CN107741504A - A kind of preparation method of parathyroid hormone detection kit - Google Patents
A kind of preparation method of parathyroid hormone detection kit Download PDFInfo
- Publication number
- CN107741504A CN107741504A CN201710917748.1A CN201710917748A CN107741504A CN 107741504 A CN107741504 A CN 107741504A CN 201710917748 A CN201710917748 A CN 201710917748A CN 107741504 A CN107741504 A CN 107741504A
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- China
- Prior art keywords
- preparation
- reagent
- buffer solutions
- latex
- polyclonal antibody
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Classifications
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
Abstract
The invention discloses a kind of preparation method of parathyroid hormone detection kit, comprise the following steps：(1) reagent R1 constituent contents are pressed, MOPSO is dissolved in purified water, pH is adjusted, obtains R1 buffer solutions；By NaCl, NaN3, Arabic gum, Tween 80, PEG 2000 be dissolved in R1 buffer solutions, obtain reagent R1；(2) reagent R2 constituent contents are pressed, MOPSO is dissolved in purified water, pH is adjusted, obtains R2 buffer solutions；By NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, obtain R2 dispersion liquids；Prepare the coated parathyroid gland polyclonal antibody of latex；R2 dispersion liquids dissolve the coated parathyroid gland polyclonal antibody of latex, obtain reagent R2.Advantage of the present invention is：Simple to operate, quick, quantitative detection is accurate；Kit high sensitivity, the range of linearity；Stabilization of kit is good, high specificity；Cost is low, it is pollution-free, for automatic clinical chemistry analyzer.
Description
Technical field
The present invention relates to technical field of medical examination, more particularly to a kind of preparation side of parathyroid hormone detection kit
Method.
Background technology
Parathyroid hormone (parathyroid hormone), it is the alkaline single chain polypeptide of chief cell secretion
Parahormone, abbreviation PTH.Parathyroid hormone is made up of 84 amino acid, and major function is calcium and phosphorus in regulation vertebrate body
Metabolism, promote calcium level to raise, serum phosphorus levels decline.
Synthesize PTH the first precursor, referred to as Pre Pro PTH first in chief cell, contain
115 amino acid, this later precursor crack as by the second precursor first shape containing 90 amino acid in the cell
Glandular hormone is former, and the latter and then in the cell cracking turn into the polypeptide containing 84 amino acid, i.e. PTH.PTH's is dense in human normal plasma
Degree is about 1 nanograms/milliliter.
At present, the method for detecting parathyroid hormone is mainly enzyme linked immunosorbent assay and radio immunoassay.Wherein,
Enzyme linked immunosorbent assay is cumbersome, and quantitative Detection results are bad, and it is larger to be as a result affected by human factors result；Radio-immunity point
Analysis rule has radial pattern pollution, and required instrument is costly.
Therefore, be badly in need of at present it is a kind of it is simple to operate, quantitatively detect the inspection of parathyroid hormone accurate, pollution-free, that cost is low
The preparation method of test agent box.
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 detection simple to operate, quantitative is accurate, nothing
The preparation method of the low parathyroid hormone detection kit of pollution, cost.
The present invention is achieved by the following technical solutions：A kind of preparation method of parathyroid hormone detection kit,
Comprise the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
1. according to mentioned reagent R1 constituent content, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, Arabic gum, Tween-80, PEG-2000 be dissolved in
In R1 buffer solutions, 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, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, stirring is equal
It is even, produce R2 dispersion liquids；
3. prepare the coated parathyroid gland polyclonal 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, parathyroid gland polyclonal antibody is added, 3-4h is reacted at 37 DEG C, centrifuged, sediment is scattered in original volume
In PBS, 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 parathyroid gland polyclonal antibody of finally required latex；
4. dissolve the obtained coated parathyroid gland polyclonal antibody of latex, ultrasonic disperse, final system with R2 dispersion liquids
Obtain reagent 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 R1 buffer solutions is 6.5.
One of preferred embodiment as the present invention, in the step (2), the pH of R2 buffer solutions is 6.5.
One of preferred embodiment as the present invention, the step a for preparing the coated parathyroid gland polyclonal antibody of latex
In, the MES buffer solutions used is 100mM MES buffer solutions.
One of preferred embodiment as the present invention, the step a for preparing the coated parathyroid gland polyclonal antibody of latex
In, the EDAC solution that uses for 50g/L EDAC solution.
One of preferred embodiment as the present invention, the step a for preparing the coated parathyroid gland polyclonal antibody of latex
In, the NHS solution that uses for 50g/L NHS solution.
One of preferred embodiment as the present invention, the step b for preparing the coated parathyroid gland polyclonal antibody of latex
In, the BSA of addition is 30g/L BSA.
The present invention compared with prior art the advantages of be：
(1) kit prepared using this method has a higher detection sensitivity, simple to operate, quantitative detection accurately,
Quickly, from detecting out that result only needs 10 minutes；
(2) in the preparation process of the coated parathyroid gland polyclonal antibody of latex, the latex microsphere of different-grain diameter is taken
It is used in mixed way, substantially increases 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 directly used on automatic clinical chemistry analyzer, be set without large-scale instrument
Standby to coordinate, cost is low, and "dead" pollution, can carry out and promote on a large 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 parathyroid hormone detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
1. according to mentioned reagent R1 constituent content, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, Arabic gum, Tween-80, PEG-2000 be dissolved in
In R1 buffer solutions, 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, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, stirring is equal
It is even, produce R2 dispersion liquids；
3. prepare the coated parathyroid gland polyclonal 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/L NHS solution to recover to original volume, mix
Even be incubated in 37 DEG C of environment mixes 1h, and 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, parathyroid gland polyclonal antibody is added, 3h is reacted at 37 DEG C, centrifuged, the PBS that sediment is scattered in original volume delays
In fliud flushing, 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 parathyroid gland polyclonal antibody of finally required latex；
4. dissolve the obtained coated parathyroid gland polyclonal antibody of latex, ultrasonic disperse, final system with R2 dispersion liquids
Obtain reagent R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 9%.
Embodiment 2
A kind of preparation method of parathyroid hormone detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
1. according to mentioned reagent R1 constituent content, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, Arabic gum, Tween-80, PEG-2000 be dissolved in
In R1 buffer solutions, 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, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, stirring is equal
It is even, produce R2 dispersion liquids；
3. prepare the coated parathyroid gland polyclonal 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/L NHS solution to recover to original volume, mix
Even be incubated in 37 DEG C of environment mixes 1h, and 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, parathyroid gland polyclonal antibody is added, 4h is reacted at 37 DEG C, centrifuged, the PBS that sediment is scattered in original volume delays
In fliud flushing, 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 parathyroid gland polyclonal antibody of finally required latex；
4. dissolve the obtained coated parathyroid gland polyclonal antibody of latex, ultrasonic disperse, final system with R2 dispersion liquids
Obtain reagent R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 11%.
Embodiment 3
A kind of preparation method of parathyroid hormone detection kit of the present embodiment, comprises the following steps：
(1) according to following component content reagent preparation R1：
Reagent R1：
1. according to mentioned reagent R1 constituent content, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R1 buffer solutions；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, Arabic gum, Tween-80, PEG-2000 be dissolved in
In R1 buffer solutions, 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, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to
R2 buffer solutions；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, stirring is equal
It is even, produce R2 dispersion liquids；
3. prepare the coated parathyroid gland polyclonal 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/L NHS solution to recover to original volume, mix
Even be incubated in 37 DEG C of environment mixes 1h, and 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, parathyroid gland polyclonal antibody is added, 3.5h is reacted at 37 DEG C, centrifuged, sediment is scattered in the PBS of original volume
In buffer solution, 3 times repeatedly, finally sediment is scattered in the NHS buffer solutions of original volume, adds BSA 30g/L；
C.4 DEG C seal 48h up for safekeeping, obtain the coated parathyroid gland polyclonal antibody of finally required latex；
4. dissolve the obtained coated parathyroid gland polyclonal antibody of latex, ultrasonic disperse, final system with R2 dispersion liquids
Obtain reagent R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 10%.
Embodiment 4
The user of the parathyroid hormone detection kit being prepared using above-described embodiment method of the present embodiment
Method, comprise the following steps：
(1) 4uL testing samples are mixed with 150uL reagents R1,37 DEG C of incubation 5min；
(2) reacted absorbance A 1 is determined at wavelength 600nm 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 600nm with automatic clinical chemistry analyzer；
(5) according to absorbance change value Δ A=A2-A1, the concentration of parathyroid hormone 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 parathyroid hormone 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, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to R1 and delays
Fliud flushing；
2. according to mentioned reagent R1 constituent content, by NaCl, NaN3, Arabic gum, Tween-80, PEG-2000 be dissolved in R1 and delay
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：
1. according to mentioned reagent R2 constituent content, MOPSO is dissolved in purified water, stirred, pH is adjusted, is configured to R2 and delays
Fliud flushing；
2. according to mentioned reagent R2 constituent content, by NaCl, NaN3, BSA, glycerine be dissolved in R2 buffer solutions, stir, i.e.,
Obtain R2 dispersion liquids；
3. prepare the coated parathyroid gland polyclonal 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 parathyroid gland polyclonal antibody, 3-4h is reacted at 37 DEG C, centrifuges, and the PBS that sediment is scattered in original volume delays
In fliud flushing, 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 parathyroid gland polyclonal antibody of finally required latex；
4. dissolving the obtained coated parathyroid gland polyclonal antibody of latex, ultrasonic disperse with R2 dispersion liquids, final be made is tried
Agent R2；Wherein, ultimate density of the polyethylene p-chloromethyl styrene copolymer in reagent R2 is 9%-11%.
2. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the step
(1) in, the pH of R1 buffer solutions is 6.5.
3. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the step
(2) in, the pH of R2 buffer solutions is 6.5.
4. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the preparation
In the step a of the coated parathyroid gland polyclonal antibody of latex, the MES buffer solutions used is 100mM MES buffer solutions.
5. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the preparation
In the step a of the coated parathyroid gland polyclonal antibody of latex, the EDAC solution that uses for 50g/L EDAC solution.
6. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the preparation
In the step a of the coated parathyroid gland polyclonal antibody of latex, the NHS solution that uses for 50g/L NHS solution.
7. the preparation method of parathyroid hormone detection kit according to claim 1, it is characterised in that the preparation
In the step b of the coated parathyroid gland polyclonal antibody of latex, the BSA of addition is 30g/L BSA.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710917748.1A CN107741504A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of parathyroid hormone detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710917748.1A CN107741504A (en) | 2017-09-30 | 2017-09-30 | A kind of preparation method of parathyroid hormone detection kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111596072A (en) * | 2020-06-11 | 2020-08-28 | 安徽大千生物工程有限公司 | Kit for determining PTH based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103134926A (en) * | 2013-02-27 | 2013-06-05 | 上海交通大学 | Magnetic microsphere carrier and its making method |
| CN105353139A (en) * | 2015-09-28 | 2016-02-24 | 成都博奥新景医学科技有限公司 | Parathyroid hormone quantitative detection kit |
| 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 CN201710917748.1A patent/CN107741504A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103134926A (en) * | 2013-02-27 | 2013-06-05 | 上海交通大学 | Magnetic microsphere carrier and its making method |
| CN105353139A (en) * | 2015-09-28 | 2016-02-24 | 成都博奥新景医学科技有限公司 | Parathyroid hormone quantitative detection kit |
| 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 (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111596072A (en) * | 2020-06-11 | 2020-08-28 | 安徽大千生物工程有限公司 | Kit for determining PTH based on latex enhanced immunoturbidimetry and preparation and use methods thereof |
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Application publication date: 20180227 |