can use following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packaging etc.
The transformant obtaining 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 mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promoter of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell a period of time again.
Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cell membrane.If needed, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant 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 processing, with the combination of protein precipitant processing (salt analysis method), centrifugal, the broken bacterium of infiltration, ultrasonic processing, 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-listeria monocytogenes monoclonal antibody 6D4H7C8B6 of the present invention and 1B4E7A6C9, its height of tiring (can reach 1:100000), can be specifically, detect efficiently listeria monocytogenes, with enterohemorrhagic Escherichia coli O 157: H7, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will is congratulated, Song Nei Shi will is congratulated, Boydii will is congratulated, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereuss etc. amount to 68 kinds of equal no cross reactions of pathogenetic bacteria, this is maximum innovative point of the present invention.Said monoclonal antibody 6D4H7C8B6 can use horseradish peroxidase, alkaline phosphatase, nanogold particle mark, uses in specific manner as detecting antibody.Said monoclonal antibody 1B4E7A6C9, in various detections are used, can use as catching antibody in specific manner.
Detection kit
The inventor is through studying widely and testing, be surprised to find that, be that the anti-listeria monocytogenes monoclonal antibody that produces of 1B4E7A6C9 (CGMCC No.8765) is as catching after antibody capture listeria monocytogenes when adopting mouse hybridoma cell, be that the anti-listeria monocytogenes monoclonal antibody that produces of 6D4H7C8B6 (CGMCC No.8002) is as detecting antibody with detectable label mark by mouse hybridoma cell again, can extremely effectively be incorporated into listeria monocytogenes, thereby detect in high sensitivity listeria monocytogenes by double antibodies sandwich 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 " are used interchangeably with " primary antibodie ", all refer to the described monoclonal antibody that can be incorporated into specifically listeria monocytogenes, it is 1B4E7A6C9 by mouse hybridoma cell, and CGMCC No.8765 produces.
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 catch antibody phase coupling (connection).For example, described solid phase carrier is enzyme reaction plate.
As used herein, described " detection antibody ", " second antibody ", " enzyme labelled antibody " are used interchangeably with " two is anti-", all refer to can specific binding in another strain monoclonal antibody of listeria monocytogenes, it is 6D4H7C8B6 by mouse hybridoma cell, and CGMCC No.8002 produces.
As used herein, described " specificity " refers to that antibody can only be incorporated into listeria monocytogenes; More particularly, refer to that those can be combined with listeria monocytogenes but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.
The 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 listeria monocytogenes in sample.The way of double antibodies sandwich method routine is that seizure antibody is fixed on to carrier, then catch antibody and antigen-reactive, after washing, (described detection antibody carries detectable with detecting antibody response again, or can be combined with the material that carries detectable), finally carry out chemiluminescence or enzyme connection chromogenic reaction detection signal.And with respect to the competition law of monoclonal antibody body, the mensuration effect of double antibody sandwich method is more good, thereby only need little sample size while measuring.So adopt double antibody sandwich method no matter to have more advantage in sensitivity, degree of accuracy, accuracy, specificity and stability.
Particularly, enzyme linked immunological kit of the present invention contains:
(a) enzyme reaction plate, on described enzyme reaction plate, be coated with as the anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 that catches antibody, described anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 is 1B4E7A6C9 by mouse hybridoma cell, and CGMCC No.8765 produces;
(b) container a, in described container a, be equipped with as the anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 that detects antibody, described anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 is 6D4H7C8B6 by mouse hybridoma cell, and CGMCC No.8002 produces.
As optimal way of the present invention, described detection antibody is with detectable label.
As used herein, whether described " detectable label " refer to existence for determining detected sample listeria monocytogenes and the mark of the amount existing.Determining the seizure antibody that kit of the present invention adopts and/or detecting after antibody, can adopt the various labels of this area routine for being combined to detect with detection antibody.The present invention has no particular limits adopted label, as long as being combined with described detection antibody, and can indicate detected sample exactly after suitably processing in listeria monocytogenes existence whether and the label of amount be all available.Described label can directly 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 can, according to the kind of adopted antibody and characteristic, select suitable label.For example, described label can be selected from: horseradish peroxidase (HRP), alkaline phosphatase vinegar enzyme (AP), glucose oxidase, beta-D-galactosidase, urase, hydrogen peroxidase or glucoamylase.
In the time adopting some enzyme labeling things as implied above, also need to adopt some and the substrate that enzyme is combined accordingly, thereby can 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 the catalysis colour developing of thing institute, detect for showing the identification signal that antibody is combined with listeria monocytogenes.Described substrate is for example: for adjacent benzene two limbs (OPD), tetramethyl biphenyl limb (TMB), the ABTS of horseradish peroxidase; Be used for p-nitrophenyl phosphoric acid vinegar (p-nitro phenyl phosphate, p-NPP) of alkaline phosphatase etc.Those skilled in the art can, according to the kind of adopted label and characteristic, 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 again with the comparison of streptavidin HRP reacting phase, directly after finishing, directly add substrate and develop the color more simple and convenient detecting mark HRP on antibody, reaction.
In order to obtain quantitative result, the standard items of knowing multiple listeria monocytogenes 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, Quality Control (contrast) also can be set in testing process.As optimal way of the present invention, the listeria monocytogenes bacterium liquid that described positive control is deactivation, the Li Shi that described negative control is sterilizing increases bacterial context soup.
In addition, in order to make kit of the present invention more convenient in the time detecting, in described kit, preferably also comprise some other auxiliary reagent, described auxiliary reagent is conventional some reagent that use 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) for example: developer, cleansing solution, stop buffer, enrichment liquid, dilution.
In addition, in described kit, also can comprise operation instructions, for the using method of the reagent wherein loading is described.
Detection 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.There is an anti-listeria monocytogenes monoclonal antibody (seizure antibody) when pre-coated on enzyme reaction plate, add after sample solution or standard items, add again with detectable label listeria monocytogenes monoclonal antibody that is as anti-in another strain of horseradish peroxidase (detection antibody), the listeria monocytogenes existing in sample or standard items will combine with seizure antibody coated on enzyme reaction plate, after detection antibody to be added, can form " antibody-antigen-enzyme labelled antibody " compound, after TMB (tetramethyl benzidine) colour developing, can form yellow substance, thereby in can judgement sample, whether and the amount existing the existence of listeria monocytogenes.
Under 450nm, measure absorbance by microplate reader.Negative control≤0.1, positive control >=0.3, experimental result is effective, otherwise that result is judged to be is invalid; Detect OD value >=0.2 o'clock, hole, be judged to be the positive; Detect hole OD value between 0.1-0.2 time, be judged to be the weak positive; Detect OD value≤0.1, hole and be judged to be feminine gender.
Test shows, listeria monocytogenes 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%.Amount to 68 kinds of equal no cross reactions of pathogenetic bacteria with enterohemorrhagic Escherichia coli O 157: H7, hog cholera sramana, mouse typhus sramana, enteritis sramana, Fu Shi will he, Song Nei Shi will he, Boydii will he, vibrio parahaemolytious, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc., this is maximum innovative point of the present invention.
In a word, kit of the present invention requires low, easy and simple to handle to the pre-treatment of sample, and detection limit is 10
5cfu/ml, specificity and good stability, and all there is no cross reaction with most of food-borne pathogens.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise number percent and umber calculate by weight.
Unless otherwise defined, the familiar meaning of all specialties that use in literary composition and scientific words and one skilled in the art is 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 embodiment 1. anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 and 6D4H7C8B6
One, the preparation of immunogene and positive criteria product
Listeria monocytogenes (ATCC No.43251) is seeded in Li Shi and increases bacterial context soup, 37 DEG C, 150r/min shaken cultivation 17h, and counting, adds 0.3% formalin room temperature deactivation 1 day.Adjust listeria monocytogenes (ATCC No.43251) concentration to 5 × 10 with physiological saline
9cfu/ml is as immunogene; Adjusting concentration with physiological saline is 10
8cfu/ml is as positive control standard items, and Li Shi increases the negative reference standards of bacterial context soup.
Two, the preparation of monoclonal antibody
1) animal used as test: select 38 week ages, body weight 20g left and right, 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) blood sampling: from tail vein blood sampling, adopt indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum titre after 3 booster immunizations.Wait to tire and no longer rise, lumbar injection is measured immunogene equally, after 3 days, carries out Fusion of Cells according to conventional method.
4) Fusion of Cells: get immune mouse spleen cell and SP2/0 myeloma cell conventional fusion under 50%PEG effect, be inoculated in respectively 96 well culture plates, be 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 cloning cultivation with limiting dilution assay.When Growth of Cells is at the bottom of being paved with hole 1/10 time, then detect with same method, strong positive hole is cloned again, 3-4 time so repeatedly, until positive rate reaches 100%.Hybridoma is expanded and cultivated, 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
Listeria monocytogenes nutrient culture media is as follows: GB: GB4789.30-2010
Li Shi increases bacterial context soup
Composition: tryptone 5.0g, multivalence peptone 5.0g, yeast extract 5.0g, sodium chloride 20.0g, potassium dihydrogen phosphate 1.4g, sodium hydrogen phosphate 12.0g, aesculin 1.0g, distilled water 1000mL
Method for making: by mentioned component heating for dissolving, regulate pH7.2～7.4, packing, 121 DEG C of autoclaving 15min, for subsequent use.
The step that antibody titer is measured is as follows:
(1) saturated culture of bacteria antigen is added in corresponding hole to 100 μ l together with nutrient culture media, 4 DEG C spend the night (about wrapper sheet 36h).
(2) turned letter liquid pat dry residual liquid, with 250 μ l cleansing solutions cleaning 3 times.
(3) in each hole, add 100 μ l1%BSA, 37 DEG C of sealing 1h.
(4) turned letter liquid pat dry residual liquid, with 250 μ l cleansing solutions cleaning 3 times.
(5) in each hole, add 100 μ l serum, hatch 1h for 37 DEG C.
(6) turned letter liquid pat dry residual liquid, adds 250 μ lPBST cleansing solutions washing 3 times in each hole.
(7) two of the HRP mark of 50 μ l anti-(sigma) in each hole, incubated at room 1h.
(8) soak 5min with cleansing solution, turned letter liquid also pats dry residual liquid, adds 250 μ lPBST cleansing solution washing 3 times in each hole.
(9) in each hole, add 100 μ l substrates, colour developing 30min, adds stop buffer 100 μ l also immediately at OD
450reading.
Table 1. liang strain antibody titer measurement result (OD
450)
The CHARACTERISTICS IDENTIFICATION of embodiment 2. monoclonal antibody 1B4E7A6C9 and 6D4H7C8B6
One, monoclonal antibody subgroup identification
1, antigen coated: with the coated mountain sheep anti mouse two anti-IgG+A+M of 0.01M PBS, every hole 50 μ l, 4 DEG C of coated spending the night, discard liquid in hole next day, wash plate 3 times.
2, sealing: every hole adds 1%BSA200 μ l, and 4 DEG C of sealings are spent the night.Pat dry plank next day and do not wash plate.
3, add monoclonal antibody hybridoma cell supernatant, 8 micropores of each sample, every hole 50 μ l.37 DEG C, hatch 1h.
4, wash after plate 4 times, add respectively the rabbit anti-mouse igg 1 of specific bond, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, hatches 1h for 37 DEG C.
5, wash after plate 4 times, every hole adds the horseradish peroxidase-labeled of the having diluted anti-IgG of anti-rabbit two (H+L), 37 DEG C, hatches 30min.
6, wash after plate 4 times, add 100 μ l substrate nitrite ions, 37 DEG C, lucifuge colour developing 10min.Reading result under 450nm wavelength.
Table 2. liang strain monoclonal antibody subgroup identification result
Two, the cross reaction of monoclonal antibody 1B4E7A6C9 and 6D4H7C8B6 test
1, antigen coated: by 78 kind 10
8the pathogenic bacteria of cfu/ml bacterial concentration join in ELISA Plate, and each pathogenic bacteria add 3 holes, every hole 100 μ l, 4 DEG C, coated spending the night.
2, sealing: wash after plate 3 times, every hole adds 3%BSA200 μ l, hatches 2h for 37 DEG C.
3, wash plate 3 times.Every hole adds the monoclonal antibody 100 μ l that diluted, and hatches 1h for 37 degrees Celsius.
4, wash plate 3 times.Add the mountain sheep anti mouse two having diluted to resist in all micropores, every hole 100 μ l, hatch 1h for 37 degrees Celsius.
5, wash plate 4 times.Add substrate nitrite ion, 37 DEG C, lucifuge colour developing 15min.Reading result under 450nm wavelength.
Table 3. liang strain monoclonal antibody cross reaction testing result
Embodiment 3. detects composition, preparation and the application thereof of listerial enzyme linked immunological kit
One, enzyme linked immunological kit is made up of following substances:
(1) ELISA Plate of pre-coated antibody: with 0.02M acetate buffer solution (pH2.0) solution dilution, the coated 96 hole ELISA Plate of anti-listeria monoclonal antibody 1B4E7A6C9, every hole 100 μ l.4 DEG C of overnight incubation, according to conventional ELISA method sealing washing.
(2) Listeria positive control standard items and negative control standard items.
(3) the monoclonal antibody 6D4H7C8B6 of the anti-listeria of horseradish peroxidase-labeled.
(4) enzyme labelled antibody dilution: 0.01M PBS, pH7.6.
(5) 10 × concentrated washing lotion: the 0.1M phosphate buffer that contains 0.5% Tween-20 and 0.2% Sodium azide, pH7.4, will concentrate 10 times of dilutions of washing lotion when 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 Listeria monoclonal antibody 1B4E7A6C9 as catching antibody coated elisa plate
Listeria monoclonal antibody 1B4E7A6C9 is diluted to 5 μ g/ml with coated damping fluid, every hole adds 100 μ l, 4 DEG C are spent the night, the coating buffer that inclines next day, washs 3 times by the washing lotion of dilute, pats dry, then in every hole, add 220 μ l confining liquids, 37 DEG C of incubation 2h, liquid in the hole of inclining, preserves with aluminium foil bag sealing after being dried.
Coated damping fluid: 0.02M acetate buffer solution, with 5M HCl adjusting pH to 2.0.
Confining liquid: the 0.01M PBS that contains 0.3% bovine serum albumin(BSA) and 10% sucrose.
(2) preparation of the monoclonal antibody 6D4H7C8B6 of horseradish peroxidase-labeled
Listeria monoclonal antibody 6D4H7C8B6 and horseradish peroxidase (HRP) are carried out to coupling, and the method for employing is the sodium periodate method of improvement, and method is as follows:
A, 5mg horseradish peroxidase (HRP) are dissolved in 0.5ml0.2M acetate buffer solution (pH5.6).
B, add the 0.06M NaIO of existing preparation
4solution 0.5ml, 4 DEG C of oxidation 20min.
C, add the 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 (pH7.4) of 2.5ml.
F, add 10mg monoclonal antibody 6D4H7C8B6, and use immediately 0.5M carbonic acid buffer (pH9.6) to regulate pH to 9.0.4 DEG C of hold over night.
G, add 10mg/ml sodium borohydride 50 μ l, 4 DEG C, leave standstill 2h.
H, 4 DEG C of dialysed overnight of use 0.01M PBS (pH7.4).
I, purification storage.
Three, the application of kit
(1) detection method
1, sample pre-treatments
Get sample 25g to be checked (ml) and add 225ml Li Shi increasing bacterial context soup homogeneous under homogenizer to mix, cultivate 16h for 36 DEG C ± 1 DEG C.Cultured sample is heated to 10min in 100 DEG C of water-baths.It is stand-by that taking-up is cooled to room temperature.
2, detect with kit
Under the micropore of taking-up requirement and all reagent normal temperature, place 30min.Get 200 μ l sample to be checked and be added in micropore, 37 DEG C, hatch 30min.Remove liquid in hole, add 200 μ l washing lotions in each micropore, rock the several seconds gently, fast upset by liquid in micropore to the greatest extent, to a folded clean thieving paper clap several under, repeat operation and wash altogether plate 3 times.Add 100 μ l monoclonal antibody linked with peroxidase 6D4H7C8B6 working fluids, 37 DEG C, hatch 60min.Remove in hole liquid 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 is judged: negative control≤0.1, positive control >=0.3, and experimental result is effective, otherwise that result is judged to be is invalid; Detect OD value >=0.2 o'clock, hole, be judged to be the positive; Detect hole OD value between 0.1-0.2 time, be judged to be the weak positive; Detect OD value≤0.1, hole and be judged to be feminine gender.
If without microplate reader, can with the naked eye judge: there is macroscopic yellow in positive control hole, negative control hole is without color, and experimental result is effective, otherwise it is invalid to be judged to be experimental result; Positive result in the time that there is obvious macroscopic yellow in detection hole; While having faint yellow, be judged to be weak positive findings; Negative result during without naked eyes visible yellow color.
(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, use the milk being polluted by Listeria to test, experiment repeats 4 times.
Precision test between plate: get 4 ELISA Plate of same batch, use the milk being polluted by Listeria to test, experiment repeats 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
Whether remove to detect 78 kinds of food-borne pathogens with kit, checking kit detects listerial specificity, observe and have cross reaction and false positive to occur with other pathogenic bacteria.
The test of table 6. kit specificity
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 result of new lot.Consider that in transport and use procedure, having improper preservation condition occurs, kit is placed 8 days under the condition of 37 DEG C of preservations, carry out accelerated aging test, result shows that the indices of kit meets the requirements completely.Therefore kit can at least can be preserved more than 12 months at 2-8 DEG C.
The preservation of biomaterial
The hybridoma cell strain 1B4E7A6C9 that produces the listeria monocytogenes monoclonal antibody of the above-mentioned qualification of process is 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) on January 9th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is anti-Listeria monocytogenes hybridoma, and preserving number is CGMCC No.8765.
The hybridoma cell strain 6D4H7C8B6 that produces the listeria monocytogenes monoclonal antibody of the above-mentioned qualification of process is 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) on July 16th, 2013, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is the hybridoma cell strain that produces anti-Listeria monocytogenes monoclonal antibody, and preserving number is CGMCC No.8002.
All documents of mentioning in the present invention are all quoted as a reference 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 after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. for detection of an enzyme linked immunological kit for listeria monocytogenes, it is characterized in that, described kit contains:
(a) solid phase carrier, on described solid phase carrier, be coated with as the anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 that catches antibody, described anti-listeria monocytogenes monoclonal antibody 1B4E7A6C9 is 1B4E7A6C9 by mouse hybridoma cell, and CGMCC No.8765 produces;
(b) container a, in described container a, be equipped with as the anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 that detects antibody, described anti-listeria monocytogenes monoclonal antibody 6D4H7C8B6 is 6D4H7C8B6 by mouse hybridoma cell, and CGMCC No.8002 produces.
2. kit as claimed in claim 1, is characterized in that, described solid phase carrier is enzyme reaction plate.
3. kit as claimed in claim 1, is characterized in that, described detection antibody is with detectable label.
4. kit as claimed in claim 3, is characterized in that, described detectable label is horseradish peroxidase.
5. kit as claimed in claim 1, is characterized in that, described kit also contains positive control and negative control.
6. kit as claimed in claim 5, is characterized in that, the listeria monocytogenes bacterium liquid that described positive control is deactivation, and the Li Shi that described negative control is sterilizing increases bacterial context soup.
Priority Applications (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527441A (en) * | 2016-01-21 | 2016-04-27 | 江南大学 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies |
| CN105842442A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for listeria monocytogenes |
| CN112034160A (en) * | 2019-06-03 | 2020-12-04 | 中国科学院福建物质结构研究所 | Circulating tumor cell detection kit based on rare earth nano material fluorescence amplification and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381777A (en) * | 2008-10-20 | 2009-03-11 | 中华人民共和国上海出入境检验检疫局 | Listeria monocytogenes detection kit and detection method thereof |
| CN101613706A (en) * | 2009-01-09 | 2009-12-30 | 何成彦 | The colloidal gold labeling lap gene monoclonal antibody measuring Listeria monocytogenes kit |
| CN103558388A (en) * | 2013-10-24 | 2014-02-05 | 江南大学 | Double-antibody sandwich method for detecting salmonella typhimurium in food based on monoclonal antibodies |
| CN103713104A (en) * | 2013-12-26 | 2014-04-09 | 中华人民共和国淮安出入境检验检疫局 | Double-antibody sandwich method for detecting enterobacter sakazakii in food |
-
2014
- 2014-04-29 CN CN201410176192.1A patent/CN103940995B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381777A (en) * | 2008-10-20 | 2009-03-11 | 中华人民共和国上海出入境检验检疫局 | Listeria monocytogenes detection kit and detection method thereof |
| CN101613706A (en) * | 2009-01-09 | 2009-12-30 | 何成彦 | The colloidal gold labeling lap gene monoclonal antibody measuring Listeria monocytogenes kit |
| CN103558388A (en) * | 2013-10-24 | 2014-02-05 | 江南大学 | Double-antibody sandwich method for detecting salmonella typhimurium in food based on monoclonal antibodies |
| CN103713104A (en) * | 2013-12-26 | 2014-04-09 | 中华人民共和国淮安出入境检验检疫局 | Double-antibody sandwich method for detecting enterobacter sakazakii in food |
Non-Patent Citations (4)
| Title |
|---|
| 段霞 等: "双抗夹心ELISA方法检测食品中单核细胞增生李斯特氏菌", 《食品科学》 * |
| 焦新安 等: "单抗夹心ELISA检测李斯特氏菌方法的建立及其初步应用", 《肉品卫生》 * |
| 范红结 等: "快速、特异检测李斯特菌单抗-夹心ELISA方法的建立及应用", 《中国畜禽传染病》 * |
| 董香梅 等: "单核细胞增生李斯特菌单克隆抗体的制备与鉴定", 《中国生物工程杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105527441A (en) * | 2016-01-21 | 2016-04-27 | 江南大学 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies |
| CN105527441B (en) * | 2016-01-21 | 2017-05-10 | 江南大学 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies |
| CN105842442A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for listeria monocytogenes |
| CN112034160A (en) * | 2019-06-03 | 2020-12-04 | 中国科学院福建物质结构研究所 | Circulating tumor cell detection kit based on rare earth nano material fluorescence amplification and application thereof |
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