CN101413955B - ELISA test box for detecting zearalenone and preparing and detecting method thereof - Google Patents
ELISA test box for detecting zearalenone and preparing and detecting method thereof Download PDFInfo
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Abstract
The invention relates to an ELISA kit for detecting zearalenone, the detection is rapid, sensitive, accurate, quantitative, simple in operation, low in requirements on sample purity and strong in specificity, thereby being particularly applicable to the detection of large quantities of samples; and the invention also provides a preparation of the kit and a detection method. The kit comprises washing liquid, color developing liquid A, color developing liquid B and stop solution, and the kit is characterized in that: the kit also comprises a coated plate, a zearalenone standard product, a zearalenone monoclonal antibody freeze-dried product and an enzyme-labeled goat anti-mouse antibody free-dried product; when in detection, the coated plate is taken, 50mu1-100mu1 of the ZEN standard product or a well processed sample is added into the respective micropores, 50mul-100mul of the anti-ZEN antibody is added, the incubation is carried out at 35 DEG C-45 DEG C for about 0.5 hour-1 hour, the washing liquid is used for washing for 3 times-5 times, 50mu1-100mu1 of the horseradish peroxidase (HRP)-goat anti-mouse antibody is added, the incubation is carried out at about 35 DEG C-45 DEG C forabout 0.5 hour-1 hour, the washing liquid is used for washing for 3 times-5 times, 50mu1-100mu1 of the color developing liquid A and 50mu1-100mu1 of the color developing liquid B are added, the mixture is placed still in the dark for 10 minutes-20 minutes, then the stop solution is added, the absorbance value is measured at 450nm, and the ZEN content in the sample is calculated from a standard curve.
Description
(1) technical field
The present invention relates to biological technical field, the ELISA that is specially zearalenone is with testing cassete and preparation and detection method.
(2) background technology
Zearalenone (ZEN) can be produced by multiple sickle-like bacteria such as Fusarium graminearum, fusarium culmorum, pink sickle-like bacteria, fusarium moniliforme, fusarium tricinctum, Fusarinm solani, Fusarium equiseti, Fusarium oxysporum.These sickle-like bacteria are mainly infected corn under state of nature, cause the virus and band poison of corn.Particularly, harvest corn more is subject to infection when overcast and rainy when running into season.Except that corn, wheat, barley, oat also can infect these sickle-like bacteria and be with poison.ZEN is a kind female hormone toxin; Can produce the hyperfunction toxin property reaction of female hormone behind the animal ingestion; Zearalenone has stronger genotoxicity and teratogenesis, can cause animal generation poisoning by estrogen disease, and cardinal symptom has vagina and mammary gland swelling, uterus enlargement, turns up, causes the infertile or miscarriage of animal; Particularly the influence of pig, ox and sheep is bigger to poultry, brings economic loss to animal husbandry; Zearalenone can directly get in the humans and animals body through the crops such as cereal that pollute; Also can pass through animal foods such as contaminated meat, milk and get into human body, that its symptom that causes that the people poisons mainly shows as is unable, the disorder of headache, dizziness, vomiting, diarrhoea and central nervous system.
Limiting the quantity of of ZEN is 60 μ g/kg in " grain hygienic standard " regulation wheat of China's revision, the corn.To limit the quantity of be 20～100 μ g/kg to the maximum of zearalenone in No. 856/2005 regulation cereal foods of EU Committee rule and the cereal, comes into force on July 1st, 2006.Also have at present many countries to formulate the limit standard of zearalenone, must not surpass 60 μ g/kg in Austria's regulation wheat, naked barley, the hard wheat, states such as Brazil, France, Romania, Russia, Uruguay have also formulated corresponding limit standard.Therefore, in order to ensure the healthy of people, must the ZEN in the food be monitored.At present; The physico-chemical analysis technology of ZEN mainly contains several different methods such as thin-layered chromatography, gas liquid chromatography, mass spectrum and nuclear magnetic resonance; Owing to need special technician, testing cost costliness when the sample pre-treatments more complicated of above all analytical approachs, operation, be unfavorable for applying.
(3) summary of the invention
To the problems referred to above; The invention provides a kind of ELISA testing cassete that detects zearalenone, it detects fast, sensitive, accurately, can be quantitative, easy and simple to handle, need not valuable instrument and equipment, and less demanding to sample purity; High specificity; Simplified sample pretreatment and purge process, be specially adapted to the detection of batch samples, the present invention also provides the preparation and the detection method of testing cassete for this reason.
A kind of ELISA testing cassete that detects zearalenone; It comprises cleansing solution, colour developing liquid A, colour developing liquid B and stop buffer, and it is characterized in that: it also comprises the sheep anti-mouse antibody dried frozen aquatic products that encapsulates plate, zearalenone standard items, zearalenone monoclonal antibody dried frozen aquatic products and enzyme labeling.
A kind of preparation that detects the ELISA testing cassete of zearalenone: it is characterized in that: it may further comprise the steps, and encapsulates the preparation of sheep anti-mouse antibody dried frozen aquatic products of preparation, the enzyme labeling of preparation, the zearalenone monoclonal antibody dried frozen aquatic products of preparation, the zearalenone standard items of plate.
It is further characterized in that: the said plate that encapsulates encapsulates solid phase antigen, and it can adopt 96 or 48 or 24 hole microwell plates, with 10mmol/L～100mmol/L pH9～10 Na
2CO
3-NaHCO
3Damping fluid as coating buffer, vomitoxin-ovalbumin (ZEN-OVA) is diluted to 0.1 μ g/mL～1.0 μ g/mL, 96 or 48 or 24 each hole of hole microwell plate add 100 μ l; 2 ℃～8 ℃ placements are spent the night, and discard coating buffer, wash 2 times～5 times; Contain 1%～5% bovine serum albumin(BSA) by adding, the phosphate buffer of pH7～8,2 ℃～8 ℃ sealings are spent the night; Discard confining liquid, dry up, rearmounted-20 ℃～-40 ℃ freezing preservations of lath sealing;
Said zearalenone standard items dilute from the pure article of zearalenone and obtain, and dilution is a deionized water, and ZEN concentration is: 0.001ng/mL～50ng/mL;
The preparation of zearalenone-bovine serum albumin(BSA) (ZEN-BSA) conjugate: ZEN is dissolved in 3mL～100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml～100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5～9.5, extracts 3 times～5 times with methylene chloride again, each 10ml～20ml; Discard methylene chloride, it is about 2.5～3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times～6 times with methylene chloride again; Each 10ml～20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add 3ml～50ml dioxane, add chloro-carbonic acid second butyl ester, 5 ℃～12 ℃ following stirring reactions 30 minutes～45 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 5ml～50ml distilled water and the 5ml～50ml dioxane then, and using sodium hydroxide solution to transfer pH is 8～9; 5 ℃～12 ℃ following stirring reactions 5 hours～6 hours then, are dialysed with distilled water; Change liquid 3 times～5 times, freeze drying, conjugate-20 ℃～-40 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=(10～1000) mg: (10～2000) mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: BSA=(10～1000) mg: (10～1000) μ l: (10-3000) mg.
The preparation of zearalenone-ovalbumin (ZEN-OVA) conjugate: ZEN is dissolved in 3mL～100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml～100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5～9.5, extracts 3 times～5 times with methylene chloride again, each 10ml～20ml; Discard methylene chloride, it is about 2.5～3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times～6 times with methylene chloride again; Each 10ml～20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add 3ml～50ml dioxane, add chloro-carbonic acid second butyl ester, 5 ℃～12 ℃ following stirring reactions 30 minutes～45 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 5ml～50ml distilled water and the 5ml～50ml dioxane then, and using sodium hydroxide solution to transfer pH is 8～9; 5 ℃～12 ℃ following stirring reactions 5 hours～6 hours then, are dialysed with distilled water; Change liquid 3 times～5 times, freeze drying, conjugate-20 ℃～-40 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=(10～1000) mg: (10～2000) mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: OVA=(10～1000) mg: (10～1000) μ l: (10-3000) mg.
The preparation of said anti-zearalenone (ZEN) monoclonal antibody and solution thereof: get the ZEN-BSA conjugate and be made into 0.1 μ g/ μ l～10 μ g/ μ l antigenic solutions with physiological saline, with the complete freund adjuvant mixing of equal-volume, fully emulsified; Confession first immunisation usefulness replaces complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, and first immunisation adopts directly injection in the mouse peritoneal; Immunizing dose is 10 μ g/～100 a μ g/ mouse, and later on whenever at a distance from all booster immunizations in 2 week～4 once, booster immunization adopts tail vein injection; Immunizing dose 10 μ g/ only～100 μ g/ only, intrasplenic injection is adopted in last immunity, gets spleen after 4 days and merges; The immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase is with the 10:1 mixed, centrifugal, remove supernatant; In 50S～90S, 0.1ml～10ml50% polyglycol (molecular weight is 1500) is added in the cell, fully mixing makes its fusion; Add 10ml～50ml DMEM nutrient solution after 1 minute～3 minutes; Stop merging, water-bath is centrifugal after static 10 minutes～30 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 5～30% calf serums, be 1 * 10 with final concentration
4～1 * 10
6Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%～10%CO
2, cultivate under 35 ℃～45 ℃ conditions, after 5 days～10 days; Every culture hole is changed the 2/3HT nutrient solution, after 10 days～20 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with vomitoxin-ovalbumin (ZEN-OVA); With the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A is blank)/(blank) > of A contrast-A; 2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask; Accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again; Inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To 10 week age～13 Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/～0.5ml/ in all ages only, after 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal deposition, and glycerol adding is in-20 ℃～-40 ℃ preservations; The ratio of above-mentioned reacted constituent is: ZEN-BSA: physiological saline=(10～1000) μ g: (0.1～10) ml.
The sheep anti-mouse antibody dried frozen aquatic products of said enzyme labeling is horseradish peroxidase-sheep anti-mouse antibody dried frozen aquatic products; Said cleansing solution is for containing tween and NaN
3PBS; Said colour developing liquid A is the citric acid-disodium hydrogen phosphate buffer solution that contains hydrogen peroxide; Said colour developing liquid B is the ethanolic solution of tetramethyl biphenyl diamines; Said stop buffer is a sulfuric acid solution.
The enzyme-linked immunosorbent assay method of zearalenone is characterized in that: get the micropore that is coated with ZEN-OVA and encapsulate plate, add the 50 μ L～ZEN standard items of 100 μ L or the sample of handling well in micropore separately; Add 50 μ L～anti-ZEN antibody of 100 μ L; Hatched 0.5 hour～1 hour for 35 ℃～45 ℃, cleansing solution is washed 3 times～5 times, adds 50 μ l～100 μ l horseradish peroxidase (HRP)-sheep anti-mouse antibodies; Hatched 0.5 hour～1 hour for 35 ℃～45 ℃; Wash 3 times～5 times with cleansing solution, add 50 μ L～100 μ L colour developing liquid A and 50 μ L～100 μ L colour developing liquid B, the dark place is left standstill after 10 minutes～20 minutes and is added stop buffer; Survey its absorbance, the ZEN content from the typical curve calculation sample at the 450nm place.
The enzyme-linked immunosorbent assay method of zearalenone of the present invention is easy and simple to handle, detects quick, sensitivity, accurate, and the special test box is easy to use, price is low, is applicable to the detection of batch samples.
(4) description of drawings
Figure
is a zearalenone ZEN canonical plotting.
(5) embodiment
A kind of ELISA testing cassete that detects zearalenone; It comprises cleansing solution 1, colour developing liquid A, colour developing liquid B and stop buffer 10, and it also comprises the sheep anti-mouse antibody dried frozen aquatic products 6 that encapsulates plate 12, zearalenone standard items 11, zearalenone monoclonal antibody dried frozen aquatic products 5 and enzyme labeling.
The preparation of testing cassete: it may further comprise the steps, and encapsulates the preparation of sheep anti-mouse antibody dried frozen aquatic products of preparation, the enzyme labeling of preparation, the zearalenone monoclonal antibody dried frozen aquatic products of preparation, the zearalenone standard items of plate.
The process of testing cassete is described below in conjunction with embodiment:
Embodiment 1
Encapsulate plate and encapsulate solid phase antigen, it can adopt 96 or 48 or 24 hole microwell plates, uses 10mmol/LpH9.5 Na
2CO
3-NaHCO
3Damping fluid as coating buffer, vomitoxin-ovalbumin (ZEN-OVA) is diluted to 1.0 μ g/mL, 96 or 48 or 24 each hole of hole microwell plate add 100 μ l; 5 ℃ of placements are spent the night, and discard coating buffer, wash 2 times; Contain 5% bovine serum albumin(BSA) by adding, the phosphate buffer of pH7.5,8 ℃ of sealings are spent the night; Discard confining liquid, dry up, rearmounted-20 ℃ of freezing preservations of lath sealing;
The zearalenone standard items dilute from the pure article of zearalenone and obtain, and dilution is a deionized water, and ZEN concentration is: 25ng/mL;
The preparation of zearalenone-bovine serum albumin(BSA) (ZEN-BSA) conjugate: ZEN is dissolved in the 100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 55ml distilled water, using sodium hydroxide solution to transfer pH is about 9, extracts 3 times with methylene chloride again, each 15ml; Discard methylene chloride, it is about 2.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 5 times with methylene chloride again; Each 20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 3ml dioxane, add chloro-carbonic acid second butyl ester, 8.5 ℃ of following stirring reaction 45min; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 5ml distilled water and the 5ml dioxane then, and using sodium hydroxide solution to transfer pH is 8.5; 5 ℃ of following stirring reactions 5.5 hours then, are dialysed with distilled water; Change liquid 3 times, freeze drying, conjugate-30 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=10mg:2000mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: BSA=10mg:1000 μ l:1450mg.
The preparation of zearalenone-ovalbumin (ZEN-OVA) conjugate: ZEN is dissolved in the 51mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5, extracts 4 times with methylene chloride again, each 15ml; Discard methylene chloride, it is about 3 that water layer uses hydrochloric acid solution to transfer pH, extracts 6 times with methylene chloride again; Each 10ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 50ml dioxane, add chloro-carbonic acid second butyl ester, 8.5 ℃ of following stirring reactions 30 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 27.5ml distilled water and the 50ml dioxane then, and using sodium hydroxide solution to transfer pH is 8.5; 12 ℃ of following stirring reactions 5 hours then, are dialysed with distilled water; Change liquid 3 times, freeze drying, conjugate-30 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=10mg:2000mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: OVA=500mg:1000 μ l:1500mg.
The preparation of said anti-zearalenone (ZEN) monoclonal antibody and solution thereof: get the ZEN-BSA conjugate and be made into 10 μ g/ μ l antigenic solutions with physiological saline, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 55 a μ g/ mouse; Every later at a distance from 4 all booster immunizations once booster immunization adopts tail vein injection, and immunizing dose 10 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 50S, 10ml 50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds 30ml DMEM nutrient solution after 1 minute; Stop merging, water-bath is centrifugal after static 20 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 30% calf serum, be 1 * 10 with final concentration
4Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 7.5%CO
2, cultivate under 40 ℃ of conditions, after 10 days; Every culture hole is changed the 2/3HT nutrient solution, after 15 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with vomitoxin-ovalbumin (ZEN-OVA); With the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank)>2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask; Accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again; Inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.4ml/ of 11.5 all age, after 8 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal deposition, and glycerol adding is in-20 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN-BSA: physiological saline=490 μ g:10ml.
Encapsulate plate and encapsulate solid phase antigen, it can adopt 96 or 48 or 24 hole microwell plates, uses 55mmol/L pH9Na
2CO
3-NaHCO
3Damping fluid as coating buffer, vomitoxin-ovalbumin (ZEN-OVA) is diluted to 0.55 μ g/mL, 96 or 48 or 24 each hole of hole microwell plate add 100 μ l; 2 ℃ of placements are spent the night, and discard coating buffer, wash 5 times; Contain 1% bovine serum albumin(BSA) by adding, the phosphate buffer of pH 7,5 ℃ of sealings are spent the night; Discard confining liquid, dry up, rearmounted-40 ℃ of freezing preservations of lath sealing;
Said zearalenone standard items dilute from the pure article of zearalenone and obtain, and dilution is a deionized water, and ZEN concentration is: 50ng/mL;
The preparation of zearalenone-bovine serum albumin(BSA) (ZEN-BSA) conjugate: ZEN is dissolved in the 3mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5, extracts 5 times with methylene chloride again, each 10ml; Discard methylene chloride, it is about 3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 6 times with methylene chloride again; Each 15ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 50ml dioxane, add chloro-carbonic acid second butyl ester, 5 ℃ of following stirring reactions 30 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 50ml distilled water and the 50ml dioxane then, and using sodium hydroxide solution to transfer pH is 8; 12 ℃ of following stirring reactions 5 hours then, are dialysed with distilled water; Change liquid 5 times, freeze drying, conjugate-20 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=1000mg:970mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: BSA=1000mg:10 μ l:3000mg.
The preparation of zearalenone-ovalbumin (ZEN-OVA) conjugate: ZEN is dissolved in the 3mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 45ml distilled water, using sodium hydroxide solution to transfer pH is about 9.5, extracts 5 times with methylene chloride again, each 20ml; Discard methylene chloride, it is about 3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times with methylene chloride again; Each 20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 3ml dioxane, add chloro-carbonic acid second butyl ester, 12 ℃ of following stirring reactions 45 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 50ml distilled water and the 27.5ml dioxane then, and using sodium hydroxide solution to transfer pH is 8; 8.5 ℃ following stirring reaction 6 hours then, is dialysed with distilled water; Change liquid 5 times, freeze drying, conjugate-20 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=505mg:1005mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: OVA=1000mg:505 μ l:3000mg.
The preparation of anti-zearalenone (ZEN) monoclonal antibody and solution thereof: get the ZEN-BSA conjugate and be made into 0.1 μ g/ μ l antigenic solution with physiological saline, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 100 a μ g/ mouse; Every later at a distance from 2 all booster immunizations once booster immunization adopts tail vein injection, and immunizing dose 100 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 90S, 0.1ml50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds the 50mlDMEM nutrient solution after 3 minutes; Stop merging, water-bath is centrifugal after static 30 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 17.5% calf serum, be 1 * 10 with final concentration
6Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 10%CO
2, cultivate under 45 ℃ of conditions, after 5 days; Every culture hole is changed the 2/3HT nutrient solution, after 10 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with vomitoxin-ovalbumin (ZEN-OVA); With the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank)>2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask; Accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again; Inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.5ml/ of 10 all age, after 10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal deposition, and glycerol adding is in-30 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN-BSA: physiological saline=10 μ g:5.05ml.
Encapsulate plate and encapsulate solid phase antigen, it can adopt 96 or 48 or 24 hole microwell plates, uses 100mmol/LpH10 Na
2CO
3-NaHCO
3Damping fluid as coating buffer, vomitoxin-ovalbumin (ZEN-OVA) is diluted to 0.1 μ g/mL, 96 or 48 or 24 each hole of hole microwell plate add 100 μ l; 8 ℃ of placements are spent the night, and discard coating buffer, wash 3 times; Contain 3% bovine serum albumin(BSA) by adding, the phosphate buffer of pH8,2 ℃ of sealings are spent the night; Discard confining liquid, dry up, rearmounted-30 ℃ of freezing preservations of lath sealing;
The zearalenone standard items dilute from the pure article of zearalenone and obtain, and dilution is a deionized water, and ZEN concentration is: 0.001ng/mL;
The preparation of zearalenone-bovine serum albumin(BSA) (ZEN-BSA) conjugate: ZEN is dissolved in the 51.5mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml distilled water, using sodium hydroxide solution to transfer pH is about 9.5, extracts 4 times with methylene chloride again, each 20ml; Discard methylene chloride, it is about 3 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times with methylene chloride again; Each 10ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 26.5ml dioxane, add chloro-carbonic acid second butyl ester, 12 ℃ of following stirring reactions 37.5 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 27.5ml distilled water and the 27.5ml dioxane then, and using sodium hydroxide solution to transfer pH is 9; 8.5 ℃ following stirring reaction 6 hours then, is dialysed with distilled water; Change liquid 4 times, freeze drying, conjugate-40 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=495mg:10mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: BSA=505mg:505 μ l:10mg.
The preparation of zearalenone-ovalbumin (ZEN-OVA) conjugate: ZEN is dissolved in the 100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml distilled water, using sodium hydroxide solution to transfer pH is about 9, extracts 3 times with methylene chloride again, each 10ml; Discard methylene chloride, it is about 2.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 5 times with methylene chloride again; Each 15ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add the 26.5ml dioxane, add chloro-carbonic acid second butyl ester, 5 ℃ of following stirring reactions 37.5 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 5ml distilled water and the 5ml dioxane then, and using sodium hydroxide solution to transfer pH is 9; 5 ℃ of following stirring reactions 5.5 hours then, are dialysed with distilled water; Change liquid 4 times, freeze drying, conjugate-40 ℃ preservation; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=1000mg:10mg, ZEN intermediate product: chloro-carbonic acid second butyl ester: OVA=10mg:10 μ l:10mg.
The preparation of anti-zearalenone (ZEN) monoclonal antibody and solution thereof: get the ZEN-BSA conjugate and be made into 5.05 μ g/ μ l antigenic solutions with physiological saline, fully emulsified with the complete freund adjuvant mixing of equal-volume, supply first immunisation to use; Replace complete freund adjuvant with the incomplete freund adjuvant with amount during booster immunization, first immunisation adopts directly injection in the mouse peritoneal, and immunizing dose is 55 a μ g/ mouse; Every later at a distance from 3 all booster immunizations once booster immunization adopts tail vein injection, and immunizing dose 100 μ g/ only; Intrasplenic injection is adopted in last immunity, get spleen after 4 days and merge, with the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase with the 10:1 mixed; Centrifugal, remove supernatant, in 70S, 5.05ml50% polyglycol (molecular weight is 1500) is added in the cell; Fully mixing makes its fusion, adds the 30mlDMEM nutrient solution after 2 minutes; Stop merging, water-bath is centrifugal after static 10 minutes, removes supernatant; After fused cell usefulness being contained the HAT selective medium suspension of 5% calf serum, be 1 * 10 with final concentration
5Feeder cells/0.1ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5%CO
2, cultivate under 35 ℃ of conditions, after 8 days; Every culture hole is changed the 2/3HT nutrient solution, after 20 days, begins microscopy is had the culture hole of hybridoma clonal growth; Getting supernatant screens; Cell to testing result is positive is cloned with limiting dilution assay immediately, and the hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out, and is envelope antigen with vomitoxin-ovalbumin (ZEN-OVA); With the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A is blank)/(blank) > of A contrast-A; 2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay, the positive colony cell is blown even, get a droplet to culture flask; Accurate counting number of cells under the inverted microscope, dilution are 70/ml, get 20 times of 1ml dilutions again; Inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To Balb/c mouse peritoneal injecting fluid paraffin 0.3ml/ of 13 all age, after 9 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 10
5Cell/only, note after 5 days observing is collected ascites, centrifugal removal deposition, and glycerol adding is in-40 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN-BSA: physiological saline=1000 μ g:0.1ml.
The sheep anti-mouse antibody dried frozen aquatic products of enzyme labeling is horseradish peroxidase-sheep anti-mouse antibody dried frozen aquatic products; Said cleansing solution is for containing tween and NaN
3PBS; Said colour developing liquid A is the citric acid-disodium hydrogen phosphate buffer solution that contains hydrogen peroxide; Said colour developing liquid B is the ethanolic solution of tetramethyl biphenyl diamines; Said stop buffer is a sulfuric acid solution.
Below in conjunction with embodiment the testing cassete detection method is described
Embodiment 1
Get the micropore that is coated with ZEN-OVA and encapsulate plate, add the ZEN standard items of 100 μ L or the sample handled well in micropore separately, add the anti-ZEN antibody of 50 μ L; Hatched 0.75 hour for 40 ℃, cleansing solution is washed 5 times, adds 50 μ l horseradish peroxidase (HRP)-sheep anti-mouse antibodies; Hatched 1 hour for 35 ℃, wash 4 times, add 50 μ L colour developing liquid A and 75 μ L colour developing liquid B with cleansing solution; The dark place is left standstill after 20 minutes and is added stop buffer, surveys its absorbance, the ZEN content from the typical curve calculation sample at the 450nm place.
Get the micropore that is coated with ZEN-OVA and encapsulate plate, add the ZEN standard items of 50 μ L or the sample handled well in micropore separately, add the anti-ZEN antibody of 100 μ L; Hatched 0.5 hour for 35 ℃, cleansing solution is washed 3 times, adds 100 μ l horseradish peroxidase (HRP)-sheep anti-mouse antibodies; Hatched 0.5 hour for 45 ℃, wash 3 times, add 75 μ L colour developing liquid A and 50 μ L colour developing liquid B with cleansing solution; The dark place is left standstill after 10 minutes and is added stop buffer, surveys its absorbance, the ZEN content from the typical curve calculation sample at the 450nm place.
Get the micropore that is coated with ZEN-OVA and encapsulate plate, add the ZEN standard items of 75 μ L or the sample handled well in micropore separately, add the anti-ZEN antibody of 75 μ L; Hatched 1 hour for 45 ℃, cleansing solution is washed 4 times, adds 75 μ l horseradish peroxidase (HRP)-sheep anti-mouse antibodies; Hatched 0.75 hour for 40 ℃, wash 5 times, add 100 μ L colour developing liquid A and 100 μ L colour developing liquid B with cleansing solution; The dark place is left standstill after 15 minutes and is added stop buffer, surveys its absorbance, the ZEN content from the typical curve calculation sample at the 450nm place.
Claims (2)
1. preparation that detects the ELISA testing cassete of zearalenone; The ELISA testing cassete of said detection zearalenone; It comprises cleansing solution, colour developing liquid A, colour developing liquid B and stop buffer; It also comprises the sheep anti-mouse antibody dried frozen aquatic products that encapsulates plate, zearalenone standard items, zearalenone monoclonal antibody dried frozen aquatic products and enzyme labeling; It is characterized in that: it may further comprise the steps, and encapsulates the preparation of sheep anti-mouse antibody dried frozen aquatic products of preparation, the enzyme labeling of preparation, the zearalenone monoclonal antibody dried frozen aquatic products of preparation, the zearalenone standard items of plate; The said plate that encapsulates encapsulates solid phase antigen, and it can adopt 96 or 48 or 24 hole microwell plates, with the damping fluid of 10 mmol/L ~ 100mmol/L pH9 ~ 10 Na2CO3-NaHCO3 as coating buffer; Zearalenone-ovalbumin (ZEN-OVA) is diluted to 0.1 μ g/m L ~ 1.0 μ g/m L, and 96 or 48 or 24 each hole of hole microwell plate add 100 μ l, and 2 ℃ ~ 8 ℃ placements are spent the night; Discard coating buffer, wash 2 times ~ 5 times, contain 1 ~ 5% bovine serum albumin(BSA) by adding; The phosphate buffer of pH 7 ~ 8,2 ℃ ~ 8 ℃ sealings are spent the night, and discard confining liquid; Dry up rearmounted-40 ℃ ~-20 ℃ freezing preservations of lath sealing; Said zearalenone standard items dilute from the pure article of zearalenone and obtain, and dilution is a deionized water, and ZEN concentration is: 0.001 ng/mL ~ 50 ng/mL; The preparation of zearalenone-bovine serum albumin(BSA) (ZEN-BSA) conjugate: ZEN is dissolved in 3mL ~ 100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml ~ 100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5 ~ 9.5, extracts 3 times ~ 5 times with methylene chloride again, each 10ml ~ 20ml; Discard methylene chloride, it is about 2.5 ~ 3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times ~ 6 times with methylene chloride again; Each 10ml ~ 20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add 3ml ~ 50ml dioxane, add isobutyl chlorocarbonate, 5 ℃ ~ 12 ℃ following stirring reactions 30 minutes～45 minutes; Reactant liquor is added drop-wise in the bovine serum albumin(BSA), and bovine serum albumin(BSA) is dissolved in 5ml ~ 50ml distilled water and the 5ml ~ 50ml dioxane then, and using sodium hydroxide solution to transfer pH is 8～9; 5 ℃ ~ 12 ℃ following stirring reactions 5 hours～6 hours then, are dialysed with distilled water; Change liquid 3 times ~ 5 times, freeze drying, conjugate-40 ℃ ~-20 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=(10 ~ 1000) mg: (10 ~ 2000) mg, ZEN intermediate product: isobutyl chlorocarbonate: BSA=(10 ~ 1000) mg: (10 ~ 1000) μ l: (10-3000) mg; The preparation of zearalenone-ovalbumin (ZEN-OVA) conjugate: ZEN is dissolved in 3mL ~ 100mL pyridine, adds the ethyloic hydroxylamine hydrochloride, stirred overnight under the room temperature, nitrogen dries up; Add 10ml ~ 100ml distilled water, using sodium hydroxide solution to transfer pH is about 8.5 ~ 9.5, extracts 3 times ~ 5 times with methylene chloride again, each 10ml ~ 20ml; Discard methylene chloride, it is about 2.5 ~ 3.5 that water layer uses hydrochloric acid solution to transfer pH, extracts 3 times ~ 6 times with methylene chloride again; Each 10ml ~ 20ml collects methylene chloride, crosses anhydrous sodium sulfate dehydration; Filter, low temperature dries up with nitrogen, obtains the ZEN intermediate product; Get the ZEN intermediate product, add 3ml ~ 50ml dioxane, add isobutyl chlorocarbonate; 5 ℃ ~ 12 ℃ following stirring reactions 30 minutes～45 minutes are added drop-wise to reactant liquor in the ovalbumin, and ovalbumin is dissolved in 5ml ~ 50ml distilled water and the 5ml ~ 50ml dioxane then; Using sodium hydroxide solution to transfer pH is 8～9,5 ℃ ~ 12 ℃ following stirring reactions 5 hours～6 hours, dialyses with distilled water then; Change liquid 3 times ~ 5 times, freeze drying, conjugate-40 ℃ ~-20 ℃ of preservations; The ratio of above-mentioned reacted constituent is: ZEN: ethyloic hydroxylamine hydrochloride=(10 ~ 1000) mg: (10 ~ 2000) mg, ZEN intermediate product: isobutyl chlorocarbonate: OVA=(10 ~ 1000) mg: (10 ~ 1000) μ l: (10-3000) mg; The sheep anti-mouse antibody dried frozen aquatic products of said enzyme labeling is horseradish peroxidase-sheep anti-mouse antibody dried frozen aquatic products; Said cleansing solution is the PBS that contains tween and NaN3; Said colour developing liquid A is the citric acid-disodium hydrogen phosphate buffer solution that contains hydrogen peroxide; Said colour developing liquid B is the ethanolic solution of tetramethyl biphenyl diamines; Said stop buffer is a sulfuric acid solution.
2. according to the said a kind of preparation that detects the ELISA testing cassete of zearalenone of claim 1: it is characterized in that the preparation of said anti-zearalenone (ZEN) monoclonal antibody and solution thereof: get the ZEN-BSA conjugate and be made into 0.1 μ g/ μ l ~ 10 μ g/ μ l antigenic solutions, with the complete freund adjuvant mixing of equal-volume with physiological saline; Fully emulsified, supply first immunisation usefulness, replace complete freund adjuvant with incomplete freund adjuvant during booster immunization with amount; Directly injection in the first immunisation employing mouse peritoneal, immunizing dose is 10 μ g/ ~ 100 a μ g/ mouse, later on whenever at a distance from all booster immunizations in 2 week ~ 4 once; Booster immunization adopts tail vein injection, immunizing dose 10 μ g/ only ~ 100 μ g/ only, intrasplenic injection is adopted in last immunity; Get spleen after 4 days and merge, the immune mouse spleen cell that separates and the myeloma cell SP-2/o that is in exponential phase is with the 10:1 mixed, centrifugal; Remove supernatant; In 50 S～90 S, be that 1500 polyglycol adds in the cell with 0.1 ~ 10 ml, 50% molecular weight, fully mixing makes its fusion; Add 10 ml ~ 50 ml DMEM nutrient solutions after 1 minute ~ 3 minutes; Stop merging, water-bath is centrifugal after static 10 minutes ~ 30 minutes, removes supernatant; After fused cell suspended with the HAT selective medium that contains 5% ~ 30% calf serum, be that 1 * 104 ~ 1 * 106 feeder cells/0.1 ml is inoculated in Turnover of Mouse Peritoneal Macrophages and does in 96 well culture plates of feeder layer, in 5% ~ 10%CO2 with final concentration; Cultivate under 35 ℃ ~ 45 ℃ conditions, after 5 days ~ 10 days, every culture hole is changed 2/3 HT nutrient solution; After 10 days～20 days; Begin microscopy is had the culture hole of hybridoma clonal growth, get supernatant and screen, the cell that testing result is positive is cloned with limiting dilution assay immediately; The hybridoma screening adopts the indirect non-competing ELISA method of solid phase antigen to carry out; With zearalenone-ovalbumin (ZEN-OVA) is envelope antigen, with the positive contrast of the serum of immune mouse, with the negative contrast of supernatant of SP-2/o myeloma cell's cultivation; The criterion in positive cell hole is (A test-A is blank)/(A contrast-A is blank)>2.1; Cell to the secretion positive antibody is cloned; Adopt limiting dilution assay; The positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope; Dilution is 70/m1; Get 20 times of 1 ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone, till the culture supernatant in all cells hole all is positive; To 10 week age～13 Balb/c mouse peritoneal injecting fluid paraffin 0.3 ml/～0.5 ml/ in all ages only; After 8 days～10 days, lumbar injection is cultured to the hybridoma of logarithmic phase, 5 * 105 cells/only; Note after 5 days observing; Collect ascites, centrifugal removal deposition, glycerol adding is in-40 ℃ ~-20 ℃ preservations; The ratio of above-mentioned reacted constituent is: ZEN-BSA: physiological saline=(10 ~ 1000) μ g: (0.1 ~ 10) m1.
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| CN102051389B (en) * | 2010-11-09 | 2013-04-03 | 北京科美生物技术有限公司 | Synthesis process of horseradish peroxidase enzymelabeled zearalenone |
| CN102478578A (en) * | 2010-11-30 | 2012-05-30 | 吉林大学 | Chemiluminescent kit for assaying zearalenone and preparation method thereof |
| CN102313810A (en) * | 2011-07-29 | 2012-01-11 | 上海交通大学 | Chemiluminescent immunoassay method for rapidly detecting zearalenone toxin |
| CN102384977A (en) * | 2011-08-19 | 2012-03-21 | 上海交通大学 | Method by utilizing glassy carbon electrode for immuno-detection zearalenone |
| CN102967709B (en) * | 2011-09-01 | 2015-08-12 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine |
| KR101280054B1 (en) * | 2012-05-31 | 2013-06-28 | 에스디 바이오센서 주식회사 | A freeze-drying conjugate-construct for point-of-care testing (poct) immunochromatography, a kit for immunoassay using the same, and a method for analysis using the kit |
| CN102841203B (en) * | 2012-08-27 | 2014-06-25 | 暨南大学 | Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN) |
| CN104849461B (en) * | 2014-02-14 | 2016-09-21 | 北京勤邦生物技术有限公司 | A kind of test strips detecting 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and application thereof |
| CN103880952B (en) * | 2014-03-31 | 2016-02-10 | 河南大学 | A kind of anti-ZEN yolk antibody and preparation method thereof |
| CN105319368A (en) * | 2014-07-23 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting zearalenone, and detection method thereof |
| CN104198711A (en) * | 2014-08-25 | 2014-12-10 | 广东省农业科学院动物卫生研究所 | Immunomagnetic bead and application of immunomagnetic bead to detection of zearalenone |
| CN107759689A (en) * | 2017-11-02 | 2018-03-06 | 南京农业大学 | A kind of preparation method of ZER monoclonal antibody and its monoclonal antibody and the application of preparation |
| CN108548918B (en) * | 2018-04-19 | 2022-04-15 | 国家食品安全风险评估中心 | Enzyme-linked immunosorbent assay blocking solution, preparation method, application and kit with enzyme-linked immunosorbent assay blocking solution |
-
2008
- 2008-11-07 CN CN200810236336A patent/CN101413955B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 王景琳.竞争间接ELISA检测谷物饲料中的玉米赤霉烯酮.《中国兽医学报》.1994,第14卷(第2期),154-157. * |
| 王玉平等.玉米赤霉烯酮ELISA 定量检测试剂盒研制.《卫生研究》.2006,第35卷(第2期),221-224. * |
| 贾红.玉米赤霉烯酮单克隆抗体的研制及初步应用.《扬州大学硕士学位论文》.2005,全文. * |
| 路戈等.玉米赤霉烯酮单克隆抗体酶联免疫测定方法的建立及初步应用.《真菌学报》.1996,第15卷(第4期),292-296. * |
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