CN108761064A - A kind of Horseradish Peroxidase Conjugates dilution and preparation method thereof - Google Patents
A kind of Horseradish Peroxidase Conjugates dilution and preparation method thereof Download PDFInfo
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- CN108761064A CN108761064A CN201810488799.1A CN201810488799A CN108761064A CN 108761064 A CN108761064 A CN 108761064A CN 201810488799 A CN201810488799 A CN 201810488799A CN 108761064 A CN108761064 A CN 108761064A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of Horseradish Peroxidase Conjugates dilutions and preparation method thereof, are matched and are formed in parts by weight by following components：51~83 parts of dipotassium hydrogen phosphate, 48~78 parts of potassium dihydrogen phosphate, 26~37 parts of sodium chloride, 19~31 parts of magnesium chloride, 4~11 parts of sucrose, 11~21 parts of goat IgG, 2~5 parts of horse serum, 2~4 parts of Tween-20,1~3 part of sodium azide, 300~400 parts of deionized water.The present invention utilizes the synergistic effect between each component, the Horseradish Peroxidase Conjugates dilution prepared that working solution is made with the dilution in antibody label horseradish peroxidase, and ensure reagent has good repeatability, accuracy.
Description
Technical field
The present invention relates to biological field more particularly to a kind of Horseradish Peroxidase Conjugates dilution and its preparations
Method.
Background technology
Immuno analytical method is widely used in the detection of biomolecule.Immunolabelling technique is can microdetermination by certain
Or the mass signatures of ultramicroassay are added in the reaction system of Ag-Ab and react in marker is made on antibody,
By detect marker content reflect indirectly sample to be tested content how much.Immunology detection based on the labelling technique with
The advantages such as its high sensitivity, accuracy be good, easy to operation, reproducible gradually become mainstream immunological test technology.It is wherein peppery
For root peroxidase because of its labelling technique maturation, method is simple, cost-effective, becomes a variety of and measures in marker preferably.Together
When, antigen to be captured jointly with the antibody to match with the antibody of horseradish peroxidase-labeled, in addition to antibody itself bound site
The correspondence of point, it is also necessary to which suitable buffer system just can guarantee repeatability, the accuracy of reagent.
Invention content
Present invention solves the technical problem that：In order to obtain a kind of Horseradish Peroxidase Conjugates dilution, ensure
After working solution is made in antibody label horseradish peroxidase, reagent has preferable repeatability, accuracy, and the present invention provides one
Kind alkali phosphatase enzyme mark antibody dilution and preparation method thereof.
Technical solution：A kind of Horseradish Peroxidase Conjugates dilution is matched in parts by weight by following components
Composition：51~83 parts of dipotassium hydrogen phosphate, 48~78 parts of potassium dihydrogen phosphate, 26~37 parts of sodium chloride, 19~31 parts of magnesium chloride, sucrose
4~11 parts, 11~21 parts of goat IgG, 2~5 parts of horse serum, 2~4 parts of Tween-20,1~3 part of sodium azide, deionized water
300~400 parts.
A kind of preferred Horseradish Peroxidase Conjugates dilution is matched in parts by weight by following components to be formed：
65 parts of dipotassium hydrogen phosphate, 58 parts of potassium dihydrogen phosphate, 31 parts of sodium chloride, 25 parts of magnesium chloride, 7 parts of sucrose, 16 parts of goat IgG, horse blood
Clear 3 parts, 3 parts of Tween-20,1 part of sodium azide, 350 parts of deionized water.
A kind of preparation method of Horseradish Peroxidase Conjugates dilution, comprises the steps of：
1st step contains deionized water in beaker, and phosphoric acid hydrogen is added while stirring with the mixing speed of 100~150rpm
Dipotassium, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose are stirred for 30~50min after being all added；
2nd step adjusts the obtained pH value of solution of the 1st step to 6.5~7.4；
Goat IgG, horse serum are added in the 2nd step acquired solution, and is stirred with the mixing speed of 50~90rpm for 3rd step
15~25min；
Tween-20, sodium azide are added in the 3rd step acquired solution, and are stirred with the mixing speed of 30~70rpm by the 4th step
Mix 20~40min, you can obtained Horseradish Peroxidase Conjugates dilution.
Preferably, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, the sucrose being added in the 1st step, need etc. upper one
Lower a reagent is added after part reagent dissolving.
Preferably, deionized water is contained in beaker in the 1st step, phosphoric acid is added with the mixing speed of 120rpm while stirring
Hydrogen dipotassium, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose are stirred for 40min after being all added.
Preferably, the obtained pH value of solution of the 1st step is adjusted in the 2nd step to 7.1.
Preferably, goat IgG, horse serum is added in the 3rd step in the 2nd step acquired solution, and with the mixing speed of 80rpm
Stir 20min.
Preferably, Tween-20, sodium azide are added in the 3rd step acquired solution in the 4th step, and with the stirring of 40rpm speed
Degree stirring 30min, you can obtained Horseradish Peroxidase Conjugates dilution.
Advantageous effect：The present invention utilizes the synergistic effect between each component, the horseradish peroxidase-labeled prepared
Antibody dilution is made working solution with the dilution in antibody label horseradish peroxidase, ensures that having for reagent is good
Repeatability, accuracy.
Specific implementation mode
Embodiment 1
A kind of Horseradish Peroxidase Conjugates dilution, is matched by following components and is formed in parts by weight：Phosphoric acid
65 parts of hydrogen dipotassium, 58 parts of potassium dihydrogen phosphate, 31 parts of sodium chloride, 25 parts of magnesium chloride, 7 parts of sucrose, 16 parts of goat IgG, horse serum 3
Part, 3 parts of Tween-20,1 part of sodium azide, 350 parts of deionized water.
A kind of preparation method of Horseradish Peroxidase Conjugates dilution, comprises the steps of：
1st step contains deionized water in beaker, with the mixing speed of 120rpm be added while stirring dipotassium hydrogen phosphate,
Potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose add lower a reagent, after being all added again after waiting a reagent dissolving
Stir 40min；
2nd step adjusts the obtained pH value of solution of the 1st step to 7.1；
Goat IgG, horse serum are added in the 2nd step acquired solution, and is stirred with the mixing speed of 80rpm for 3rd step
20min；
Tween-20, sodium azide are added in the 3rd step acquired solution, and are stirred with the mixing speed of 40rpm by the 4th step
30min, you can obtained Horseradish Peroxidase Conjugates dilution.,
Embodiment 2
A kind of Horseradish Peroxidase Conjugates dilution, is matched by following components and is formed in parts by weight：Phosphoric acid
56 parts of hydrogen dipotassium, 58 parts of potassium dihydrogen phosphate, 30 parts of sodium chloride, 27 parts of magnesium chloride, 6 parts of sucrose, 17 parts of goat IgG, horse serum 3
Part, 3 parts of Tween-20,1 part of sodium azide, 300 parts of deionized water.
A kind of preparation method of Horseradish Peroxidase Conjugates dilution, comprises the steps of：
1st step contains deionized water in beaker, with the mixing speed of 100rpm be added while stirring dipotassium hydrogen phosphate,
Potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose add lower a reagent, after being all added again after waiting a reagent dissolving
Stir 50min；
2nd step adjusts the obtained pH value of solution of the 1st step to 6.9；
Goat IgG, horse serum are added in the 2nd step acquired solution, and is stirred with the mixing speed of 80rpm for 3rd step
20min；
Tween-20, sodium azide are added in the 3rd step acquired solution, and are stirred with the mixing speed of 50rpm by the 4th step
30min, you can obtained Horseradish Peroxidase Conjugates dilution.
Embodiment 3
A kind of Horseradish Peroxidase Conjugates dilution, is matched by following components and is formed in parts by weight：Phosphoric acid
68 parts of hydrogen dipotassium, 71 parts of potassium dihydrogen phosphate, 31 parts of sodium chloride, 27 parts of magnesium chloride, 8 parts of sucrose, 17 parts of goat IgG, horse serum 4
Part, 4 parts of Tween-20,1 part of sodium azide, 380 parts of deionized water.
A kind of preparation method of Horseradish Peroxidase Conjugates dilution, comprises the steps of：
1st step contains deionized water in beaker, with the mixing speed of 120rpm be added while stirring dipotassium hydrogen phosphate,
Potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose add lower a reagent, after being all added again after waiting a reagent dissolving
Stir 35min；
2nd step adjusts the obtained pH value of solution of the 1st step to 6.8；
Goat IgG, horse serum are added in the 2nd step acquired solution, and is stirred with the mixing speed of 80rpm for 3rd step
20min；
Tween-20, sodium azide are added in the 3rd step acquired solution, and are stirred with the mixing speed of 60rpm by the 4th step
35min, you can obtained Horseradish Peroxidase Conjugates dilution.
Embodiment 4
The Horseradish Peroxidase Conjugates that Examples 1 to 3 is prepared are diluted with dilution for antibody, to it
Repetitive test and accuracy test are carried out, it is as a result as follows：
| Repetitive test (%) | Accuracy test (%) | |
| Embodiment 1 | 4.9 | 7.7 |
| Embodiment 2 | 6.8 | 8.1 |
| Embodiment 3 | 5.7 | 9.5 |
Claims (8)
1. a kind of Horseradish Peroxidase Conjugates dilution, which is characterized in that matched in parts by weight by following components
Composition：51~83 parts of dipotassium hydrogen phosphate, 48~78 parts of potassium dihydrogen phosphate, 26~37 parts of sodium chloride, 19~31 parts of magnesium chloride, sucrose
4~11 parts, 11~21 parts of goat IgG, 2~5 parts of horse serum, 2~4 parts of Tween-20,1~3 part of sodium azide, deionized water
300~400 parts.
2. a kind of Horseradish Peroxidase Conjugates dilution according to claim 1, which is characterized in that by with the following group
Divide proportioning composition in parts by weight：65 parts of dipotassium hydrogen phosphate, 58 parts of potassium dihydrogen phosphate, 31 parts of sodium chloride, 25 parts of magnesium chloride, sucrose
7 parts, 16 parts of goat IgG, 3 parts of horse serum, 3 parts of Tween-20,1 part of sodium azide, 350 parts of deionized water.
3. a kind of preparation method of Horseradish Peroxidase Conjugates dilution described in claim 1, which is characterized in that packet
Containing following steps：
1st step contains deionized water in beaker, with the mixing speed of 100~150rpm be added while stirring dipotassium hydrogen phosphate,
Potassium dihydrogen phosphate, sodium chloride, magnesium chloride, sucrose are stirred for 30~50min after being all added；
2nd step adjusts the obtained pH value of solution of the 1st step to 6.5~7.4；
3rd step, in the 2nd step acquired solution be added goat IgG, horse serum, and with the mixing speed of 50~90rpm stirring 15~
25min；
Tween-20, sodium azide are added in the 3rd step acquired solution, and stir 20 with the mixing speed of 30~70rpm by the 4th step
~40min, you can obtained Horseradish Peroxidase Conjugates dilution.
4. a kind of preparation method of Horseradish Peroxidase Conjugates dilution, feature exist according to claim 3
In dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, the sucrose being added in the 1st step, after upper a reagent dissolving such as need
Add lower a reagent.
5. a kind of preparation method of Horseradish Peroxidase Conjugates dilution, feature exist according to claim 3
In deionized water is contained in beaker in the 1st step, dipotassium hydrogen phosphate, phosphoric acid are added while stirring with the mixing speed of 120rpm
Potassium dihydrogen, sodium chloride, magnesium chloride, sucrose are stirred for 40min after being all added.
6. a kind of preparation method of Horseradish Peroxidase Conjugates dilution, feature exist according to claim 3
In the pH value of solution that the 1st step of adjusting obtains in the 2nd step to 7.1.
7. a kind of preparation method of Horseradish Peroxidase Conjugates dilution, feature exist according to claim 3
In, in the 3rd step in the 2nd step acquired solution be added goat IgG, horse serum, and with the mixing speed of 80rpm stir 20min.
8. a kind of preparation method of Horseradish Peroxidase Conjugates dilution, feature exist according to claim 3
In, Tween-20, sodium azide are added in the 3rd step acquired solution in the 4th step, and 30min is stirred with the mixing speed of 40rpm,
It can be prepared by Horseradish Peroxidase Conjugates dilution.
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Citations (6)
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| IE20040661A1 (en) * | 2004-09-29 | 2006-04-05 | Biotrin Intellectual Pty Ltd | Stabilising diluent for a protein-horseradish peroxidase conjugate |
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| EP1710305A4 (en) * | 2003-12-26 | 2007-09-12 | Dainippon Sumitomo Pharma Co | Fraction comprising guinea pig immunoglobulin e and antibody |
| CN101236206A (en) * | 2008-01-17 | 2008-08-06 | 中国兽医药品监察所 | A kind of Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit |
| CN101424686A (en) * | 2008-11-14 | 2009-05-06 | 深圳市绿诗源生物技术有限公司 | ELISA reagent for detecting malachite green and method |
| CN107741496A (en) * | 2017-09-20 | 2018-02-27 | 何诚 | A kind of kit for detecting nose tracheae bird bacillus and preparation method and application |
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2018
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1710305A4 (en) * | 2003-12-26 | 2007-09-12 | Dainippon Sumitomo Pharma Co | Fraction comprising guinea pig immunoglobulin e and antibody |
| CN1969189A (en) * | 2004-06-14 | 2007-05-23 | 协和梅迪克斯株式会社 | Method of immunoassay having nonspecific reaction inhibited and reagent therefor |
| IE20040661A1 (en) * | 2004-09-29 | 2006-04-05 | Biotrin Intellectual Pty Ltd | Stabilising diluent for a protein-horseradish peroxidase conjugate |
| CN101236206A (en) * | 2008-01-17 | 2008-08-06 | 中国兽医药品监察所 | A kind of Mycoplasma hyopneumoniae recombinant antigen ELISA detection kit |
| CN101424686A (en) * | 2008-11-14 | 2009-05-06 | 深圳市绿诗源生物技术有限公司 | ELISA reagent for detecting malachite green and method |
| CN107741496A (en) * | 2017-09-20 | 2018-02-27 | 何诚 | A kind of kit for detecting nose tracheae bird bacillus and preparation method and application |
Non-Patent Citations (2)
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| K THIRUMALA-DEVI ET AL.: "Occurrence of Ochratoxin A in Black Pepper, Coriander, Ginger and Turmeric in India", 《FOOD ADDIT CONTAM》 * |
| 李亚璞: "不同酶标抗体稀释液对酶标抗体稳定性的影响", 《湖南农业科学》 * |
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