CN103969444B - Detect immune chromatography test paper and the preparation method of ochratoxin A based on M13 bacteriophage - Google Patents
Detect immune chromatography test paper and the preparation method of ochratoxin A based on M13 bacteriophage Download PDFInfo
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- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 title claims abstract description 95
- 238000012360 testing method Methods 0.000 title claims abstract description 74
- 241001515965 unidentified phage Species 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 52
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 22
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 title claims abstract description 10
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 title claims abstract description 10
- 229920002678 cellulose Polymers 0.000 claims abstract description 54
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- 238000000034 method Methods 0.000 claims abstract description 30
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 29
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- 102000004169 proteins and genes Human genes 0.000 claims description 5
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- 239000000725 suspension Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
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- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 claims description 4
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
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- CEYULKASIQJZGP-UHFFFAOYSA-L disodium;2-(carboxymethyl)-2-hydroxybutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O CEYULKASIQJZGP-UHFFFAOYSA-L 0.000 claims description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims 2
- BRSVJNYNWNMJKC-UHFFFAOYSA-N [Cl].[Au] Chemical compound [Cl].[Au] BRSVJNYNWNMJKC-UHFFFAOYSA-N 0.000 claims 1
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- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 54
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- 231100000252 nontoxic Toxicity 0.000 description 2
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- 229930183344 ochratoxin Natural products 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
-
- 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
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/385—Assays involving biological materials from specific organisms or of a specific nature from fungi from Penicillium
Abstract
The present invention discloses a kind of immune chromatography test paper and preparation method based on M13 bacteriophage fast detecting ochratoxin A, test paper adsorption layer comprises adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and absorbent material layer, and cellulose rete is provided with uses the detection trace of the M13 bacteriophage printing of showing OTA mimic epitope and the contrast trace with goat anti-mouse igg antibody solution; OTA monoclonal antibody or polyclonal antibody that gold labeling antibody is colloid gold label. The present invention has realized the application in fast detecting OTA is residual of immunochromatography technique based on M13 bacteriophage, there is the M13 bacteriophage of OTA mimic epitope to replace the OTA conjugate of printing on traditional ELISA test strip marking with displaying, avoided the harm of OTA conjugate for the test paper producer, experimenter and environment; The high specificity of this detection paper, highly sensitive, it is easy, directly perceived, accurate to detect, and cost is low, applied widely.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper, particularly relate to a kind of to show the M13 phagocytosis that has OTA mimic epitopeBody is as detecting the marking, for immune chromatography test paper of fast detecting ochratoxin A and preparation method thereof.
Background technology
Ochratoxin (Ochratoxin) is the secondary metabolite that some bacterial strain of aspergillus and Penicillium produces, itComprise seven kinds of similar compounds of structure, its Poisoning maximum, distribute the widest, produce poison amount the highest, the heaviest to crop pollution, withThat human health is the closest is ochratoxin A (OchratoxinA, OTA), and the target organ of its effect is mainly kidneyAnd liver, be a kind of strong kidney, hepatotoxin, and there is stronger carcinogenic, teratogenesis and mutagenesis. Because OTA extensively depositsBe agricultural product and livestock products, and can enter human body through food chain, human life's health is worked the mischief therefore, how to realizeQuick, accurate Clinical significance of detecting to OTA is great.
At present, OTA detection method is had to chemical analysis, instrumental method and immune analysis method etc. Wherein immunologyThe advantage that analytic approach is high with susceptibility, detection limit is low extensive use in OTA qualitative and quantitative analysis, has realized OTA quick, simpleJust, highly sensitive detection, to ensure agricultural products in China have safely positive effect. But, immunological detection method be based upon withMark toxin or coupling toxin are the poisonous detection architecture on competition antigen basis, have toxin mark product costliness, to producing and behaviourThe health of doing personnel is harmful, and the defect such as coupling reaction poor repeatability, has restricted its application and popularization.
In recent years, utilize the mimic epitope peptide that phage random peptide library technology obtains to substitute toxin as competition antigen, buildVertical nontoxic detection architecture, has promoted the fast development of OTA immunology detection. Phage random peptide library technology is with a large amount of randomThe peptide sequence of coding inserts Vector for Phage Display, forms phage display library. Each bacteriophage particles or bacteriophageOnly show a kind of external source peptide chain of sequence, the not homotactic external source peptide chain of different bacteriophage particles or phage display.
Immune test paper method has sxemiquantitative and certain quantitation capabilities, and the preliminary information of determinand can be provided, this method spiritSensitivity is high, and analytic process is simple, has unique advantage as the detection of OTA, is the detection technique that need to first develop.
Summary of the invention
The technical problem to be solved in the present invention: the deficiency existing for prior art, provide a kind of safe, special, sensitive,Immune chromatography test paper of easy, fast detecting ochratoxin A and preparation method thereof.
Technical scheme of the present invention:
Detect an immune chromatography test paper for ochratoxin A based on M13 bacteriophage, comprise supporting layer, the centre of bottomLayer adsorption layer and be fixed on the protective layer on adsorption layer, adsorption layer is followed successively by adsorbing fiber layer, golden labeling antibody fibre from test leadThe absorbent material layer of dimension layer, cellulose rete and handle end is provided with the M13 that shows OTA mimic epitope on cellulose reteThe stealth that bacteriophage is printed detects trace, is also provided with print with goat-anti or the anti-mouse IgG of rabbit or goat anti-rabbit igg antibody solution hiddenShape contrast trace; Described golden labeling antibody fibrage adopts the glass fibre cotton of ADSORPTION OF GOLD labeling antibody to make, and golden labeling antibody adopts glueMonoclonal antibody or the polyclonal antibody of the OTA of body gold nano grain mark.
The peptide sequence of described OTA mimic epitope is MPLWXDL, and X is arbitrary amino acid.
Described M13 bacteriophage is screened by following methods, and concrete steps are:
(1) affine screening M13 phage random seven peptide storehouses
Get the elisa plate that is coated with OTA monoclonal antibody, every hole adds the phage peptide library 100 μ L after dilution, Qi ZhonghanBacteriophage 1 × 1011~2×1011Pfu, under room temperature, jog concussion 1h, discards liquid; Precipitation TBST solution washing 10 times, wash awayAfter unconjugated bacteriophage, every hole adds the eluent Gly-HCl of 100 μ LpH2.2, jog concussion 8min under room temperature, and sucking-off is washedDe-liquid, adds neutralization buffer to neutral; Eluent after neutralization is except staying a part to do titer determination, and remaining eluent connectsPlant in the LB nutrient solution in logarithmic growth E.coliER2738 in earlier stage, carry out amplification cultivation; Cultivate 4-in 37 DEG C of concussions5h, 4 DEG C, the centrifugal 10min of 12000rpm add the PEG/NaCl solution that accounts for supernatant 1/6 volume, 4 DEG C of precipitations in supernatantSpend the night; After precipitation, 4 DEG C, the centrifugal 15min of 12000rpm, abandon supernatant, then use TBST solution suspension bacteriophage, adds that to account for TBST moltenThe PEG/NaCl of liquid 1/6 volume, hatches 60min on ice; 4 DEG C of centrifugal 15min, remove supernatant again, and precipitation suspends with TBST solution;
Measure after titre, carry out the 2nd time, the 3rd time and the affine screening of the 4th; The amount that at every turn adds bacteriophage is 1 × 1011~2×1011Pfu, coated antibody amount is respectively 75,50 and 25 μ g/ml; In TBST solution, the content of Tween-20 is from the 2nd sieveSelected and increased to 0.5%, it is identical that all the other steps and the 1st are taken turns screening, and the eluted product after the 4th screening is after titer determination, from total amountOn flat board less than 100 plaques, select some blue plaques, inoculate respectively in the exponential phase E.coli in early stageIn the LB nutrient solution of ER2738, carry out amplification cultivation, purifying is also measured titre;
(2) qualification phage clone
Get the elisa plate that is coated with OTA monoclonal antibody, add the phage clone that will identify, 37 DEG C of effect 1h, usePBST solution washing 6 times, adds the monoclonal antibody of the anti-M13 of horseradish peroxidase-labeled, and 37 DEG C of effect 1h are molten with PBSTLiquid washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction, is determined at the absorbance OD value under 450nm wavelength,Blank is made in the hole that does not add phage clone, to treat 2.1 times of OD value in the absorbance OD value >=blank of gaging hole, judgementPositive;
Then take the sensitiveness of competitive ELISA method qualification positive phage clones, add the OTA dilution of variable concentrationsLiquid and positive bacteriophage, in 37 DEG C of effect 1h, use PBST solution washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4StopReaction, is determined at the absorbance OD value under 450nm wavelength, and blank is made in the hole that does not add phage clone, to treat the suction of gaging holeIn luminosity OD value >=blank 2.1 times of OD value, be judged as the positive; Calculate the combination rate of bacteriophage under each concentration, and markDirectrix curve judges the sensitiveness of phage clone;
(3) DNA sequencing and peptide sequence analysis
Bacteriophage after amplification is checked order, translate amino acid sequence ,-96 of employing according to the DNA sequence dna insertingG III sequencing primer is 5 '-HOCCCTCATAGTTAGCGTAACG-3 ', the amino acid sequence of mimic epitope isMPLWXDL, X is arbitrary amino acid.
Described adsorbing fiber for layer glass fibre cotton, nylon membrane, PVDF membrane or polyester film make; Absorbent materialLayer is made with absorbent filter, and supporting layer is made with the toughness material not absorbing water; Cellulose is nitrocellulose filter, pure fiber for reteElement film or carboxylation cellulose membrane are made.
The described stealthy trace that detects is " ︱ ︱ " orthoscopic trace, " 10 " font row who is arranged in parallel with stealthy contrast tracePrint mark,
" ┬ ┬ " font is arranged trace, " ┴ ┴ " font is arranged trace, " ├ ├ " font arrangement trace or " ┤ ┤ " wordType is arranged trace.
Described protective layer is the diaphragm covering on adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer,On the adsorbing fiber layer diaphragm corresponding with golden labeling antibody fibrage intersection, be printed with sample mark line, this mark line deflection is inhaledAttached fibrage one side 0.3-0.7cm.
In described PEG/NaCl solution, the concentration of PEG-8000 is 20% (w/v), and the concentration of NaCl is 2.5mol/L.
A preparation method for immune chromatography test paper, comprises the following steps:
(1) preparation of OTA monoclonal antibody or polyclonal antibody;
(2) screening of M13 bacteriophage;
(3) preparation of golden labeling antibody: at 0.01 ~ 0.02%(w/v of boiling) add 1%(w/v in aqueous solution of chloraurate) newThe sodium citrate solution of preparation, the colloidal gold solution of acquisition diameter 15 ~ 20nm after reaction, with the K of 0.1mol/L2CO3Regulate pH valueTo 8.5 ~ 9.5, be placed in 2 ~ 8 DEG C and save backup;
With the mark ratio of 1:2000, OTA monoclonal antibody to be marked or polyclonal antibody are added to the glue of pH8.5 ~ 9.5In body gold solution, after mark 10min, add 20%(w/v) PEG10000 to final concentration be 0.05%, in 4 DEG C, 1500 ~The centrifugal 20min of 3000rpm, removes unconjugated colloid gold particle, and 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtains tentatively pureThe golden labeling antibody protein mixture of changing, then use propylene glucan S-400 column chromatography, obtains colloid gold label after separation and purificationOTA antibody;
(4) preparation of adsorbing fiber layer: adsorbing fiber is glass fibre cotton, nylon membrane, PVDF membrane or poly-for layerEster film is made;
(5) preparation of cellulose rete: cellulose is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane for reteMake, with point sample instrument diverse location specking detection trace and contrast trace respectively on cellulose membrane, then dry;
(6) assembling of test paper: by adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete, absorbent material layer from a left side extremelyRight being attached to successively on the supporting layer that scribbles adhesive, is assembled into test paper by supporting layer, adsorption layer and protective layer successively.
The screening of described M13 bacteriophage is carried out by the following method:
(1) affine screening M13 phage random seven peptide storehouses
Get the elisa plate that is coated with OTA monoclonal antibody, every hole adds the phage peptide library 100 μ L after dilution, Qi ZhonghanBacteriophage 1 × 1011~2×1011Pfu, under room temperature, jog concussion 1h, discards liquid; Precipitation TBST solution washing 10 times, wash awayAfter unconjugated bacteriophage, every hole adds the eluent Gly-HCl of 100 μ LpH2.2, jog concussion 8min under room temperature, and sucking-off is washedDe-liquid, adds neutralization buffer to neutral; Eluent after neutralization is except staying a part to do titer determination, and remaining eluent connectsPlant in the LB nutrient solution in logarithmic growth E.coliER2738 in earlier stage, carry out amplification cultivation; Cultivate 4-in 37 DEG C of concussions5h, 4 DEG C, the centrifugal 10min of 12000rpm add the PEG/NaCl solution that accounts for supernatant 1/6 volume, 4 DEG C of precipitations in supernatantSpend the night; After precipitation, 4 DEG C, the centrifugal 15min of 12000rpm, abandon supernatant, then use TBST solution suspension bacteriophage, adds that to account for TBST moltenThe PEG/NaCl of liquid 1/6 volume, hatches 60min on ice; 4 DEG C of centrifugal 15min, remove supernatant again, and precipitation suspends with TBST solution;
Measure after titre, carry out the 2nd time, the 3rd time and the affine screening of the 4th; The amount that at every turn adds bacteriophage is 1 × 1011~2×1011Pfu, coated antibody amount is respectively 75,50 and 25 μ g/ml; In TBST solution, the content of Tween-20 is from the 2nd sieveSelected and increased to 0.5%, it is identical that all the other steps and the 1st are taken turns screening, and the eluted product after the 4th screening is after titer determination, from total amountOn flat board less than 100 plaques, select some blue plaques, inoculate respectively in the exponential phase E.coli in early stageIn the LB nutrient solution of ER2738, carry out amplification cultivation, purifying is also measured titre;
(2) qualification phage clone
Get the elisa plate that is coated with OTA monoclonal antibody, add the phage clone that will identify, 37 DEG C of effect 1h, usePBST solution washing 6 times, adds the monoclonal antibody of the anti-M13 of horseradish peroxidase-labeled, and 37 DEG C of effect 1h are molten with PBSTLiquid washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction, is determined at the absorbance OD value under 450nm wavelength,Blank is made in the hole that does not add phage clone, to treat 2.1 times of OD value in the absorbance OD value >=blank of gaging hole, judgementPositive;
Then take the sensitiveness of competitive ELISA method qualification positive phage clones, add the OTA dilution of variable concentrationsLiquid and positive bacteriophage, in 37 DEG C of effect 1h, use PBST solution washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4StopReaction, is determined at the absorbance OD value under 450nm wavelength, and blank is made in the hole that does not add phage clone, to treat the suction of gaging holeIn luminosity OD value >=blank 2.1 times of OD value, be judged as the positive; Calculate the combination rate of bacteriophage under each concentration, and markDirectrix curve judges the sensitiveness of phage clone;
(3) DNA sequencing and peptide sequence analysis
Bacteriophage after amplification is checked order, translate amino acid sequence ,-96 of employing according to the DNA sequence dna insertingG III sequencing primer is 5 '-HOCCCTCATAGTTAGCGTAACG-3 ', the amino acid sequence of mimic epitope isMPLWXDL, X is arbitrary amino acid.
Positive beneficial effect of the present invention:
(1) the present invention has realized the application in fast detecting OTA is residual of immunochromatography technique based on M13 bacteriophage,The maximum feature of this test paper is that it is with showing that having the M13 bacteriophage of OTA mimic epitope to replace on traditional detection paper marking printsThe OTA conjugate of system, has avoided the harm of OTA conjugate for the test paper producer, experimenter and environment.
In the present invention, the screening of M13 bacteriophage is taking the monoclonal antibody of anti-OTA as aglucon, from phage random seven peptide storehousesThe mimic epitope of affine screening OTA, and utilize and show have the M13 bacteriophage of OTA mimic epitope to substitute artificial antigen.
(2) high specificity, highly sensitive. Test paper of the present invention is with the monoclonal antibody of colloid gold label high-affinity or many gramsGrand antibody is that basis is prepared from, and in golden labeling antibody, between gold grain and antibody molecule, forms without covalent bond, and the two is by the opposite sexVan der Waals force between electric charge combines, and the specificity of colloid gold label antagonist and affinity impact are very little, and have higherMark rate. Therefore, this test paper has stronger specificity and higher sensitivity, the micro-Aspergillus ochraceus poison of 5ng/ml can be detectedElement A.
(3) security is good. In this test paper, show and have the M13 bacteriophage of OTA mimic epitope to there is nontoxic feature, makeTest paper that must be based on M13 bacteriophage assembling for the test paper producer, tester and environment all without any harm, safety.
(4) easy, quick. Use test paper of the present invention, without other any reagent and instrument, can execute-in-place. Only need byTest paper inserts in test sample 10 ~ 20 seconds, takes out in latter 5 minutes and can judge testing result.
(5) result shows image, directly perceived, accurate. Test paper of the present invention with show redness " ︱ " and " ︱ ︱ " (or " ten ", " ┬ "," ┴ ", " ├ ", " ┤ ") trace as detect the positive and negative marker, on cellulose membrane, show a redness " ︱ " printMark, represents to contain OTA in test sample; Show two redness " ︱ ︱ " trace, represent in test sample not containing OTA. This result canWith the naked eye directly observation, judges image, directly perceived, accurate, simple and clear, is not prone to the artificial erroneous judgement such as false positive and false negative.
(6) cost saving. Use this test paper without separately joining instrument and other reagent, can detect expense whenever and wherever possibleCheap, can save a large amount of expensive instruments and cost of equipment.
(7) applied widely, easy to utilize. The immunochromatography technique that the present invention has realized based on M13 bacteriophage existsApplication in fast detecting OTA, makes the detection of OTA disturb without background; Easy and simple to handle, can meet different levels personnel's needs,Comprise professional chemical examination, customs quarantine control, health quarantine, quality-monitoring, livestock products processing, raiser and consumer individual etc. ThisInvention ensuring food safety, Protection of consumer is extremely important aspect healthy, will have obvious economic benefitAnd social benefit.
Brief description of the drawings
Fig. 1 is the structural representation of immune chromatography test paper of the present invention.
Fig. 2 is the plan structure schematic diagram of immune chromatography test paper in Fig. 1.
In figure, 1 is supporting layer, and 2 is adsorbing fiber layer, and 3 is golden labeling antibody fibrage, and 4 is cellulose rete, and 5 is water suctionMaterial layer, 6 is the stealthy marking that detects, and 7 is the stealthy contrast marking, and 8-1 is test lead diaphragm, and 8-2 is handle end diaphragm, 9For sample mark line.
Detailed description of the invention
The preparation process of test paper of the present invention comprises: preparation, the displaying of OTA monoclonal or polyclonal antibody have OTA simulation tableThe preparation of screening, the fibrolaminar preparation of golden labeling antibody of the M13 bacteriophage of position, the preparation of adsorbing fiber layer, cellulose rete andThe steps such as the assembling of test paper.
1, the preparation of OTA monoclonal antibody or polyclonal antibody
The preparation of monoclonal antibody: the preparation, immune animal (mouse), Fusion of Cells, the monoclonal that comprise OTA artificial antigenThe screening of antibody, the preparation of monoclonal antibody; The preparation of polyclonal antibody: the preparation, the immune animal that comprise OTA artificial antigenThe screening of (white rabbit), polyclonal antibody and the preparation of polyclonal antibody.
(1) preparation of OTA artificial antigen
Get 1mgOTA and be dissolved in ethanol, add 3mgDCC and 2mgNHS, 4 DEG C of lucifuge stirring reaction 12h,The centrifugal 15min of 10000rpm, abandons precipitation, and dry supernatant, is dissolved in desciccate in dimethyl sulfoxide (DMSO), obtains activation and producesThing; Take 10mgBSA, be fully dissolved in the NaHCO of 0.13mol/L3In solution, in 4 DEG C of precoolings; Activation products are dropwise slowSlowly add in the BSA solution that is pre-chilled to 4 DEG C, 4 DEG C of lucifuge concuss 4h, the product 72h that dialyses in PBS, 5000rpm is centrifugal10min, getting supernatant is immunizing antigen OTA-BSA, packing ,-20 DEG C save backup. With legal system for coating antigen.
(2) preparation of OTA monoclonal antibody
With the OTA artificial antigen making with 50 μ g/ consumption immunity BALB/c mouse in 6～8 weeks age only 3～4 times, at every turnImmunity interval 3～4 weeks, determine antibody titer meet the requirements after superpower immunity, 3～4 days afterwards by hole blood sampling under immune mouse socket of the eye,Separate positive serum; De-neck is lethal, and with alcohol-pickled mouse 5～10min sterilization body surface of 75%, aseptic its spleen of getting, by spleenShred and grind, filter through 120 order nylon gauzes, the centrifugal 10min of 1000rpm, collects splenocyte. By 1 × 108Splenocyte withNS0 myeloma cell mixes in the ratio of 10:1, and the centrifugal 10min of 1000rpm, abandons supernatant, cell precipitation thing in 37 DEG C of water-baths,Slowly add the PEG4000 effect 1min of 0.7～1.0mL50%, then slowly add serum-free 1640 culture medium 15mL, withStop the effect of PEG; 37 DEG C of water-bath 5～10min, the centrifugal 10min of 1000rpm, abandons supernatant, and cell precipitation thing is resuspended in to HATSelect in culture medium, and add 96 porocytes to cultivate plate hole (100 μ L/ hole, μ L～200), be placed in 37 DEG C, 5%CO2In incubator, trainSupport 10～14 days, carry out positive hole sizer choosing with indirect elisa method, select strong positive, inhibiting rate is high, Growth of Cells is vigorous hole to enter3 limited dilution clonings of row, then expand and cultivate, and set up hybridoma cell strain. The list of prepared hybridoma secretionClonal antibody can react with OTA specifically, and affinity constant reaches 1010～1012, hypotype is IgG1, for the preparation of golden labeling antibodyGlass fibre cotton.
(3) preparation of OTA polyclonal antibody
With the OTA artificial antigen immunity NZw making, immunizing dose is 200 μ g～500 μ g/ time, and back is subcutaneousDivide 4～6 injections. Immunity 4～5 times, 2～3 weeks, each immune interval, surveys with ELISA method after last immunity for 10～15 daysSurely tire, and identify its sensitiveness and specificity, blood sampling separated and collected serum. Extract IgG with saturated ammonium sulfate salting out method anti-Body, gets 1 part of serum and adds 2 parts of PBS(pH7.2) mix, add equal-volume saturated ammonium sulfate solution and mix, put 4 DEG C of refrigerator 12h, 4DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, then with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to dense eventuallyDegree is 33%, puts 4 DEG C of refrigerator 2h, and 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put 4In DEG C refrigerator with PBS(pH7.2) dialysis 48～72h, liquid is changed for several times in centre, 4 DEG C, the centrifugal 15min of 12000rpm collect supernatant,The anti-OTA polyclonal antibody that obtains purifying ,-20 DEG C frozen, for the preparation of golden labeling antibody glass fibre cotton.
2, show the screening of the M13 bacteriophage that has OTA mimic epitope
(1) affine screening M13 phage random seven peptide storehouses
Get the elisa plate that is coated with OTA monoclonal antibody, every hole adds the phage peptide library 100 μ L after dilution, Qi ZhongyueContaining bacteriophage 1 × 1011~2×1011Pfu, under room temperature, jog concussion 1h, discards liquid; Precipitation TBST solution washing 10 times, washGo after unconjugated bacteriophage, every hole adds the eluent Gly-HCl of 100 μ LpH2.2, jog concussion 8min under room temperature, sucking-offEluent, adds neutralization buffer to neutral; Eluent after neutralization is except staying a part to do titer determination, remaining eluentIn the LB nutrient solution of inoculation in logarithmic growth E.coliER2738 in earlier stage, carry out amplification cultivation; Cultivate in 37 DEG C of concussions4-5h, 4 DEG C, the centrifugal 10min of 12000rpm add the PEG/NaCl solution that accounts for supernatant 1/6 volume in supernatant, and 4 DEG C are heavySpend the night in shallow lake; After precipitation, 4 DEG C, the centrifugal 15min of 12000rpm, abandon supernatant, then use TBST solution suspension bacteriophage, adds and account for TBSTThe PEG/NaCl of solution 1/6 volume, hatches 60min on ice; 4 DEG C of centrifugal 15min, remove supernatant again, and precipitation suspends with TBST solution;
Measure after titre, carry out the 2nd time, the 3rd time and the affine screening of the 4th; The amount that at every turn adds bacteriophage is 1 × 1011~2×1011Pfu, coated antibody amount is respectively 75,50 and 25 μ g/ml; In TBST solution, the content of Tween-20 is from the 2nd sieveSelected and increased to 0.5%, it is identical that all the other steps and the 1st are taken turns screening, and the eluted product after the 4th screening is after titer determination, from total amountOn flat board less than 100 plaques, select some blue plaques, inoculate respectively in the exponential phase E.coli in early stageIn the LB nutrient solution of ER2738, carry out amplification cultivation, purifying is also measured titre;
(2) qualification phage clone
Get the elisa plate that is coated with OTA monoclonal antibody, add the phage clone that will identify, 37 DEG C of effect 1h, usePBST solution washing 6 times, adds the monoclonal antibody of the anti-M13 of horseradish peroxidase-labeled, and 37 DEG C of effect 1h are molten with PBSTLiquid washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction, is determined at the absorbance OD value under 450nm wavelength,Blank is made in the hole that does not add phage clone, to treat 2.1 times of OD value in the absorbance OD value >=blank of gaging hole, judgementPositive;
Then take the sensitiveness of competitive ELISA method qualification positive phage clones, add the OTA dilution of variable concentrationsLiquid and positive bacteriophage, in 37 DEG C of effect 1h, use PBST solution washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4StopReaction, is determined at the absorbance OD value under 450nm wavelength, and blank is made in the hole that does not add phage clone, to treat the suction of gaging holeIn luminosity OD value >=blank 2.1 times of OD value, be judged as the positive; Calculate the combination rate of bacteriophage under each concentration, and markDirectrix curve judges the sensitiveness of phage clone;
(3) DNA sequencing and peptide sequence analysis
Bacteriophage after amplification is checked order, translate amino acid sequence ,-96 of employing according to the DNA sequence dna insertingG III sequencing primer is 5 '-HOCCCTCATAGTTAGCGTAACG-3 ', the amino acid sequence of mimic epitope isMPLWXDL, X is arbitrary amino acid.
3, the fibrolaminar preparation of golden labeling antibody
Adopt natrium citricum reducing process to prepare colloidal gold solution. At 0.01 ~ 0.02%(w/v of boiling) gold chloride is water-solubleIn liquid, add 1%(w/v) the new sodium citrate solution of preparing, after reaction, the colloidal gold solution of acquisition diameter 15 ~ 20nm, usesThe K of 0.1mol/L2CO3Regulate pH value to 8.5 ~ 9.5, be placed in 2 ~ 8 DEG C and save backup.
With the mark ratio of 1:2000, OTA monoclonal antibody to be marked or polyclonal antibody are added to the glue of pH8.5 ~ 9.5In body gold solution, after mark 10min, add 20%(w/v) PEG10000 to final concentration be 0.05%, 4 DEG C, 1500 ~ 3000rpmCentrifugal 20min, removes unconjugated colloid gold particle, and 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtains the gold mark of preliminary purificationAntibody protein mixture, then use propylene glucan S-400 column chromatography, after separation and purification, obtain the OTA antibody of colloid gold label. WillThe colloidal gold labeled monoclonal antibody of 1:100 ~ 1:500 dilution is adsorbed in processed glass fiber cotton, 4 DEG C of low-temperature vacuum dryings, preparation goldLabeling antibody glass fibre cotton.
4, the preparation of adsorbing fiber layer
Test lead adsorbing fiber is glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film preparation for layer, willFibrous material is cut into the band of wide 1.5cm specification, puts it in sample pad confining liquid and soaks 30min, in 37 DEG C of oven dry, standbyWith.
5, the preparation of cellulose rete
Cellulose for rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make, cut into wide 1.5cm ruleThe band of lattice, with point sample instrument on cellulose membrane diverse location respectively specking show have M13 bacteriophage and the sheep of OTA mimic epitopeAnti-mouse IgG antibody (or the anti-mouse IgG of rabbit, goat anti-rabbit igg antibody), makes stealthy detection trace band and contrast trace band,In 37 DEG C of dry for standby.
Wherein the preparation method of the anti-mouse IgG of goat-anti or rabbit (or goat anti-rabbit igg) antibody is as follows:
Extract the negative mice serum IgG(of OTA or negative rabbit anteserum IgG with saturated ammonium sulfate), get 1 part of mice serum (orRabbit anteserum), add 2 parts of PBS(pH7.2) mix, add isopyknic saturated ammonium sulfate solution and mix, put 4 DEG C of refrigerator 12h, 4 DEG C,The centrifugal 15min of 2500rpm, abandons supernatant; Again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration and be33%, put 4 DEG C of refrigerator 2h, 4 DEG C, the centrifugal 15min of 2500rpm, abandon supernatant; With appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of iceIn case, with PBS(pH7.2) dialysis 48h, liquid is changed 3 times in centre, and 4 DEG C, the centrifugal 15min of 12000rpm collect supernatant, divide with ultravioletIts protein concentration of light photometric determination; With mice serum (or rabbit anteserum) IgG of 50 μ g～100 μ g/kg body weight through subcutaneous and fleshMeat is injected healthy goat or rabbit 3～4 times, and last injection is after 10 days, measures more than its serum titer reaches 1:2000 with ELISATime, heart or arterial blood drawing, separated and collected hyper-immune serum, extracts goat-anti or the anti-mouse IgG of rabbit (or goat-anti rabbit with saturated ammonium sulfateIgG) antibody (method is identical with extraction mice serum IgG, no longer repeats), for the preparation of OTA Test paper contrast trace.
6, the assembling of immune chromatography test paper
By adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete, absorbent material layer be attached to successively from left to right withOn the supporting layer of adhesive, and be cut into the wide test paper of 3-4cm.
7, the detection reaction principle of immune chromatography test paper
When test paper test lead inserts after testing sample solution, solution to be measured drives OTA to be measured and gold mark by siphonageGolden labeling antibody in antibody fibrage spreads to cellulose rete together, and finally infiltrates the absorbent material layer of handle end. ExpandingIn loose process, OTA to be measured can combine with golden labeling antibody, and then seals the antigen-combining site of OTA on golden labeling antibody, stops gold markAntibody is shown on cellulose membrane has the detection trace of the M13 of OTA mimic epitope bacteriophage to be combined, and can not show detection trace,The anti-mouse IgG of sheep or rabbit (or goat anti-rabbit igg) antibody can be combined with golden labeling antibody, forms red contrast trace band " ︱ ",, a positive expression of red zone trace; Otherwise during without OTA, can not stop on golden labeling antibody and cellulose membrane in sample solutionDisplaying have the detection trace combination of the M13 bacteriophage of OTA mimic epitope, show the red trace band that detects, same goat-anti or rabbitAnti-mouse IgG (or goat anti-rabbit igg) antibody is also combined with golden labeling antibody, shows red contrast trace band " ︱ ", forms two rednessBand " ︱ ︱ " negative expression. If show without any red trace band on cellulose membrane, show that test paper lost efficacy.
Illustrate by the following examples the structure of test paper of the present invention.
Embodiment mono-: based on the immune chromatography test paper of M13 bacteriophage fast detecting ochratoxin A, referring to Fig. 1, Fig. 2.In figure, 1 is supporting layer, makes with plastic slice bar, and 2 is adsorbing fiber layer, makes golden labeling antibody fibrage 3 with glass fibre cottonUpper absorption has the golden labeling antibody glass fibre cotton of anti-OTA monoclonal antibody, and cellulose rete 4 adopts nitrocellulose filter, handleThe absorbent material layer 5 use absorbent filters of end are made, by adsorbing fiber layer 2, golden labeling antibody fibrage 3, cellulose rete 4, water suctionEach layer of material layer 5 is pasted and fixed on supporting layer 1 from left to right successively, the intersection fiber infiltration that crosses one another each other. ?Cellulose rete 4 is provided with the stealthy trace 6 that detects, and the stealthy trace that detects is with showing the M13 phagocytosis system that has OTA mimic epitopeBecome; Stealthy contrast trace 7 use goat anti-mouse igg antibody solution trace on cellulose membrane is made, and two trace bands are arranged in parallel,Form combination trace band " ︱ ︱ ".
8-1 covers adsorbing fiber layer 2 and golden labeling antibody fibrage 3 sample end diaphragm (white) above, and 8-2 isHandle end diaphragm, covers above absorbent material layer 5, is other color (as yellow), and 9 is sample mark line, this mark lineBe positioned at the white diaphragm that adsorbing fiber layer 2 is corresponding with golden labeling antibody fibrage 3 intersections and be partial to adsorbing fiber layer 2 one side approximately0.5cm place is printed on arrow and Max printed words on the diaphragm of mark line right side.
The preparation of testing sample and detecting step:
Detect corn: by sample pulverize, levigate, take 5g sample, dissolve play with methyl alcohol-PBS solution of 12.5ml70%Violent shock swings 5 minutes, after extract is filtered with whatman1 filter paper, gets 1ml filtrate and adds in 1mlPBS, stand-by.
Method of operating: OTA test paper sample end is inserted in testing sample, and insertion depth is no more than mark line, approximately 10～20Take out test paper second, horizontal positioned, observes in 5min and judges testing result.
Result is judged: if (a) positive shows a red trace band " ︱ " on cellulose membrane, represent testing resultPositive, illustrate and in testing sample, contain OTA; (b) if feminine gender shows two red trace bands on cellulose membrane" ︱ ︱ ", represents that testing result is negative, illustrates in testing sample not containing OTA; (c) do not have on cellulose membrane if lost efficacyRed zone shows, shows that test paper lost efficacy.
Embodiment bis-: test paper structure and embodiment mono-are basic identical, and difference is: golden labeling antibody fibrage 3 adsorbsHave the polyclonal antibody of anti-OTA, adsorbing fiber layer 2 use nylon membrane are made, and cellulose rete 4 adopts pure cellulose film, stealthy inspectionSurvey trace band and be " ten " with stealthy contrast trace band, handle end diaphragm 8-2 is blue look.
Detect flour: sample preparation methods is with embodiment mono-.
Result is judged: if (a) positive shows a red trace band " ten " on cellulose membrane, represent to detect knotFruit is positive, illustrates and in testing sample, contains OTA; (b) if feminine gender shows two red trace bands on cellulose membrane" ten ", represent that testing result is negative, illustrate in testing sample not containing OTA; (c) do not have on cellulose membrane if lost efficacyRed zone shows, shows that test paper lost efficacy. Method of operating is with embodiment mono-.
Embodiment tri-: test paper structure and embodiment mono-are basic identical, and difference is: adsorbing fiber layer 2 use poly-inclined to one side twoPVF pvdf membrane is made, and the anti-mouse IgG antibody solution of stealthy contrast trace 7 use rabbit is made on cellulose membrane, cellulose membraneLayer 4 adopts carboxylation cellulose membrane, and handle end diaphragm 8-2 is green, and stealthy detection trace band and stealthy contrast trace band are“┬”。
Detect feed: sample preparation methods, result judgement and method of operating are all with embodiment mono-.
Embodiment tetra-: test paper structure and embodiment mono-are basic identical, and difference is: adsorbing fiber layer 2 use polyester filmMake, cellulose rete 4 adopts carboxylation cellulose membrane, detects trace band and contrast trace band to be " ┴ ". Method of operating and knotFruit decision method is with example one.
Embodiment five: test paper structure and embodiment mono-are basic identical, and difference is: adsorbing fiber layer 2 use nylon membraneMake, stealthy contrast trace 7 use goat anti-rabbit igg antibody solution are made on cellulose membrane. Detect trace band and contrast trace bandBe " ├ ". Detect sample, result judgement and method of operating with example one.
Embodiment six: and embodiment mono-is basic identical, difference is: golden labeling antibody fibrage 3 absorption have anti-OTA'sPolyclonal antibody, detects trace band and contrast trace band is " ┤ ".
SEQUENCELISTING
<110>Henan Academy of Agricultural Sciences
<120>immune chromatography test paper and the preparation method based on M13 bacteriophage detection ochratoxin A
<130>immunochromatography technique
<160>2
<170>PatentInversion3.4
<210>1
<211>7
<212>PRT
<213>artificial sequence
<400>1
MetProLeuTrpXaaAspLeu
15
<210>2
<211>20
<212>DNA
<213>artificial sequence
<400>2
ccctcatagttagcgtaacg20
Claims (9)
1. detect an immune chromatography test paper for ochratoxin A based on M13 bacteriophage, comprise supporting layer, the intermediate layer of bottomAdsorption layer and be fixed on the protective layer on adsorption layer, adsorption layer is followed successively by adsorbing fiber layer, golden labeling antibody fiber from test leadThe absorbent material layer of layer, cellulose rete and handle end, is characterized in that: on cellulose rete, be provided with showing OTA simulationThe stealth that the M13 bacteriophage of epi-position is printed detects trace, is also provided with goat-anti or the anti-mouse IgG of rabbit or goat anti-rabbit igg antibody moltenThe stealth contrast trace that liquid is printed; Described golden labeling antibody fibrage adopts the glass fibre cotton of ADSORPTION OF GOLD labeling antibody to make, Jin BiaoAntibody adopts monoclonal antibody or the polyclonal antibody of the OTA of Au colloidal nanoparticles mark; Described golden labeling antibody is fibrolaminarPreparation method is as follows: adopt natrium citricum reducing process to prepare colloidal gold solution, 0.01～0.02% (w/v) chlorine gold seething with excitementIn aqueous acid, add the sodium citrate solution of the new preparation of 1% (w/v), the collaurum that obtains diameter 15～20nm after reaction is moltenLiquid, with the K of 0.1mol/L2CO3Regulate pH value to 8.5～9.5, be placed in 2～8 DEG C and save backup;
With the mark ratio of 1:2000, OTA monoclonal antibody to be marked or polyclonal antibody are added to the colloid of pH8.5～9.5In gold solution, after mark 10min, the PEG10000 that adds 20% (w/v) to final concentration be 0.05%, 4 DEG C, 1500～The centrifugal 20min of 3000rpm, removes unconjugated colloid gold particle, and 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtains tentatively pureThe golden labeling antibody protein mixture of changing, then use propylene glucan S-400 column chromatography, obtains colloid gold label after separation and purificationOTA antibody;
The preparation of OTA monoclonal antibody or polyclonal antibody:
The preparation of monoclonal antibody: comprise preparation, immune animal, the Fusion of Cells of OTA artificial antigen, the sieve of monoclonal antibodyThe preparation of choosing, monoclonal antibody; The preparation of polyclonal antibody: the preparation, immune animal, the Anti-TNF-α that comprise OTA artificial antigenThe screening of body and the preparation of polyclonal antibody;
Wherein, the preparation of OTA artificial antigen:
Get 1mgOTA and be dissolved in ethanol, add 3mgDCC and 2mgNHS, 4 DEG C of lucifuge stirring reaction 12h, 10000rpm fromHeart 15min, abandons precipitation, and dry supernatant, is dissolved in desciccate in dimethyl sulfoxide (DMSO), obtains activation products; Take 10mgBSA, is fully dissolved in the NaHCO of 0.13mol/L3In solution, in 4 DEG C of precoolings; Activation products are dropwise slowly added and are pre-chilled to 4DEG C BSA solution in, 4 DEG C of lucifuge concuss 4h, the product 72h that dialyses in PBS, the centrifugal 10min of 5000rpm, gets supernatant and isImmunizing antigen OTA-BSA, packing ,-20 DEG C save backup.
2. immune chromatography test paper according to claim 1, is characterized in that: the peptide sequence of described OTA mimic epitope isMPLWXDL, X is arbitrary amino acid.
3. immune chromatography test paper according to claim 1 and 2, is characterized in that: described M13 bacteriophage is sieved by following methodsChoosing, concrete steps are:
(1) affine screening M13 phage random seven peptide storehouses
Get the elisa plate that is coated with OTA monoclonal antibody, every hole adds the phage peptide library 100 μ L after dilution, wherein containing phagocytosisBody 1 × 1011～2×1011Pfu, under room temperature, jog concussion 1h, discards liquid; Precipitation TBST solution washing 10 times, wash away notIn conjunction with bacteriophage after, every hole adds the eluent Gly-HCl of 100 μ LpH2.2, jog concussion 8min under room temperature, sucking-off wash-outLiquid, adds neutralization buffer to neutral; Eluent after neutralization is except staying a part to do titer determination, and remaining eluent is inoculatedIn LB nutrient solution in logarithmic growth E.coliER2738 in earlier stage, carry out amplification cultivation; Cultivate 4-5h, 4 in 37 DEG C of concussionsDEG C, the centrifugal 10min of 12000rpm, in supernatant, add the PEG/NaCl solution that accounts for supernatant 1/6 volume, 4 DEG C of precipitations are spent the night;After precipitation, 4 DEG C, the centrifugal 15min of 12000rpm, abandon supernatant, then use TBST solution suspension bacteriophage, adds and account for TBST solution 1/6The PEG/NaCl of volume, hatches 60min on ice; 4 DEG C of centrifugal 15min, remove supernatant again, and precipitation suspends with TBST solution;
Measure after titre, carry out the 2nd time, the 3rd time and the affine screening of the 4th; The amount that at every turn adds bacteriophage is 1 × 1011～2×1011Pfu, coated antibody amount is respectively 75,50 and 25 μ g/ml; In TBST solution, the content of Tween-20 is from the 2nd screeningIncrease to 0.5%, it is identical that all the other steps and the 1st are taken turns screening, and the eluted product after the 4th screening is after titer determination, from total amount notTo the flat board of 100 plaques, select some blue plaques, inoculate respectively in the exponential phase E.coli in early stageIn the LB nutrient solution of ER2738, carry out amplification cultivation, purifying is also measured titre;
(2) qualification phage clone
Get the elisa plate that is coated with OTA monoclonal antibody, add the phage clone that will identify, 37 DEG C of effect 1h, molten with PBSTLiquid washs 6 times, adds the monoclonal antibody of the anti-M13 of horseradish peroxidase-labeled, and 37 DEG C of effect 1h, use PBST solution washing6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction, is determined at the absorbance OD value under 450nm wavelength, does not add and bitesBlank is made in thalline clone's hole, to treat 2.1 times of OD value in the absorbance OD value >=blank of gaging hole, is judged as sunProperty;
Then take the sensitiveness of competitive ELISA method qualification positive phage clones, add variable concentrations OTA dilution andPositive bacteriophage, in 37 DEG C of effect 1h, uses PBST solution washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction,Be determined at the absorbance OD value under 450nm wavelength, blank is made in the hole that does not add phage clone, to treat the absorbance OD of gaging holeIn value >=blank 2.1 of OD value times, be judged as the positive; Calculate the combination rate of bacteriophage under each concentration, and make calibration curveJudge the sensitiveness of phage clone;
(3) DNA sequencing and peptide sequence analysis
Bacteriophage after amplification is checked order, translate amino acid sequence according to the DNA sequence dna inserting, employing-survey of 96g IIIOrder primer is 5 '-HOCCCTCATAGTTAGCGTAACG-3 ', the amino acid sequence of mimic epitope is MPLWXDL, X is for appointingMeaning amino acid.
4. immune chromatography test paper according to claim 1 and 2, is characterized in that: described adsorbing fiber glass fibre cotton for layer,Nylon membrane, PVDF membrane or polyester film are made; Absorbent material layer is made with absorbent filter, and supporting layer is tough with what do not absorb waterProperty material is made; Cellulose for rete nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane make.
5. immune chromatography test paper according to claim 1 and 2, is characterized in that: described stealthy detect trace and stealthy contrastTrace is that " ︱ ︱ " orthoscopic trace, " 10 " font of being arranged in parallel are arranged trace, " ┬ ┬ " font is arranged trace, " ┴ ┴ " wordType is arranged trace, " ├ ├ " font arranges trace or " ┤ ┤ " font is arranged trace.
6. immune chromatography test paper according to claim 1 and 2, is characterized in that: described protective layer is for covering adsorbing fiberDiaphragm on layer, golden labeling antibody fibrage and absorbent material layer, at adsorbing fiber layer and golden labeling antibody fibrage intersection pairOn the diaphragm of answering, be printed with sample mark line, this mark line deflection adsorbing fiber layer one side 0.3-0.7cm.
7. immune chromatography test paper according to claim 3, is characterized in that: in described PEG/NaCl solution, PEG-8000's is denseDegree is 20% (w/v), and the concentration of NaCl is 2.5mol/L.
8. a preparation method for immune chromatography test paper described in claim 1, is characterized in that: the method comprises the following steps:
(1) preparation of OTA monoclonal antibody or polyclonal antibody;
(2) screening of M13 bacteriophage;
(3) preparation of golden labeling antibody: add 1% (w/v) newly to join in 0.01～0.02% (w/v) aqueous solution of chloraurate of boilingThe sodium citrate solution of system, the colloidal gold solution of acquisition diameter 15～20nm after reaction, with the K of 0.1mol/L2CO3Regulate pH valueTo 8.5～9.5, be placed in 2～8 DEG C and save backup;
With the mark ratio of 1:2000, OTA monoclonal antibody to be marked or polyclonal antibody are added to the colloid of pH8.5～9.5In gold solution, the PEG10000 that adds 20% (w/v) after mark 10min to final concentration be 0.05%, in 4 DEG C, 1500～The centrifugal 20min of 3000rpm, removes unconjugated colloid gold particle, and 4 DEG C, the centrifugal 1h of 15000rpm, abandon supernatant, obtains tentatively pureThe golden labeling antibody protein mixture of changing, then use propylene glucan S-400 column chromatography, obtains colloid gold label after separation and purificationOTA antibody;
(4) preparation of adsorbing fiber layer: adsorbing fiber is glass fibre cotton, nylon membrane, PVDF membrane or polyester film for layerMake;
(5) preparation of cellulose rete: cellulose is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane system for reteBecome, with point sample instrument diverse location specking detection trace and contrast trace respectively on cellulose membrane, then dry;
(6) assembling of test paper: adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete, absorbent material layer are complied with from left to rightInferior being attached on the supporting layer that scribbles adhesive, is assembled into test paper by supporting layer, adsorption layer and protective layer successively.
9. preparation method according to claim 8, is characterized in that: the screening of described M13 bacteriophage is to enter by the following methodRow:
(1) affine screening M13 phage random seven peptide storehouses
Get the elisa plate that is coated with OTA monoclonal antibody, every hole adds the phage peptide library 100 μ L after dilution, wherein containing phagocytosisBody 1 × 1011～2×1011Pfu, under room temperature, jog concussion 1h, discards liquid; Precipitation TBST solution washing 10 times, wash away notIn conjunction with bacteriophage after, every hole adds the eluent Gly-HCl of 100 μ LpH2.2, jog concussion 8min under room temperature, sucking-off wash-outLiquid, adds neutralization buffer to neutral; Eluent after neutralization is except staying a part to do titer determination, and remaining eluent is inoculatedIn LB nutrient solution in logarithmic growth E.coliER2738 in earlier stage, carry out amplification cultivation; Cultivate 4-5h, 4 in 37 DEG C of concussionsDEG C, the centrifugal 10min of 12000rpm, in supernatant, add the PEG/NaCl solution that accounts for supernatant 1/6 volume, 4 DEG C of precipitations are spent the night;After precipitation, 4 DEG C, the centrifugal 15min of 12000rpm, abandon supernatant, then use TBST solution suspension bacteriophage, adds and account for TBST solution 1/6The PEG/NaCl of volume, hatches 60min on ice; 4 DEG C of centrifugal 15min, remove supernatant again, and precipitation suspends with TBST solution;
Measure after titre, carry out the 2nd time, the 3rd time and the affine screening of the 4th; The amount that at every turn adds bacteriophage is 1 × 1011～2×1011Pfu, coated antibody amount is respectively 75,50 and 25 μ g/ml; In TBST solution, the content of Tween-20 is from the 2nd screeningIncrease to 0.5%, it is identical that all the other steps and the 1st are taken turns screening, and the eluted product after the 4th screening is after titer determination, from total amount notTo the flat board of 100 plaques, select some blue plaques, inoculate respectively in the exponential phase E.coli in early stageIn the LB nutrient solution of ER2738, carry out amplification cultivation, purifying is also measured titre;
(2) qualification phage clone
Get the elisa plate that is coated with OTA monoclonal antibody, add the phage clone that will identify, 37 DEG C of effect 1h, molten with PBSTLiquid washs 6 times, adds the monoclonal antibody of the anti-M13 of horseradish peroxidase-labeled, and 37 DEG C of effect 1h, use PBST solution washing6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction, is determined at the absorbance OD value under 450nm wavelength, does not add and bitesBlank is made in thalline clone's hole, to treat 2.1 times of OD value in the absorbance OD value >=blank of gaging hole, is judged as sunProperty;
Then take the sensitiveness of competitive ELISA method qualification positive phage clones, add variable concentrations OTA dilution andPositive bacteriophage, in 37 DEG C of effect 1h, uses PBST solution washing 6 times; Add TMB nitrite ion, add the H of 2mol/L2SO4Cessation reaction,Be determined at the absorbance OD value under 450nm wavelength, blank is made in the hole that does not add phage clone, to treat the absorbance OD of gaging holeIn value >=blank 2.1 of OD value times, be judged as the positive; Calculate the combination rate of bacteriophage under each concentration, and make calibration curveJudge the sensitiveness of phage clone;
(3) DNA sequencing and peptide sequence analysis
Bacteriophage after amplification is checked order, translate amino acid sequence according to the DNA sequence dna inserting, employing-survey of 96g IIIOrder primer is 5 '-HOCCCTCATAGTTAGCGTAACG-3 ', the amino acid sequence of mimic epitope is MPLWXDL, X is for appointingMeaning amino acid.
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| CN108802381B (en) * | 2018-06-13 | 2021-03-12 | 河南中泽生物工程有限公司 | Bovine viral diarrhea virus identification and detection test strip and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN202814981U (en) * | 2012-08-09 | 2013-03-20 | 河南省农业科学院 | Test strip for quickly detecting trace amount of chlorothalonil |
| CN103145808A (en) * | 2013-03-06 | 2013-06-12 | 南昌大学 | Antigen mimic epitope of ochratoxin A and application thereof |
| CN103728451A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN203881773U (en) * | 2014-04-29 | 2014-10-15 | 河南省农业科学院 | M13-phage-based immunochromatography test paper for detecting ochratoxin A |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202814981U (en) * | 2012-08-09 | 2013-03-20 | 河南省农业科学院 | Test strip for quickly detecting trace amount of chlorothalonil |
| CN103145808A (en) * | 2013-03-06 | 2013-06-12 | 南昌大学 | Antigen mimic epitope of ochratoxin A and application thereof |
| CN103728451A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN203881773U (en) * | 2014-04-29 | 2014-10-15 | 河南省农业科学院 | M13-phage-based immunochromatography test paper for detecting ochratoxin A |
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| Development of a colloidal gold strip for rapid detection of ochratoxin A;Weihua Lai et al;《Food Control》;20091231;第791–795页 * |
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