CN107741494A - A kind of preparation method of CER detection kit - Google Patents
A kind of preparation method of CER detection kit Download PDFInfo
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- CN107741494A CN107741494A CN201710915773.6A CN201710915773A CN107741494A CN 107741494 A CN107741494 A CN 107741494A CN 201710915773 A CN201710915773 A CN 201710915773A CN 107741494 A CN107741494 A CN 107741494A
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- cer
- reagent
- buffer solutions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Abstract
The invention discloses a kind of preparation method of CER detection kit, comprise the following steps：(1) reagent R1 constituent contents are pressed, prepare PBS, pH is adjusted, makees R1 buffer solutions；By NaCl, NaN3, trehalose, Qula be logical, PEG 8000 is dissolved in R1 buffer solutions, obtain reagent R1；(2) reagent R2 constituent contents are pressed, prepare PBS, pH is adjusted, makees R2 buffer solutions；By NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, obtain R2 dispersion liquids；The coated CER antibody of glue breast；R2 dispersion liquids dissolve the coated CER antibody of latex, obtain reagent R2.Advantages of the present invention is：(1) it is simple to operate, quick；(2) kit sensitivity, the degree of accuracy are high；(3) different-grain diameter latex microsphere is taken, improves kit sensitivity and linear measurement range；(4) stabilization of kit is good, high specificity；(5) it is adapted to full-automatic testing.
Description
Technical field
The present invention relates to technical field of medical examination, more particularly to a kind of preparation method of CER detection kit.
Background technology
CER (ceruloplasmin, CER) is also known as CuO-2 layer, is a kind of glycoprotein of α 2 of cupric, molecular weight is about
For 12-16 ten thousand, not easy purification.Current known, CER is a single chain polypeptide, and per molecule contains 6-7 copper atom, due to containing
Copper and in blueness, containing sugar about 10%, terminal sialic acid and polypeptide chain link, there is gene polymorphy genetically.It act as
Adjust copper each position of body distribution, synthesize the zymoprotein of cupric, play the role of antioxidant, and with oxidation enzyme activity
Property, there is the ability for being catalyzed its oxidation to polyphenol and more amine substrates.It is generally believed that CER is synthesized by liver, a part by
Biliary excretion, content is little in urine.Diagnosis of the measure of CER to diseases such as some livers, courage, kidneys has the certain significance.
At present, the method for detecting CER is mainly enzyme linked immunosorbent assay, but the specific behaviour of enzyme linked immunosorbent assay
Make relatively cumbersome, and quantitative Detection results are bad, and it is larger to be as a result affected by human factors result.
Therefore, it is badly in need of a kind of simple to operate, result at present and detects accurate CER detection kit.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of simple to operate, result detection accurately
The preparation method of CER detection kit.
The present invention is achieved by the following technical solutions：A kind of preparation method of CER detection kit, including
Following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, PBS is prepared, pH is adjusted, as R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, trehalose, Qula be logical, PEG-8000 is dissolved in R1 and delayed
In fliud flushing, stir, treat that all raw materials fully dissolve, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, PBS is prepared, pH is adjusted, as R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, stir
Mix uniformly, produce R2 dispersion liquids；
3. prepare the coated CER antibody of latex：
A. the latex microsphere that particle diameter is 80nm and 120nm is taken in MES buffer solutions, is added EDAC solution and is mixed after 37 DEG C
It is incubated in environment and mixes 1h, supernatant is removed in centrifugation；Add NHS solution to recover to original volume, mix and be incubated in 37 DEG C of environment
1h is mixed, centrifugation removes supernatant, must mix microballoon emulsion；
B. polyethylene p-chloromethyl styrene copolymer is added in microballoon emulsion is mixed, is copolymerized 80nm and 120nm particle diameters
Latex；Centrifugation, surplus materials is scattered in MES buffer solutions, 2-4 times repeatedly, most after recover in MES buffer solutions to
Original volume, CER antibody is added, 3-4h is reacted at 37 DEG C, centrifuged, the PBS that sediment is scattered in original volume is buffered
In liquid, 2-4 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA；
C.2-8 DEG C seal 45-50h up for safekeeping, obtain the coated CER antibody of finally required latex；
4. dissolving the obtained coated CER antibody of latex with R2 dispersion liquids, ultrasonic disperse, reagent is finally made
R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 9%-11%.
One of preferred embodiment as the present invention, in the step (1), the pH of reagent R1 PBS is 8.3.
One of preferred embodiment as the present invention, in the step (2), the pH of reagent R2 PBS is 8.3.
One of preferred embodiment as the present invention, in the step a for preparing the coated CER antibody of latex, use
MES buffer solutions be 100mM MES buffer solutions.
One of preferred embodiment as the present invention, in the step a for preparing the coated CER antibody of latex, use
EDAC solution be 50g/L EDAC solution.
One of preferred embodiment as the present invention, in the step a for preparing the coated CER antibody of latex, use
NHS solution be 50g/L NHS solution.
One of preferred embodiment as the present invention, in the step b for preparing the coated CER antibody of latex, add
BSA be 30g/L BSA.
The present invention compared with prior art the advantages of be：
(1) kit prepared using this method has higher detection sensitivity and a degree of accuracy, and use it is simple to operate,
Quickly, from detecting out that result only needs 10 minutes；
(2) in the preparation process of the coated CER antibody of breast, the latex microsphere for taking different-grain diameter is used in mixed way,
Substantially increase the sensitivity and linear measurement range of kit；
(3) antigen antibody complex formed using kit prepared by this method with sample, good stability, specific
There are certain absorbance, high specificity under wavelength；
(4) kit prepared using this method can be used on automatic clinical chemistry analyzer, be adapted to full-automatic testing, can be big
The development and popularization of scale.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
A kind of preparation method of CER detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, PBS is prepared, pH is adjusted, as R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, trehalose, Qula be logical, PEG-8000 is dissolved in R1 and delayed
In fliud flushing, stir, treat that all raw materials fully dissolve, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, PBS is prepared, pH is adjusted, as R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, stir
Mix uniformly, produce R2 dispersion liquids；
3. prepare the coated CER antibody of latex：
A. the latex microsphere that particle diameter is 80nm and 120nm is taken to add 50g/L EDAC solution in 100mM MES buffer solutions
Mix after being incubated mixing 1h in 37 DEG C of environment, supernatant is removed in centrifugation；Add 50g/LNHS solution to recover to original volume, mix
It is incubated in 37 DEG C of environment and mixes 1h, centrifugation removes supernatant, must mix microballoon emulsion；
B. polyethylene p-chloromethyl styrene copolymer is added in microballoon emulsion is mixed, is copolymerized 80nm and 120nm particle diameters
Latex；Centrifugation, surplus materials is scattered in MES buffer solutions, 2 times repeatedly, most after recovery in MES buffer solutions to original
Volume, CER antibody is added, 3h is reacted at 37 DEG C, centrifuged, sediment is scattered in the PBS of original volume
In, 2 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA 30g/L；
C.2 DEG C seal 45h up for safekeeping, obtain the coated CER antibody of finally required latex；
4. dissolving the obtained coated CER antibody of latex with R2 dispersion liquids, ultrasonic disperse, reagent is finally made
R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 9%.
Embodiment 2
A kind of preparation method of CER detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, PBS is prepared, pH is adjusted, as R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, trehalose, Qula be logical, PEG-8000 is dissolved in R1 and delayed
In fliud flushing, stir, treat that all raw materials fully dissolve, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, PBS is prepared, pH is adjusted, as R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, stir
Mix uniformly, produce R2 dispersion liquids；
3. prepare the coated CER antibody of latex：
A. the latex microsphere that particle diameter is 80nm and 120nm is taken to add 50g/L EDAC solution in 100mM MES buffer solutions
Mix after being incubated mixing 1h in 37 DEG C of environment, supernatant is removed in centrifugation；Add 50g/LNHS solution to recover to original volume, mix
It is incubated in 37 DEG C of environment and mixes 1h, centrifugation removes supernatant, must mix microballoon emulsion；
B. polyethylene p-chloromethyl styrene copolymer is added in microballoon emulsion is mixed, is copolymerized 80nm and 120nm particle diameters
Latex；Centrifugation, surplus materials is scattered in MES buffer solutions, 4 times repeatedly, most after recovery in MES buffer solutions to original
Volume, CER antibody is added, 4h is reacted at 37 DEG C, centrifuged, sediment is scattered in the PBS of original volume
In, 4 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA 30g/L；
C.8 DEG C seal 50h up for safekeeping, obtain the coated CER antibody of finally required latex；
4. dissolving the obtained coated CER antibody of latex with R2 dispersion liquids, ultrasonic disperse, reagent is finally made
R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 11%.
Embodiment 3
A kind of preparation method of CER detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
Its solvent is purified water；
1. according to mentioned reagent R1 constituent content, PBS is prepared, pH is adjusted, as R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, trehalose, Qula be logical, PEG-8000 is dissolved in R1 and delayed
In fliud flushing, stir, treat that all raw materials fully dissolve, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
Its solvent is purified water；
1. according to mentioned reagent R2 constituent content, PBS is prepared, pH is adjusted, as R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, stir
Mix uniformly, produce R2 dispersion liquids；
3. prepare the coated CER antibody of latex：
A. the latex microsphere that particle diameter is 80nm and 120nm is taken to add 50g/L EDAC solution in 100mM MES buffer solutions
Mix after being incubated mixing 1h in 37 DEG C of environment, supernatant is removed in centrifugation；Add 50g/LNHS solution to recover to original volume, mix
It is incubated in 37 DEG C of environment and mixes 1h, centrifugation removes supernatant, must mix microballoon emulsion；
B. polyethylene p-chloromethyl styrene copolymer is added in microballoon emulsion is mixed, is copolymerized 80nm and 120nm particle diameters
Latex；Centrifugation, surplus materials is scattered in MES buffer solutions, 3 times repeatedly, most after recovery in MES buffer solutions to original
Volume, CER antibody is added, 3.5h is reacted at 37 DEG C, centrifuged, sediment is scattered in the PBS of original volume
In, 3 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA 30g/L；
C.5 DEG C seal 48h up for safekeeping, obtain the coated CER antibody of finally required latex；
4. dissolving the obtained coated CER antibody of latex with R2 dispersion liquids, ultrasonic disperse, reagent is finally made
R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 10%.
Embodiment 4
The application method of the CER detection kit being prepared using above-described embodiment method of the present embodiment, bag
Include following steps：
(1) 5uL testing samples are mixed with 200uL reagents R1,37 DEG C of incubation 5min；
(2) reacted absorbance A 1 is determined at wavelength 660nm with automatic clinical chemistry analyzer；
(3) mixed again with 50uL reagents R2,37 DEG C of reaction 5min；
(4) reacted absorbance A 2 is determined at wavelength 660nm with automatic clinical chemistry analyzer；
(5) according to absorbance change value Δ A=A2-A1, the concentration of CER in sample is calculated.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (7)
1. a kind of preparation method of CER detection kit, it is characterised in that comprise the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
1. according to mentioned reagent R1 constituent content, PBS is prepared, pH is adjusted, as R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, trehalose, Qula be logical, PEG-8000 is dissolved in R1 buffer solutions
In, stir, treat that all raw materials fully dissolve, that is, reagent R1 is made；
(2) according to following component content reagent preparation R2：
Reagent R2：
1. according to mentioned reagent R2 constituent content, PBS is prepared, pH is adjusted, as R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, Arabic gum be dissolved in R2 buffer solutions, stirring is equal
It is even, produce R2 dispersion liquids；
3. prepare the coated CER antibody of latex：
A. the latex microsphere that particle diameter is 80nm and 120nm is taken in MES buffer solutions, is added EDAC solution and is mixed after 37 DEG C of environment
Middle be incubated mixes 1h, and supernatant is removed in centrifugation；Add NHS solution to recover to original volume, mix and mixing is incubated in 37 DEG C of environment
1h, centrifugation remove supernatant, must mix microballoon emulsion；
B. polyethylene p-chloromethyl styrene copolymer is added in microballoon emulsion is mixed, is copolymerized the glue of 80nm and 120nm particle diameters
Breast；Centrifugation, surplus materials is scattered in MES buffer solutions, 2-4 times repeatedly, most after recovery in MES buffer solutions to substance
Product, adds CER antibody, 3-4h is reacted at 37 DEG C, centrifuges, and sediment is scattered in the PBS of original volume
In, 2-4 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA；
C.2-8 DEG C seal 45-50h up for safekeeping, obtain the coated CER antibody of finally required latex；
4. dissolving the obtained coated CER antibody of latex with R2 dispersion liquids, ultrasonic disperse, reagent R2 is finally made；Its
In, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 9%-11%.
2. the preparation method of CER detection kit according to claim 1, it is characterised in that the step (1)
In, the pH of reagent R1 PBS is 8.3.
3. the preparation method of CER detection kit according to claim 1, it is characterised in that the step (2)
In, the pH of reagent R2 PBS is 8.3.
4. the preparation method of CER detection kit according to claim 1, it is characterised in that described to prepare latex
In the step a of coated CER antibody, the MES buffer solutions used is 100mMMES buffer solutions.
5. the preparation method of CER detection kit according to claim 1, it is characterised in that described to prepare latex
In the step a of coated CER antibody, the EDAC solution that uses for 50g/L EDAC solution.
6. the preparation method of CER detection kit according to claim 1, it is characterised in that described to prepare latex
In the step a of coated CER antibody, the NHS solution that uses for 50g/L NHS solution.
7. the preparation method of CER detection kit according to claim 1, it is characterised in that described to prepare latex
In the step b of coated CER antibody, the BSA of addition is 30g/L BSA.
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| CN201710915773.6A CN107741494A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of CER detection kit |
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| CN201710915773.6A CN107741494A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of CER detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122882A (en) * | 2020-01-02 | 2020-05-08 | 四川纳海川生物科技有限公司 | Total iron detection kit and preparation method thereof |
| CN111141913A (en) * | 2020-01-02 | 2020-05-12 | 四川纳海川生物科技有限公司 | Ceruloplasmin detection kit and preparation method thereof |
| CN112730833A (en) * | 2020-11-23 | 2021-04-30 | 迪瑞医疗科技股份有限公司 | Ceruloplasmin determination kit by using immuno-transmission turbidimetry |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
| CN103134926A (en) * | 2013-02-27 | 2013-06-05 | 上海交通大学 | Magnetic microsphere carrier and its making method |
| CN106932588A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Detection α1Kit of-microglobulin and preparation method thereof |
-
2017
- 2017-09-30 CN CN201710915773.6A patent/CN107741494A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
| CN103134926A (en) * | 2013-02-27 | 2013-06-05 | 上海交通大学 | Magnetic microsphere carrier and its making method |
| CN106932588A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Detection α1Kit of-microglobulin and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111122882A (en) * | 2020-01-02 | 2020-05-08 | 四川纳海川生物科技有限公司 | Total iron detection kit and preparation method thereof |
| CN111141913A (en) * | 2020-01-02 | 2020-05-12 | 四川纳海川生物科技有限公司 | Ceruloplasmin detection kit and preparation method thereof |
| CN112730833A (en) * | 2020-11-23 | 2021-04-30 | 迪瑞医疗科技股份有限公司 | Ceruloplasmin determination kit by using immuno-transmission turbidimetry |
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