CN103616515A - Chemiluminescence detection kit for zearalenone - Google Patents
Chemiluminescence detection kit for zearalenone Download PDFInfo
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- CN103616515A CN103616515A CN201310601069.5A CN201310601069A CN103616515A CN 103616515 A CN103616515 A CN 103616515A CN 201310601069 A CN201310601069 A CN 201310601069A CN 103616515 A CN103616515 A CN 103616515A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones
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Abstract
The invention belongs to the field of immunological detection and particularly relates to a chemiluminescence detection kit for zearalenone. The kit consists of a luminescent plate, a horseradish peroxidase labeled zearalenone polyclonal antibody, a zearalenone standard solution, a concentrated phosphate buffer solution, a concentrated washing solution, a chemiluminescent substrate solution and an envelope antigen. Through detecting by adopting the kit, the sensitivity is high and can reach 5-500ng/mL, and substances which can not be detected by methods, such as radioimmunoassay, enzyme-linked immunoassay and the like, can be detected, so that the kit has a very important significance in detection of antibiotic residues; the luminescent intensity of the kit is of 4-6 orders of magnitude, and the luminescent intensity and the concentration of a detected substance are of a linear relationship; the light signal duration of the kit is long, and an analytical method is simple, convenient and rapid.
Description
Technical field
The invention belongs to immunology detection field, be specifically related to a kind of zearalenone chemiluminescence detection kit.
Background technology
The residual technology of mycotoxin in cereal that detects at present mainly contains following several method:
(1) physics and chemistry detection method: after the nineties in 20th century, in most mensuration cereal, the residual physics and chemistry detection method of mycotoxin mainly relies on liquid chromatography technology to carry out separation, next is look/mass spectrometric hyphenated technique, the detection method such as vapor-phase chromatography, high performance thin layer chromatography, because of its distinctive performance separately, in mycotoxin context of detection, slightly apply.Chromatography detect mycotoxin in cereal residual to pass through sample preparation (comprise sample extraction, take off the steps such as albumen, centrifugal, chromatographic column purification, derivatization), the separation of left drug and the detection of left drug.Physics and chemistry detection method utilizes special reaction or the character that the group in lps molecule has to measure its content, can carry out qualitative and quantitative analysis and drug identification, can be used as the confirmation method of mycotoxin residue detection in cereal.This method detection sensitivity is higher, but instrument and testing cost are high, trace routine is complicated, compared with time-consuming etc.
(2) immune analysis method: immune analysis method is that to take specificity, the reversibility association reaction of antigen and antibody be basic analytical approach, and the residual immuno analytical method of mycotoxin is mainly divided three major types at present: method, the immunity receptor method of relatively independent analytical approach, immuno analytical method and the coupling of conventional physical and chemical analysis technology.Dutch van weeman, schurrs in 1973 and Sweden Engvall, Perlmann propose respectively enzyme linked immunosorbent assay, and over more than 30 year, Chinese scholars detects in cereal to immunological method that mycotoxin is residual has carried out large quantity research.But because toxin is haptens, antigen constructing technology difficulty is large, immunologic opsonin is strong, testing cost is high, the research aspect immunoassay such as current Idexx Lab Inc. is comparatively ripe, and domestic this technology is still in research and development.But enzyme linked immunosorbent assay is compared with luminescence method, enzyme linked immunosorbent assay sensitivity is lower, testing result out of true, and impact is research further.
Summary of the invention
The object of this invention is to provide the red enzyme ketenes of a kind of corn chemiluminescence detection kit, not only highly sensitive, sensing range is wide, easy and simple to handle fast, nontoxic pollution-free, and equipment used, cheap, is easy to universal use.
The technical solution adopted in the present invention is, the red enzyme ketenes of a kind of corn chemiluminescence detection kit, by luminous plaque, reagent and box body, formed, reagent is placed in box body, described reagent is comprised of the red enzyme ketenes of corn polyclonal antibody, the red enzyme ketenes of corn standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, Chemoluminescent substrate and the envelope antigen of horseradish peroxidase mark, and the red enzyme ketenes of the corn polyclonal antibody of described horseradish peroxidase mark is to using the red enzyme ketenes of corn and ovalbumin coupling as immunogenic White Rabbit antibody;
The immunogene preparation method of the red enzyme ketenes of described corn and ovalbumin coupling, comprises the following steps:
(1) p-aminobenzoic acid that takes 10mg joins in the ammonium chloride solution that volume is 1.1mL, makes p-aminobenzoic acid solution; Take afterwards the NaNO of 6mg
2joining in the distilled water that volume is 0.35mL, is to stir under the condition of 0-4 ℃ in temperature, makes NaNO
2solution; By NaNO
2solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains solution A;
(2) measure the red enzyme ketenes of 2.4 μ L corn solution and join in the borate buffer solution that volume is 10mL, and be to stir at 0-4 ℃ in temperature, afterwards to the solution A that dropwise adds 0.9mL in this solution, lucifuge reaction 2 hours, obtains orange colour solution B; The concentration of described borate buffer solution is 0.05mol/L, and pH value is 8.5, contains the NaCl that concentration is 0.15mol/L in borate buffer solution;
(3) in solution B, add H
3bO
3crystal is until the pH value of solution B is 7.4, adds 94mgBSA afterwards to adjusting in the solution B of pH value, 80mgEDC, and 24mgNHS, stirs 2 hours at ambient temperature, obtains orange colour solution C;
(4) solution C is transferred in bag filter, with PBS damping fluid, in temperature, be to dialyse five days under the condition of 0-4 ℃ afterwards, within every 12 hours, change a PBS damping fluid, by the solution freeze-drying after dialysis, obtain faint yellow solid powder and be immunogene, immunogene is placed under the condition that temperature is-20 ℃ and is preserved, standby; The pH value of described PBS damping fluid is 7.4, and concentration is 0.01mol/L;
The preparation method of described envelope antigen, comprises the following steps:
(1) take the NaNO of 9mg
2join in the distilled water that volume is 3mL, make NaNO2 solution, it is in 1.6mL, the concentration hydrochloric acid solution that is 0.2mol/L that the red enzyme ketenes of corn that then takes 10mg joins volume, is to stir under the condition of 0-4 ℃ in temperature, make red enzyme ketenes solution, by NaNO
2solution dropwise adds in red enzyme ketenes solution, and lucifuge reaction 0.5 hour adds 25mg ammonium sulfate until emit without nitrogen afterwards in this solution, makes solution D;
(2) take in the PBS buffer solution that 70mg ovalbumin joins 4mL, the concentration of described PBS buffer solution is 0.1mol/L, pH value is 7.5, then solution D is dropwise joined in this solution, make mixed liquor, then the sodium hydroxide solution that is 1mol/L by concentration is adjusted to 7.5 by the pH value of mixed liquor, and in temperature, is lucifuge reaction 18 hours under the condition of 0-4 ℃, makes solution E;
(3) solution E is transferred in bag filter, and in temperature, be under the condition of 0-4 ℃, by concentration, be 0.01mol/L, pH value is 7.4 PBS damping fluid dialysis five days, within every 12 hours, changes a PBS damping fluid, afterwards dislysate is placed in to rotating speed and is centrifugal 15 minutes of the hydro-extractor of 3000r/min, extract supernatant freeze-drying, obtain faint yellow solid powder and be envelope antigen, afterwards envelope antigen is placed in to temperature and preserves under the condition of-20 ℃, standby.
Described Chemoluminescent substrate, comprises A liquid and B liquid, and the preparation method of described A liquid is: it is that 0.01M, pH make in 8.8 Tris damping fluid that the luminol that is 0.01mol/L by concentration is dissolved into concentration; The preparation method of described B liquid is: by concentration, to be first 0.001mol/L be dissolved into concentration to iodophenol is that 0.01mol/L, pH are in 8.8 Tris damping fluid, adds the sterilized water that hydrogen peroxide and water ratio are 3:10000 more afterwards in damping fluid.
The concentration of the red enzyme ketenes of described corn titer is respectively 30ng/ml, 60ng/ml, 200ng/ml, 500ng/ml.
Described concentrated cleaning solution is by NaCl 80g, KH
2p0
42.0g, Na
2hP0
412H
2o 229.0g, KC1 2.0g and Tween-20 make after being dissolved in and stirring in 1000mL distilled water, and in the concentrated cleaning solution making, Tween-20 mass percent concentration is 0.5%.
Described concentrated cleaning solution: before use, by 10 times of distilled water dilutings for the concentrated cleaning solution providing in kit.
Beneficial effect of the present invention has following five aspects:
One, in reagent of the present invention by adopting polyclonal antibody, be not only beneficial to precipitation and the aggegation of testing result, and while detecting response intensity large, be easy to observe.And the present invention is owing to adopting polyclonal antibody, and not only preparation method is simple, and cheap, greatly reduces production cost.
Two, of the present invention highly sensitive, sensitivity is the key of chemiluminescence immune assay, the conjugate of the present invention by adopting the red enzyme ketenes of corn and ovalbumin as the red enzyme ketenes of the corn polyclonal antibody of envelope antigen, horseradish peroxidase mark as antibody, sensitivity can reach 5-500 ng/mL, can detect the material that the methods such as radiommunoassay and enzyme-linked immuno assay cannot detect, significant to the detection of antibiotic residue.
Three, wide linear dynamics scope, luminous intensity of the present invention, between 4-6 magnitude, and and is measured between material concentration linear.Compare with the scope that the EIA enzyme immunoassay absorbance (OD value) of colour developing is 2.0, with the obvious advantage.
Four, this product luminous substrate is stablized, and the light signal duration is long.
Five, good, the long shelf-life of security, the present invention has avoided the use of radiomaterial, does not find harmfulness, and stable reagent of the present invention, and storage life can reach 1 year.
Embodiment
The red enzyme ketenes of a kind of corn chemiluminescence detection kit, by luminous plaque, reagent and box body, formed, reagent is placed in box body, described reagent is comprised of the red enzyme ketenes of corn polyclonal antibody, the red enzyme ketenes of corn standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, Chemoluminescent substrate and the envelope antigen of horseradish peroxidase mark, and the red enzyme ketenes of the corn polyclonal antibody of described horseradish peroxidase mark is to using the red enzyme ketenes of corn and ovalbumin coupling as immunogenic White Rabbit antibody;
The immunogene preparation method of the red enzyme ketenes of described corn and ovalbumin coupling, comprises the following steps:
(1) p-aminobenzoic acid that takes 10mg joins in the ammonium chloride solution that volume is 1.1mL, makes p-aminobenzoic acid solution; Take afterwards the NaNO of 6mg
2joining in the distilled water that volume is 0.35mL, is to stir under the condition of 0-4 ℃ in temperature, makes NaNO
2solution; By NaNO
2solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains solution A;
(2) measure the red enzyme ketenes of 2.4 μ L corn solution and join in the borate buffer solution that volume is 10mL, and be to stir at 0-4 ℃ in temperature, afterwards to the solution A that dropwise adds 0.9mL in this solution, lucifuge reaction 2 hours, obtains orange colour solution B; The concentration of described borate buffer solution is 0.05mol/L, and pH value is 8.5, contains the NaCl that concentration is 0.15mol/L in borate buffer solution;
(3) in solution B, add H
3bO
3crystal is until the pH value of solution B is 7.4, adds 94mgBSA afterwards to adjusting in the solution B of pH value, 80mgEDC, and 24mgNHS, stirs 2 hours at ambient temperature, obtains orange colour solution C;
(4) solution C is transferred in bag filter, with PBS damping fluid, in temperature, be to dialyse five days under the condition of 0-4 ℃ afterwards, within every 12 hours, change a PBS damping fluid, by the solution freeze-drying after dialysis, obtain faint yellow solid powder and be immunogene, immunogene is placed under the condition that temperature is-20 ℃ and is preserved, standby; The pH value of described PBS damping fluid is 7.4, and concentration is 0.01mol/L;
The preparation method of described envelope antigen, comprises the following steps:
(1) take the NaNO of 9mg
2join in the distilled water that volume is 3mL, make NaNO2 solution, it is in 1.6mL, the concentration hydrochloric acid solution that is 0.2mol/L that the red enzyme ketenes of corn that then takes 10mg joins volume, is to stir under the condition of 0-4 ℃ in temperature, make red enzyme ketenes solution, by NaNO
2solution dropwise adds in red enzyme ketenes solution, and lucifuge reaction 0.5 hour adds 25mg ammonium sulfate until emit without nitrogen afterwards in this solution, makes solution D;
(2) take in the PBS buffer solution that 70mg ovalbumin joins 4mL, the concentration of described PBS buffer solution is 0.1mol/L, pH value is 7.5, then solution D is dropwise joined in this solution, make mixed liquor, then the sodium hydroxide solution that is 1mol/L by concentration is adjusted to 7.5 by the pH value of mixed liquor, and in temperature, is lucifuge reaction 18 hours under the condition of 0-4 ℃, makes solution E;
(3) solution E is transferred in bag filter, and in temperature, be under the condition of 0-4 ℃, by concentration, be 0.01mol/L, pH value is 7.4 PBS damping fluid dialysis five days, within every 12 hours, changes a PBS damping fluid, afterwards dislysate is placed in to rotating speed and is centrifugal 15 minutes of the hydro-extractor of 3000r/min, extract supernatant freeze-drying, obtain faint yellow solid powder and be envelope antigen, afterwards envelope antigen is placed in to temperature and preserves under the condition of-20 ℃, standby.
Described Chemoluminescent substrate, comprises A liquid and B liquid, and the preparation method of described A liquid is: it is that 0.01M, pH make in 8.8 Tris damping fluid that the luminol that is 0.01mol/L by concentration is dissolved into concentration; The preparation method of described B liquid is: by concentration, to be first 0.001mol/L be dissolved into concentration to iodophenol is that 0.01mol/L, pH are in 8.8 Tris damping fluid, adds the sterilized water that hydrogen peroxide and water ratio are 3:10000 more afterwards in damping fluid.
The concentration of the red enzyme ketenes of described corn titer is respectively 30ng/ml, 60ng/ml, 200ng/ml, 500ng/ml.
Described concentrated cleaning solution is by NaCl 80g, KH
2p0
42.0g, Na
2hP0
412H
2o 229.0g, KC1 2.0g and Tween-20 make after being dissolved in and stirring in 1000mL distilled water, and in the concentrated cleaning solution making, Tween-20 mass percent concentration is 0.5%.
embodiment 1
The red enzyme ketenes of a kind of corn chemiluminescence detection kit, by luminous plaque, reagent and box body, formed, reagent is placed in box body, described reagent is comprised of the red enzyme ketenes of corn polyclonal antibody, the red enzyme ketenes of corn standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, Chemoluminescent substrate and the envelope antigen of horseradish peroxidase mark, and the red enzyme ketenes of the corn polyclonal antibody of described horseradish peroxidase mark is to using the red enzyme ketenes of corn and ovalbumin coupling as immunogenic White Rabbit antibody;
The immunogene preparation method of the red enzyme ketenes of described corn and ovalbumin coupling, comprises the following steps:
(1) p-aminobenzoic acid that takes 10mg joins in the ammonium chloride solution that volume is 1.1mL, makes p-aminobenzoic acid solution; Take afterwards the NaNO of 6mg
2joining in the distilled water that volume is 0.35mL, is to stir under the condition of 0-4 ℃ in temperature, makes NaNO
2solution; By NaNO
2solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains solution A;
(2) measure the red enzyme ketenes of 2.4 μ L corn solution and join in the borate buffer solution that volume is 10mL, and be to stir at 0-4 ℃ in temperature, afterwards to the solution A that dropwise adds 0.9mL in this solution, lucifuge reaction 2 hours, obtains orange colour solution B; The concentration of described borate buffer solution is 0.05mol/L, and pH value is 8.5, contains the NaCl that concentration is 0.15mol/L in borate buffer solution;
(3) in solution B, add H
3bO
3crystal is until the pH value of solution B is 7.4, adds 94mgBSA afterwards to adjusting in the solution B of pH value, 80mgEDC, and 24mgNHS, stirs 2 hours at ambient temperature, obtains orange colour solution C;
(4) solution C is transferred in bag filter, with PBS damping fluid, in temperature, be to dialyse five days under the condition of 0-4 ℃ afterwards, within every 12 hours, change a PBS damping fluid, by the solution freeze-drying after dialysis, obtain faint yellow solid powder and be immunogene, immunogene is placed under the condition that temperature is-20 ℃ and is preserved, standby; The pH value of described PBS damping fluid is 7.4, and concentration is 0.01mol/L;
The preparation method of described envelope antigen, comprises the following steps:
(1) take the NaNO of 9mg
2join in the distilled water that volume is 3mL, make NaNO2 solution, it is in 1.6mL, the concentration hydrochloric acid solution that is 0.2mol/L that the red enzyme ketenes of corn that then takes 10mg joins volume, is to stir under the condition of 0-4 ℃ in temperature, make red enzyme ketenes solution, by NaNO
2solution dropwise adds in red enzyme ketenes solution, and lucifuge reaction 0.5 hour adds 25mg ammonium sulfate until emit without nitrogen afterwards in this solution, makes solution D;
(2) take in the PBS buffer solution that 70mg ovalbumin joins 4mL, the concentration of described PBS buffer solution is 0.1mol/L, pH value is 7.5, then solution D is dropwise joined in this solution, make mixed liquor, then the sodium hydroxide solution that is 1mol/L by concentration is adjusted to 7.5 by the pH value of mixed liquor, and in temperature, is lucifuge reaction 18 hours under the condition of 0-4 ℃, makes solution E;
(3) solution E is transferred in bag filter, and in temperature, be under the condition of 0-4 ℃, by concentration, be 0.01mol/L, pH value is 7.4 PBS damping fluid dialysis five days, within every 12 hours, changes a PBS damping fluid, afterwards dislysate is placed in to rotating speed and is centrifugal 15 minutes of the hydro-extractor of 3000r/min, extract supernatant freeze-drying, obtain faint yellow solid powder and be envelope antigen, afterwards envelope antigen is placed in to temperature and preserves under the condition of-20 ℃, standby.
Described Chemoluminescent substrate, comprises A liquid and B liquid, and the preparation method of described A liquid is: it is that 0.01M, pH make in 8.8 Tris damping fluid that the luminol that is 0.01mol/L by concentration is dissolved into concentration; The preparation method of described B liquid is: by concentration, to be first 0.001mol/L be dissolved into concentration to iodophenol is that 0.01mol/L, pH are in 8.8 Tris damping fluid, adds the sterilized water that hydrogen peroxide and water ratio are 3:10000 more afterwards in damping fluid.
The concentration of the red enzyme ketenes of described corn titer is respectively 30ng/ml, 60ng/ml, 200ng/ml, 500ng/ml.
Described concentrated cleaning solution is by NaCl 80g, KH
2p0
42.0g, Na
2hP0
412H
2o 229.0g, KC1 2.0g and Tween-20 make after being dissolved in and stirring in 1000mL distilled water, and in the concentrated cleaning solution making, Tween-20 mass percent concentration is 0.5%.
Process the early stage of sample:
(1) Preparatory work of experiment: the methanol aqueous solution that the methanol aqueous solution that dose volume percent concentration is 60% and concentration of volume percent are 20%.
(2) take 5 g testing samples pulverizing and be placed in the band plug triangular flask of 100mL, in triangular flask, add 25 mL concentration of volume percent 60% methanol aqueous solutions afterwards.
(3) sample of step (2) being processed is put into oscillator, fully vibrates after 3 minutes, with No. 1 Filter paper filtering of whatman, collects filtrate.
(4) get the filtrate 2mL of step (3), and add the methanol aqueous solution that 6 mL concentration of volume percent are 20%, after stirring and evenly mixing, with No. 1 Filter paper filtering of whatman, this is sample liquid to be checked.
2, testing process:
Select and the red enzyme ketenes of the corn of horseradish peroxidase mark polyclonal antibody is diluted to 8000 times, envelope antigen concentration is the mensuration that 1.5 μ g/mL carry out kit parameter.
(1) coated: with concentration, be 0.05M, the carbonate that pH value is 9.6 is coated with solution and envelope antigen is made into the solution of 1.5 μ g/mL, adds 100 μ L in the reacting hole of each polystyrene board, in temperature, be the preservation of spending the night under the condition of 4 ℃.Next day, remove solution in reacting hole, with lavation buffer solution, to wash 3 times, 300 μ L/ holes, each 5 minutes, pat dry.
(2) sealing: with the above-mentioned coated ELISA Plate of lock solution sealing, 250 μ L/ holes, be to hatch 1 hour under the condition of 37 ℃ in temperature, then wash 3 times 300 μ L/ holes, each 5 minutes with lavation buffer solution.
(3) application of sample: add 50 μ L mass concentrations to be respectively 30ng/ml in the reacting hole having sealed in step (2), 60ng/ml, 200ng/ml, the red enzyme ketenes standard solution of 500ng/ml.Then to the red enzyme ketenes antibody that adds the horseradish peroxidase mark after 8000 times of dilutions in each reacting hole, 100 μ L/ holes, are to react 0.5 hour under the condition of 37 ℃ in temperature, then with lavation buffer solution, wash 3 times 300 μ L/ holes, each 5 minutes.
(4) luminous: in each reacting hole, to add the Chemoluminescent substrate 100 μ L/ holes of interim preparation, and with chemical illumination immunity analysis instrument, detect immediately.
(5) testing result: get standard items concentration logarithm and make horizontal ordinate, standard items detect luminous value logarithm and make ordinate, do typical curve, and the concentration of each sample can be calculated from typical curve.
Testing result is as shown in the table:
Claims (4)
1. the red enzyme ketenes of a corn chemiluminescence detection kit, by luminous plaque, reagent and box body, formed, reagent is placed in box body, it is characterized in that: described reagent is comprised of the red enzyme ketenes of corn polyclonal antibody, the red enzyme ketenes of corn standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, Chemoluminescent substrate and the envelope antigen of horseradish peroxidase mark, the red enzyme ketenes of the corn polyclonal antibody of described horseradish peroxidase mark is to using the red enzyme ketenes of corn and ovalbumin coupling as immunogenic White Rabbit antibody;
The immunogene preparation method of the red enzyme ketenes of described corn and ovalbumin coupling, comprises the following steps:
(1) p-aminobenzoic acid that takes 10mg joins in the ammonium chloride solution that volume is 1.1mL, makes p-aminobenzoic acid solution; Take afterwards the NaNO of 6mg
2joining in the distilled water that volume is 0.35mL, is to stir under the condition of 0-4 ℃ in temperature, makes NaNO
2solution; By NaNO
2solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains solution A;
(2) measure the red enzyme ketenes of 2.4 μ L corn solution and join in the borate buffer solution that volume is 10mL, and be to stir at 0-4 ℃ in temperature, afterwards to the solution A that dropwise adds 0.9mL in this solution, lucifuge reaction 2 hours, obtains orange colour solution B; The concentration of described borate buffer solution is 0.05mol/L, and pH value is 8.5, contains the NaCl that concentration is 0.15mol/L in borate buffer solution;
(3) in solution B, add H
3bO
3crystal is until the pH value of solution B is 7.4, adds 94mgBSA afterwards to adjusting in the solution B of pH value, 80mgEDC, and 24mgNHS, stirs 2 hours at ambient temperature, obtains orange colour solution C;
(4) solution C is transferred in bag filter, with PBS damping fluid, in temperature, be to dialyse five days under the condition of 0-4 ℃ afterwards, within every 12 hours, change a PBS damping fluid, by the solution freeze-drying after dialysis, obtain faint yellow solid powder and be immunogene, immunogene is placed under the condition that temperature is-20 ℃ and is preserved, standby; The pH value of described PBS damping fluid is 7.4, and concentration is 0.01mol/L;
The preparation method of described envelope antigen, comprises the following steps:
(1) take the NaNO of 9mg
2join in the distilled water that volume is 3mL, make NaNO2 solution, it is in 1.6mL, the concentration hydrochloric acid solution that is 0.2mol/L that the red enzyme ketenes of corn that then takes 10mg joins volume, is to stir under the condition of 0-4 ℃ in temperature, make red enzyme ketenes solution, by NaNO
2solution dropwise adds in red enzyme ketenes solution, and lucifuge reaction 0.5 hour adds 25mg ammonium sulfate until emit without nitrogen afterwards in this solution, makes solution D;
(2) take in the PBS buffer solution that 70mg ovalbumin joins 4mL, the concentration of described PBS buffer solution is 0.1mol/L, pH value is 7.5, then solution D is dropwise joined in this solution, make mixed liquor, then the sodium hydroxide solution that is 1mol/L by concentration is adjusted to 7.5 by the pH value of mixed liquor, and in temperature, is lucifuge reaction 18 hours under the condition of 0-4 ℃, makes solution E;
(3) solution E is transferred in bag filter, and in temperature, be under the condition of 0-4 ℃, by concentration, be 0.01mol/L, pH value is 7.4 PBS damping fluid dialysis five days, within every 12 hours, changes a PBS damping fluid, afterwards dislysate is placed in to rotating speed and is centrifugal 15 minutes of the hydro-extractor of 3000r/min, extract supernatant freeze-drying, obtain faint yellow solid powder and be envelope antigen, afterwards envelope antigen is placed in to temperature and preserves under the condition of-20 ℃, standby.
2. the red enzyme ketenes of a kind of corn according to claim 1 chemiluminescence detection kit, it is characterized in that: described Chemoluminescent substrate, comprise A liquid and B liquid, the preparation method of described A liquid is: it is that 0.01M, pH make in 8.8 Tris damping fluid that the luminol that is 0.01mol/L by concentration is dissolved into concentration; The preparation method of described B liquid is: by concentration, to be first 0.001mol/L be dissolved into concentration to iodophenol is that 0.01mol/L, pH are in 8.8 Tris damping fluid, adds the sterilized water that hydrogen peroxide and water ratio are 3:10000 more afterwards in damping fluid.
3. the red enzyme ketenes of a kind of corn according to claim 1 chemiluminescence detection kit, is characterized in that: the concentration of the red enzyme ketenes of described corn titer is respectively 30ng/ml, 60ng/ml, 200ng/ml, 500ng/ml.
4. the red enzyme ketenes of a kind of corn according to claim 1 chemiluminescence detection kit, is characterized in that: described concentrated cleaning solution is by NaCl 80g, KH
2p0
42.0g, Na
2hP0
412H
2o 229.0g, KC1 2.0g and Tween-20 make after being dissolved in and stirring in 1000mL distilled water, and in the concentrated cleaning solution making, Tween-20 mass percent concentration is 0.5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310601069.5A CN103616515A (en) | 2013-11-25 | 2013-11-25 | Chemiluminescence detection kit for zearalenone |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310601069.5A CN103616515A (en) | 2013-11-25 | 2013-11-25 | Chemiluminescence detection kit for zearalenone |
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| CN103616515A true CN103616515A (en) | 2014-03-05 |
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| CN201310601069.5A Pending CN103616515A (en) | 2013-11-25 | 2013-11-25 | Chemiluminescence detection kit for zearalenone |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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