CN103869074B - Enzyme-linked immunoassay kit for staphylococcus aureus - Google Patents

Enzyme-linked immunoassay kit for staphylococcus aureus Download PDF

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CN103869074B
CN103869074B CN201410130674.3A CN201410130674A CN103869074B CN 103869074 B CN103869074 B CN 103869074B CN 201410130674 A CN201410130674 A CN 201410130674A CN 103869074 B CN103869074 B CN 103869074B
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staphylococcus aureus
kit
antibody
monoclonal antibody
5d2g2d9c1
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CN103869074A (en
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刘箐
郭慧琴
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University of Shanghai for Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • 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
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

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Abstract

The invention discloses an enzyme-linked immunoassay kit for staphylococcus aureus. The kit contains two monoclonal antibodies which can be specifically combined to the staphylococcus aureus, wherein one is a monoclonal antibody 5D6D9G3B4 specifically used as a capture antibody, and the other is a monoclonal antibody 5D2G2D9C1 used as a detection antibody. A large number of tests confirm that the kit can specifically and efficiently test the staphylococcus aureus, but does not have any cross reaction with the other 74 kinds of common pathogenic bacteria, thus being is a pathogenic bacteria assay product with good performance.

Description

Staphylococcus aureus 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 staphylococcus aureus.
Background technology
Staphylococcus aureus (Staphylococcus aureus) is modal food-borne pathogens, is extensively present in physical environment.Staphylococcus aureus mainly pollutes the animal products such as milk, meat, egg, fish and goods thereof, comprise beverage, cold drink, cake that milk makes, cold cuts, meat products can, leftovers, fried egg, glutinous rice cakes, bean jelly, surplus rice and rice wine etc., popular name " addicted to meat bacterium ".Staphylococcus aureus mainly can cause human body illness after infecting human body, first affecting conditions as caused pyogenic infection, as wound suppuration, cellulitis, pneumonia, meningitis, pericarditis, septicemia etc.; It two is the toxin diseases produced by staphylococcus aureus, cause dizziness after people is edible, feel sick, diarrhoea, the food poisoning symptoms such as acute gastroenteritis such as vomiting, and the shock-syndrome caused by toxin shows as high heat, low blood pressure, diarrhoea, fash, shock.
The detection of current staphylococcus aureus 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 staphylococcus aureus, and technology required for protection relates to the quick testing product of staphylococcus aureus.
Summary of the invention
The object of this invention is to provide a kind of kit detecting staphylococcus aureus.
And then the invention provides a kind of enzyme linked immunological kit for detecting staphylococcus aureus, described kit contains:
(a) solid phase carrier, described solid phase carrier is coated with the anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 as catching antibody, described anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 is produced by mouse hybridoma cell system 5D6D9G3B4, CGMCC No.8764;
(b) container a, anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 as detecting antibody is housed in described container a, described anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 is produced by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763.
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 staphylococcus aureus bacterium liquid of deactivation, and described negative control is the brain heart leachate meat soup 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
Lane1：5D2G2D9C1，Lane2：5D6D9G3B4
Embodiment
The research of the present inventor shows, using staphylococcus aureus as immunogene, immunity Balb/c mouse, separation and purification obtains two strain anti-Staphylococcus aureus monoclonal antibodies, i.e. 5D6D9G3B4, preserving number CGMCC No.8764 and 5D2G2D9C1, preserving number CGMCC No.8763, the antibody titer of above-mentioned two strain monoclonal antibodies can reach 1:100000, its can specificity, be combined with staphylococcus aureus efficiently.Using said monoclonal antibody 5D6D9G3B4 bag by enzyme reaction plate as seizure antibody, with the said monoclonal antibody 5D2G2D9C1 of horseradish peroxidase-labeled as detection antibody, make enzyme-linked immunologic detecting kit.Result shows, and above-mentioned staphylococcus aureus 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, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, 74 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 staphylococcus aureus.
Antibody
The present invention includes two strain anti-Staphylococcus aureus monoclonal antibodies.Two strain anti-Staphylococcus aureus monoclonal antibodies of the present invention can utilize mouse hybridoma cell system 5D6D9G3B4(CGMCC No.8764) and mouse hybridoma cell system 5D2G2D9C1(CGMCC No.8763) secrete generation respectively.
The present invention includes the monoclonal antibody of the corresponding amino acid sequence with anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 and 5D2G2D9C1, 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-Staphylococcus aureus 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-Staphylococcus aureus monoclonal antibody or its fragment.
For anti-Staphylococcus aureus monoclonal antibody heavy of the present invention and sequence of light chain, can measure by conventional method.The hypervariable region of anti-Staphylococcus aureus 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-Staphylococcus aureus 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 ') 2 fragment; 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 5D6D9G3B4(CGMCC No.8764) and 5D2G2D9C1(CGMCC No.8763) 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 staphylococcus aureus time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, and with the process of CaC12 method, step used is well-known in this area.Another kind method uses MgC12.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-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 and 5D6D9G3B4 of the present invention, its height of tiring (can 1:100000 be reached), staphylococcus aureus 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, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc. amount to 74 kinds of equal no cross reactions of pathogenetic bacteria, this is maximum innovative point of the present invention.Said monoclonal antibody 5D2G2D9C1 can use horseradish peroxidase, alkaline phosphatase, nanogold particle to mark, and uses in specific manner as detecting antibody.Said monoclonal antibody 5D6D9G3B4, 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, when adopting mouse hybridoma cell system 5D6D9G3B4(CGMCC No.8764) the anti-Staphylococcus aureus monoclonal antibody that produces is as after seizure antibody capture staphylococcus aureus, again with detectable label mark by mouse hybridoma cell system 5D2G2D9C1(CGMCC No.8763) the anti-Staphylococcus aureus monoclonal antibody that produces is as detection antibody, extremely effectively can be incorporated into staphylococcus aureus, thus detect staphylococcus aureus 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 staphylococcus aureus specifically, it is produced by mouse hybridoma cell system 5D6D9G3B4, CGMCC No.8764.
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 staphylococcus aureus, it is produced by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763.
As used herein, described " specificity " refers to that antibody can only be incorporated into staphylococcus aureus; More particularly, refer to that those can be combined with staphylococcus aureus 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 Gold Samples staphylococcus aureus.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-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 as catching antibody, described anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 is produced by mouse hybridoma cell system 5D6D9G3B4, CGMCC No.8764;
(b) container a, anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 as detecting antibody is housed in described container a, described anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 is produced by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763.
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 staphylococcus aureus 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 staphylococcus aureus 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 staphylococcus aureus 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 staphylococcus aureuses 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 staphylococcus aureus bacterium liquid of deactivation, and described negative control is the brain heart leachate meat soup 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-Staphylococcus aureus monoclonal antibody (seizure antibody) is had when pre-coated on enzyme reaction plate, after adding sample solution or standard items, add with detectable label another strain anti-Staphylococcus aureus monoclonal antibody (detection antibody) as horseradish peroxidase again, the staphylococcus aureus 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, through TMB(tetramethyl benzidine) colour developing after can form yellow substance, thus can in judgement sample staphylococcus aureus 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, staphylococcus aureus 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, enterohemorrhagic Escherichia coli O 157: H7, Enterobacter sakazakii, small intestine Yersinia ruckeri, streptococcus pneumonia, bacillus cereus etc. amount to 74 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 embodiment 1. anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 and 5D2G2D9C1
One, the preparation of immunogene and positive criteria product
Staphylococcus aureus (ATCC No.27660) is inoculated in brain heart leachate meat soup (BHI), 150r/min shaken cultivation 17h, and counting, adds 0.3% formalin room temperature deactivation 1 day.Staphylococcus aureus (ATCC No.27660) 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 brain heart leachate meat soup (BHI) 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, the cultivation of 5%CO2 incubator.
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
Staphylococcus aureus culture medium is as follows: GB: GB4789.10-2010
Brain heart leachate meat soup (BHI)
Composition: tryptose matter peptone 10.0g, sodium chloride 5.0g, sodium hydrogen phosphate (Na 2hPO 412H 2o) 2.5g, glucose 2.0g, OX-heart leachate 500mL.
Method for making: heating for dissolving, regulates pH7.4 ± 0.2, puts 121 DEG C, 15min sterilizing.
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. antibody titer measurement result
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 5D2G2D9C1 and 5D6D9G3B4.The heavy chain molecule amount of 5D2G2D9C1 is 48kDa, and light chain molecule amount is 26kDa; The heavy chain molecule amount of 5D6D9G3B4 is 48kDa, and light chain molecule amount is 26kDa.
The CHARACTERISTICS IDENTIFICATION of embodiment 2. monoclonal antibody 5D6D9G3B4 and 5D2G2D9C1
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%BSA200 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 5D6D9G3B4 and 5D2G2D9C1
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%BSA200 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
Sequence number Strain name Strain number 5D2G2 D9C1（ OD 450） 5D6D9 G3B4（ OD 450） Cross reaction
01 Enterohemorrhagic Escherichia coli O 157: H7 ATCC43895 0.055 0.057 0.052 0.053 0.063 0.056 －
02 Enterohemorrhagic Escherichia coli O 157: H7 ATCC43889 0.057 0.055 0.056 0.061 0.043 0.043 －
03 Enterobacter cloacae CMCC（B）45301 0.061 0.051 0.059 0.051 0.045 0.049 －
04 Enterococcus faecalis ATCC29212 0.057 0.059 0.055 0.057 0.049 0.040 －
05 Colon bacillus ATCC8739 0.045 0.058 0.052 0.053 0.050 0.054 －
04 Colon bacillus ATCC25922 0.057 0.059 0.056 0.045 0.054 0.047 －
07 Colon bacillus CMCC（B）44102 0.058 0.046 0.051 0.059 0.048 0.059 －
08 Colon bacillus 8099 0.047 0.059 0.053 0.041 0.056 0.045 －
09 Listeria monocytogenes ATCC43251 0.051 0.055 0.054 0.055 0.058 0.042 －
10 Listeria monocytogenes ATCC19115 0.041 0.057 0.056 0.055 0.055 0.054 －
11 Listeria monocytogenes CMCC（B）54002 0.047 0.059 0.057 0.055 0.055 0.048 －
12 Listeria monocytogenes ATCC13932 0.057 0.053 0.052 0.057 0.059 0.047 －
13 Listeria monocytogenes ATCC19112 0.044 0.056 0.057 0.051 0.051 0.045 －
14 Listeria monocytogenes ATCC19114 0.055 0.059 0.053 0.057 0.059 0.048 －
15 Listeria monocytogenes ATCC19116 0.044 0.055 0.050 0.051 0.051 0.043 －
16 Listeria monocytogenes ATCC19117 0.047 0.056 0.055 0.053 0.059 0.047 －
17 Listeria monocytogenes ATCC19115 0.057 0.057 0.059 0.057 0.059 0.047 －
18 Singly increase listeria spp ATCC19111 0.047 0.059 0.058 0.057 0.059 0.047 －
19 Yi Shi listeria spp ATCC19119 0.046 0.049 0.051 0.047 0.047 0.057 －
20 Grignard listeria spp ATCC25401 0.043 0.046 0.041 0.046 0.047 0.048 －
21 Wei Shi listeria spp ATCC35897 0.045 0.047 0.051 0.041 0.048 0.047 －
22 Harmless Listeria monocytogenes CICC10297 0.048 0.050 0.054 0.047 0.049 0.047 －
23 Bacterium enteritidis ATCC13056 0.041 0.041 0.046 0.051 0.054 0.77 －
24 Salmonella choleraesuls CICC21493 0.048 0.050 0.047 0.049 0.045 0.044 －
25 Salmonella typhimurium CMCC（B）50115 0.047 0.049 0.048 0.049 0.043 0.047 －
26 Salmonella typhimurium CTCC22956 0.046 0.040 0.043 0.042 0.049 0.67 －
27 Salmonella typhimurium ATCC14028 0.048 0.048 0.054 0.055 0.041 0.054 －
28 Salmonella paratyphi A CMCC（B）50093 0.051 0.052 0.044 0.047 0.049 0.049 －
29 Moscow' paratyphi B CMCC（B）50094 0.047 0.049 0.047 0.047 0.049 0.047 －
30 Salmonella typhi CMCC（B）50071 0.047 0.049 0.047 0.047 0.041 0.046 －
31 Moscow' saint paul IQCC10529 0.046 0.056 0.040 0.049 0.049 0.056 －
32 Salmonella IQCC10531 0.051 0.047 0.057 0.055 0.048 0.051 －
33 Salmonella kentucky IQCC10430 0.051 0.061 0.054 0.061 0.055 0.057 －
34 Bu Luokeli salmonella IQCC10432 0.047 0.062 0.057 0.064 0.067 0.058 －
36 Salmonella aberdeen IQCC10411 0.047 0.065 0.067 0.051 0.061 0.067 －
36 Moscow' paratyphi B IQCC10404 0.058 0.055 0.057 0.052 0.069 0.087 －
37 Salmonella dublin IQCC10423 0.051 0.055 0.058 0.057 0.067 0.065 －
38 Salmonella anatis IQCC10409 0.051 0.051 0.054 0.066 0.058 0.067 －
39 Sick ox salmonella IQCC10410 0.057 0.061 0.067 0.057 0.054 0.067 －
40 Salmonella paratyphi A IQCC10418 0.057 0.069 0.053 0.067 0.057 0.052 －
41 Salmonella gallinarum IQCC10426 0.045 0.045 0.047 0.055 0.047 0.044 －
42 Turkey salmonella IQCC10426 0.041 0.046 0.056 0.044 0.049 0.047 －
43 Moscow' paratyphi C IQCC10427 0.051 0.041 0.054 0.047 0.041 0.057 －
44 Bacterium enteritidis IQCC10428 0.057 0.049 0.057 0.047 0.042 0.041 －
46 Salmonella paratyphi A IQCC10480 0.057 0.049 0.047 0.046 0.059 0.058 －
46 Salmonella paratyphi A IQCC30437 0.046 0.046 0.047 0.059 0.049 0.047 －
47 Salmonella paratyphi A IQCC30438 0.041 0.041 0.044 0.048 0.089 0.047 －
48 Moscow' paratyphi B CMCC60004 0.047 0.049 0.047 0.057 0.058 0.047 －
49 Moscow' paratyphi C CMCC60118 0.047 0.049 0.055 0.057 0.046 0.057 －
60 Shigella flexneri ATCC12022 0.041 0.042 0.049 0.087 0.045 0.087 －
61 Bacillus ceylonensis A ATCC26931 0.056 0.048 0.043 0.049 0.042 0.042 －
62 Shigella bogdii ATCC8700 0.051 0.050 0.049 0.042 0.041 0.057 －
63 Shigella bogdii ATCC9207 0.041 0.059 0.040 0.051 0.049 0.051 －
64 Shigella bogdii CMCC61346 0.044 0.049 0.057 0.047 0.042 0.047 －
66 Shigella dysenteriae CMCC（B）61104 0.056 0.047 0.057 0.047 0.052 0.057 －
66 Shigella dysenteriae Field isolates 0.042 0.058 0.052 0.041 0.049 0.057 －
67 Shigella flexneri CMCC（B）61672 0.048 0.054 0.056 0.042 0.049 0.057 －
68 Shigella flexneri ATCC12022 0.051 0.087 0.051 0.061 0.056 0.058 －
69 Bacillus ceylonensis A CMCC（B）61692 0.053 0.056 0.061 0.064 0.056 0.068 －
60 Bacillus ceylonensis A ATCC26931 0.048 0.069 0.064 0.062 0.069 0.060 －
61 Vibrio parahaemolytious ATCC17802 0.041 0.065 0.068 0.061 0.067 0.068 －
62 Enterobacter sakazakii ATCC29004 0.044 0.060 0.069 0.062 0.060 0.058 －
63 Enterobacter sakazakii ATCC29664 0.055 0.058 0.067 0.066 0.065 0.056 －
64 Enterobacter sakazakii ATCC12868 0.057 0.069 0.056 0.068 0.069 0.056 －
66 Small intestine Yersinia ruckeri ATCC23716 0.058 0.062 0.068 0.058 0.053 0.056 －
66 Small intestine Yersinia ruckeri CMCC62207 0.057 0.050 0.061 0.057 0.052 0.063 －
67 Small intestine Yersinia ruckeri CMCC（B）62204 0.054 0.042 0.048 0.058 0.053 0.056 －
68 Beta hemolytic streptococcus ATCC21049 0.047 0.048 0.052 0.051 0.054 0.046 －
69 Beta hemolytic streptococcus ATCC21040 0.057 0.056 0.053 0.057 0.049 0.086 －
70 Bacillus cereus ATCC11778 0.058 0.053 0.056 0.058 0.049 0.086 －
71 Bacillus cereus CMCC（B）63301 0.057 0.049 0.053 0.058 0.051 0.053 －
72 Bacillus cereus CMCC（B）63303 0.058 0.056 0.052 0.058 0.055 0.058 －
73 Staphylococcus aureus ATCC6638 0.871 0.876 0.762 0.778 0.736 0.740 +
77 Staphylococcus aureus ATCC26923 0.878 0.826 0.866 0.717 0.789 0.786 +
76 Staphylococcus aureus ATCC23213 0.817 0.879 0.816 0.647 0.686 0.616 +
76 Staphylococcus aureus ATCC27660 0.778 0.786 0.766 0.687 0.586 0.656 +
77 Staphylococcus epidermidis CMCC（B）26049 0.048 0.056 0.051 0.053 0.042 0.059 －
78 Campylobacter jejuni CICC22937 0.044 0.051 0.052 0.055 0.057 0.059 －
Embodiment 3. detects the composition of the enzyme linked immunological kit of staphylococcus aureus, 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 (pH2.0) solution dilution, anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4, every hole 100 μ l.4 DEG C of overnight incubation, conveniently ELISA method closes washing.
(2) S. aureus-positive reference standards and negative control standard items.
(3) the monoclonal antibody 5D2G2D9C1 of the anti-Staphylococcus aureus of horseradish peroxidase-labeled.
(4) enzyme labelled antibody dilution: 0.01M PBS, pH7.6.
(5) 10 × concentrated washing lotion: the 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% Sodium azide, pH7.4, 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 staphylococcus aureus monoclonal antibody 5D6D9G3B4 as seizure antibody coated elisa plate
Be buffered liquid with bag and staphylococcus aureus monoclonal antibody 5D6D9G3B4 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 5D2G2D9C1 of horseradish peroxidase-labeled
Staphylococcus aureus monoclonal antibody 5D2G2D9C1 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 0.5ml0.2M 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 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, will precipitation be dissolved in the 0.01M PBS(pH7.4 of 2.5ml) in.
F, add 10mg monoclonal antibody 5D2G2D9C1, and use 0.5M carbonic acid buffer (pH9.6) 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(pH7.4) 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) add 225ml brain heart leachate meat soup (BHI) 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 5D2G2D9C1 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 staphylococcus aureus, experiment repetition 4 times.
Precision test between plate: 4 pieces of ELISA Plate of getting same batch, tests with the milk polluted by staphylococcus aureus, 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 68 kinds of food-borne pathogens with kit, whether checking kit detects the specificity of staphylococcus aureus, 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 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 5D2G2D9C1 of the staphylococcus aureus monoclonal antibody of above-mentioned qualification on January 9th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is the hybridoma cell strain producing anti-Staphylococcus aureus monoclonal antibody, and preserving number is CGMCC No.8763.
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 5D6D9G3B4 of the staphylococcus aureus monoclonal antibody of above-mentioned qualification on January 9th, 2014, Institute of Microorganism, Academia Sinica, postcode: 100101), Classification And Nomenclature is anti-Staphylococcus aureus hybridoma, and preserving number is CGMCC No.8764.
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 staphylococcus aureus, it is characterized in that, described kit contains:
(a) solid phase carrier, described solid phase carrier is coated with the anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 as catching antibody, described anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4 is produced by mouse hybridoma cell system 5D6D9G3B4, CGMCC No.8764;
(b) container a, anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 as detecting antibody is housed in described container a, described anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 is produced by mouse hybridoma cell system 5D2G2D9C1, CGMCC No.8763.
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 staphylococcus aureus bacterium liquid of deactivation, and described negative control is the brain heart leachate meat soup of sterilizing.
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