CN103642781A - Horse radish peroxidase protective agent - Google Patents
Horse radish peroxidase protective agent Download PDFInfo
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- CN103642781A CN103642781A CN201310673542.0A CN201310673542A CN103642781A CN 103642781 A CN103642781 A CN 103642781A CN 201310673542 A CN201310673542 A CN 201310673542A CN 103642781 A CN103642781 A CN 103642781A
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- hrp
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- horse radish
- horseradish peroxidase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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Abstract
The invention relates to a peroxidase protective agent. The peroxidase protective agent comprises the following components: 50mM of HEPES buffering liquid with pH value of 7.4, 1g/L to 2g/L BSA, 8mM to 10mM of MgSO4, 0.5mM to 2mM of NaCl, 0.2mM to 1mM of ZnSO4, 0.1 to 0.5 of beta-cyclodextrin, 0.5g/L to 2g/L trehalose, 0.5g/L to 2g/L glycerin, 1g/L to 3g/L dodecyl dimethyl betaine and 0.5g/L to 1g/L preservative Proclin300. By adopting the horse radish peroxidase protective agent, the activity of the enzyme can be maintained for a long time, and the spatial configuration of a HRP (horse radish peroxidase) coupler can be maintained for a long time; moreover, the stability is good, the shelf life of a test kit can be remarkably prolonged, and the commercial value of the test kit can be improved.
Description
Technical field
The present invention relates to a kind of horseradish peroxidase protective material, belong to clinical vitro detection technical field.
Background technology
Enzymoimmunoassay (ELISA) is a kind of immunoassay of the efficient catalytic of high degree of specificity based on antigen-antibody combination and enzyme, because the natural existence of animal body produces the ability of various antibody, therefore, this immune analysis method of establishing according to antigen and antibody specific combination principle, almost can be applied to the detection of all soluble antigens.And along with the development and application of monoclonal antibody technique, ELISA has been widely used in the fields such as clinical medicine, Animal Quarantine, Food science, plant virus, drug residue, the prevention and control of plant diseases, pest control, its I measured value can reach even pg level of ng.Compare with radioimmunoassay (RIA), in enzyme immunoassay, the content of determinand is that enzyme catalysis color reaction by enzyme labelling thing judges, its advantage is that labelled reagent is more stable, and cold harm, can objectively show result, so its range of application is wider.
The marker enzyme that ELISA is conventional has horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-D-galactosidase (Gal) etc.Wherein HRP is easy to extract, relative low price, and after antigen or antibody coupling, active little impaired is lost.Therefore, be the mark enzyme being most widely used up to now in ELISA.
Popularizing of enzymoimmunoassay (ELISA) test kit is inseparable with the high quality of itself, and stability is one of the important indicator of the quality of test kit.There are accuracy and reliability that good stability can guaranteed reagent,G.R. box measurement result before the deadline, and easy to utilize.The stability of enzymoimmunoassay (ELISA) test kit is affected by many factors, and wherein the activity of marker enzyme is one of important factor.And horseradish peroxidase (HRP) chemical nature is protein, a lot of physical factors or chemical factor all can cause its deactivation, all these can make the reduction of protein biological activity even lose its function, particularly in the situation that protein concentration is lower.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of horseradish peroxidase protective material, it can increase the stability of horseradish peroxidase.
HRP(horseradish peroxidase provided by the invention) protective material comprises component and content thereof are as follows:
HEPES damping fluid (pH=7.4) 50mM
BSA 1~2g/L
MgSO4 8~10mM
NaCl 0.5~2mM
ZnSO4 0.2~1mM
Beta-cyclodextrin 0.1 ~ 0.5g/L
Trehalose 0.5 ~ 2g/L
Glycerine 0.5 ~ 2g/L
Varion CDG-K 1 ~ 3g/L
Sanitas Proclin300 0.5 ~ 1g/L
Preparation method: first compound concentration is 50mM HEPES damping fluid (pH=7.4), then will remain each component and dissolve one by one with 50mM HEPES damping fluid (pH=7.4), make it into above-mentioned concentration.
Beneficial effect of the present invention is as follows:
1) the present invention can keep activity and the HRP(horseradish peroxidase of enzyme for a long time) space conformation of conjugate, and good stability, can significantly improve the shelf-life of test kit, improves its commercial value.Reduce the impact of transportation on enzymoimmunoassay (ELISA) test kit, the experimental repeatability of guaranteed reagent,G.R. box.
2) the present invention is that liquid use reagent, convenient to use, has reduced manual operation error, improves accuracy and the reliability of test result, can be widely used in the mark analysis experiment of enzyme of various HRP.The present invention designed HRP(horseradish peroxidase) protective material can directly be kept at HRP conjugate under low-down working concentration.Not only can save the front prediluted step of experiment, can also keep the activity of the neutral avidin of HRP antibody coupling matter and HRP coupling.
Accompanying drawing explanation
Fig. 1 is protective material of the present invention and other protectant protection effect contrast figures.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
Embodiment 1
Preparation HRP(horseradish peroxidase) protective material, its component and concentration are:
HEPES damping fluid (pH=7.4) 50mM
BSA 1g/L
MgSO4 10mM
NaCl 1mM
ZnSO4 0.5mM
Beta-cyclodextrin 0.3g/L
Trehalose 1g/L
Glycerine 1g/L
Varion CDG-K 2g/L
Sanitas Proclin300 0.5g/L
Preparation method: first compound concentration is 50mM HEPES damping fluid (pH=7.4), then will remain each component and dissolve one by one with 50mM HEPES damping fluid (pH=7.4), make it into above-mentioned concentration.
Embodiment 2
HRP(horseradish peroxidase provided by the invention) protective material comprises component and content thereof are as follows:
HEPES damping fluid (pH=7.4) 50mM
BSA 1g/L
MgSO4 8mM
NaCl 0.5mM
ZnSO4 0.2mM
Beta-cyclodextrin 0.1g/L
Trehalose 0.5g/L
Glycerine 0.5g/L
Varion CDG-K 1g/L
Sanitas Proclin300 0.5g/L
Preparation method: first compound concentration is 50mM HEPES damping fluid (pH=7.4), then will remain each component and dissolve one by one with 50mM HEPES damping fluid (pH=7.4), make it into above-mentioned concentration.
Embodiment 3
HRP(horseradish peroxidase provided by the invention) protective material comprises component and content thereof are as follows:
HEPES damping fluid (pH=7.4) 50mM
BSA 2g/L
MgSO4 10mM
NaCl 2mM
ZnSO4 1mM
Beta-cyclodextrin 0.5g/L
Trehalose 2g/L
Glycerine 2g/L
Varion CDG-K 3g/L
Sanitas Proclin300 1g/L
Preparation method: first compound concentration is 50mM HEPES damping fluid (pH=7.4), then will remain each component and dissolve one by one with 50mM HEPES damping fluid (pH=7.4), make it into above-mentioned concentration.
The HRP antibody coupling matter quality guaranteed period can be reflected HRP(horseradish peroxidase indirectly) protectant protection effect.
Concrete grammar is as follows:
After the antibody of horseradish peroxidase (HRP) mark is combined with assay, by ELISA, detect the peroxidase signal judgement HRP antibody coupling matter quality guaranteed period (numerical value represents with the per-cent of the HRP antibody coupling matter signal of fresh dilution).Length by the quality guaranteed period reflects the protectant effect of HRP.
Be used in respectively the Kan De biotechnology company enzyme stabilizers product that obtains extensively approval on market (be called for short: comparative group) and the HRP protective material that obtains of embodiment 1 stablize HRP coupling antibody.As Fig. 1, after the antibody of horseradish peroxidase (HRP) mark is combined with assay, the quality guaranteed period of the peroxidase signal of mensuration judgement HRP antibody coupling matter.Numerical value represents with the per-cent of the HRP antibody coupling matter signal of fresh dilution.45 ℃ of continuance tests 30 days, by ELISA, detect the signal of peroxidase, measure the active residue of the combination per-cent of HRP coupling antibody and assay.
As shown in Figure 1, with the antibody coupling matter of the HRP enzymatic protective reagent dilution of embodiment 1 gained, hatch after 30 days at 45 ℃, HRP enzymic activity still maintains 85% left and right.This shows, according to Arrhenius(Arrhenius) formula, conservative estimation, enzyme 4 ℃ preserve 2 years after enzyme live and still can keep more than 80%.And the enzyme of comparative group is alive, only maintain 51%.
In sum, HRP(horseradish peroxidase involved in the present invention) protective material, can improve the stability of HRP conjugate greatly, extends the shelf-life of test kit; Improved the anti-qualitative change ability of reagent under transportation and condition of storage; Reducing the unstable that experiment repeats, is a kind of HRP(horseradish peroxidase of efficient stable) protective material.
Claims (1)
1. a horseradish peroxidase protective material, is characterized in that, the component that comprises following content:
HEPES damping fluid, pH=7.4 50mM
BSA 1~2g/L
MgSO4 8~10mM
NaCl 0.5~2mM
ZnSO4 0.2~1mM
Beta-cyclodextrin 0.1 ~ 0.5g/L
Trehalose 0.5 ~ 2g/L
Glycerine 0.5 ~ 2g/L
Varion CDG-K 1 ~ 3g/L
Sanitas Proclin300 0.5 ~ 1g/L.
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| CN201310673542.0A CN103642781B (en) | 2013-12-12 | 2013-12-12 | A kind of Horse radish peroxidase protective agent |
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| CN201310673542.0A CN103642781B (en) | 2013-12-12 | 2013-12-12 | A kind of Horse radish peroxidase protective agent |
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| CN103642781B CN103642781B (en) | 2016-04-20 |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195222A (en) * | 2014-08-18 | 2014-12-10 | 苏州康铭诚业医用科技有限公司 | Compound stabilizer for total cholesterol measurement kits |
| CN105777858A (en) * | 2016-03-22 | 2016-07-20 | 威特曼生物科技（南京）有限公司 | Composite stabilizer of various antibodies and application method of composite stabilizer |
| CN106771216A (en) * | 2016-12-01 | 2017-05-31 | 上海科华生物工程股份有限公司 | Improve immunoreagent and detect specific method and its application |
| CN107831162A (en) * | 2017-09-27 | 2018-03-23 | 北京仪诺益生科技有限公司 | A kind of oophoroma AnnexinA3 detection kits |
| CN108120713A (en) * | 2017-12-20 | 2018-06-05 | 青岛汉唐生物科技有限公司 | A kind of Tes-Tape and preparation method thereof |
| CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
| CN108398564A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgG types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN108398554A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN108469519A (en) * | 2018-03-07 | 2018-08-31 | 深圳市伯劳特生物制品有限公司 | It is a kind of for the composition of enzyme linked immunological kit and diabetes antibody repertoire detection kit and preparation method thereof |
| CN108486090A (en) * | 2018-02-27 | 2018-09-04 | 山东博科诊断科技有限公司 | A kind of serum inorganic phosphorus that stability is strong (enzyme process) detection kit |
| CN110846303A (en) * | 2019-11-23 | 2020-02-28 | 吉林省富生医疗器械有限公司 | Peroxidase protective agent |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195222A (en) * | 2014-08-18 | 2014-12-10 | 苏州康铭诚业医用科技有限公司 | Compound stabilizer for total cholesterol measurement kits |
| CN105777858A (en) * | 2016-03-22 | 2016-07-20 | 威特曼生物科技（南京）有限公司 | Composite stabilizer of various antibodies and application method of composite stabilizer |
| CN105777858B (en) * | 2016-03-22 | 2019-09-10 | 东软威特曼生物科技(南京)有限公司 | The compound stabilizer and its application method of Multiple Antibodies |
| CN106771216A (en) * | 2016-12-01 | 2017-05-31 | 上海科华生物工程股份有限公司 | Improve immunoreagent and detect specific method and its application |
| CN106771216B (en) * | 2016-12-01 | 2019-06-25 | 上海科华生物工程股份有限公司 | Improve the method and its application of immunoreagent detection specificity |
| CN108279302A (en) * | 2017-07-11 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of composition and pylori spiral bacilli antibody for enzyme linked immunological kit composes detection kit and preparation method thereof |
| CN108279302B (en) * | 2017-07-11 | 2020-05-26 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, helicobacter pylori antibody spectrum detection kit and preparation method thereof |
| CN107831162A (en) * | 2017-09-27 | 2018-03-23 | 北京仪诺益生科技有限公司 | A kind of oophoroma AnnexinA3 detection kits |
| CN108120713A (en) * | 2017-12-20 | 2018-06-05 | 青岛汉唐生物科技有限公司 | A kind of Tes-Tape and preparation method thereof |
| CN108486090A (en) * | 2018-02-27 | 2018-09-04 | 山东博科诊断科技有限公司 | A kind of serum inorganic phosphorus that stability is strong (enzyme process) detection kit |
| CN108469519A (en) * | 2018-03-07 | 2018-08-31 | 深圳市伯劳特生物制品有限公司 | It is a kind of for the composition of enzyme linked immunological kit and diabetes antibody repertoire detection kit and preparation method thereof |
| CN108398554A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgM types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN108469519B (en) * | 2018-03-07 | 2020-03-24 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof |
| CN108398564A (en) * | 2018-03-07 | 2018-08-14 | 深圳市伯劳特生物制品有限公司 | A kind of kit of anti-torch-IgG types antibody spectrum chip and preparation method thereof and TORCH detections |
| CN110846303A (en) * | 2019-11-23 | 2020-02-28 | 吉林省富生医疗器械有限公司 | Peroxidase protective agent |
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| CN103642781B (en) | 2016-04-20 |
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