CN101871935A - Covalent cross-linking method for carboxylic polystyrene microsphere and C reaction protein antibody - Google Patents
Covalent cross-linking method for carboxylic polystyrene microsphere and C reaction protein antibody Download PDFInfo
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- CN101871935A CN101871935A CN201010192601A CN201010192601A CN101871935A CN 101871935 A CN101871935 A CN 101871935A CN 201010192601 A CN201010192601 A CN 201010192601A CN 201010192601 A CN201010192601 A CN 201010192601A CN 101871935 A CN101871935 A CN 101871935A
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Abstract
The invention provides a covalent cross-linking method for carboxylic polystyrene microspheres and C reaction protein antibody. The method comprises the following steps: selecting carboxylic polystyrene microspheres with proper grain diameters; centrifugally cleaning the carboxylic polystyrene microspheres with buffer solution, and resuspending the microspheres; uniformly mixing the microspheres with cross-linking agent, and centrifugally cleaning the microspheres after incubation; adding the C reaction protein antibody and sealant into the microspheres after pH regulating agent is added into the microspheres for incubating; cleaning the carboxylic polystyrene microspheres; and adding the buffer solution into the microspheres, resuspending the microspheres, and storing the microspheres in the buffer solution. The method solves the problems of low cross-linking efficiency, poor stability and the like existing in the current conventional cross-linking method.
Description
Technical field
The invention belongs to the Medical Immunology field, relate to a kind of preparation method of immunologic function test reagent, particularly relate to the covalent cross-linking method of a kind of carboxylic polystyrene microsphere and c reactive protein antibody.
Background technology
The detection method of c reactive protein is used at present many have latex agglutination test, immuno-precipitation and label immunoassay method etc., but, use conventional c reactive protein assay method often sensitivity lower, can't be accurately quantitative, therefore be not suitable for the detection of major diseases such as myocardial infarction, tumour.By with carboxylic polystyrene microsphere and c reactive protein antibody covalent cross-linking, thereby utilize the rate scattering turbidimetry c reactive protein in the sample to be measured the sensitivity and the accuracy that can improve detection.But traditional cross-linking method is low, the poor stability of cross-linking efficiency often, is unsuitable for long preservation.
Summary of the invention
The covalent cross-linking method that the purpose of this invention is to provide a kind of carboxylic polystyrene microsphere and c reactive protein antibody, problems such as the cross-linking efficiency that it can solve at present existing method existence is low, poor stability.
To achieve these goals, the technical scheme of the present invention's proposition is: the covalent cross-linking method of a kind of carboxylic polystyrene microsphere and c reactive protein antibody may further comprise the steps:
(1) selects the carboxylic polystyrene microsphere of appropriate particle size as required;
(2) carboxylic polystyrene microsphere is used damping fluid eccentric cleaning, resuspension;
(3) mixing behind the adding crosslinking chemical, eccentric cleaning behind the incubation;
(4) add c reactive protein antibody and sealer, incubation behind the adding acid-base modifier;
(5) clean carboxylic polystyrene microsphere;
(6) add damping fluid, resuspension is stored in the damping fluid.
The particle diameter of the described carboxylic polystyrene microsphere of step (1) is 50～300nm.The described damping fluid of step (2) is acetate buffer or phosphate buffer, and the ion concentration of damping fluid is 20～80mM, and pH value is 7.5～8.The described crosslinking chemical of step (3) comprises the carbodiimide class.The concentration of crosslinking chemical was 1～20mg/mL after step (3) was finished.The temperature of the described incubation of step (3) is 15～45 ℃, and the time is 15～60 minutes.The described acid-base modifier of step (4) is the organic acid of 2 to 6 C atoms, comprises acetate.The described sealer of step (4) is a non-ionics, comprises triton x-100, Tween 80.The temperature of the described incubation of step (4) is 15～45 ℃, and the time is 1.5～20 hours.The described damping fluid of step (6) is nonionic both sexes damping fluids, comprises hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid.The described damping fluid ion concentration of step (6) is 20～100mM, and pH value is 4.5～8.
Step of the present invention (1) will be as required, selects the carboxylic polystyrene microsphere of appropriate particle size, and this is related to prepared detection sensitivity and the stability that goes out immunoturbidimetry reagent.The reagent of in particle diameter is 50～300nm scope, preparing, detection sensitivity can reach 1.0ng/mL, and reagent can be preserved more than 1 year at normal temperatures.Step (2) is with carboxylic polystyrene microsphere damping fluid eccentric cleaning, resuspension.Cleaning step wherein is very crucial, temperature preferably is controlled at 4 ℃, and the most handy ultrasonic method of microballoon after eccentric cleaning is suspended in the damping fluid again, the most handy acetate buffer of damping fluid or phosphate buffer, the ion concentration of damping fluid is 20～80mM, and pH value is 7.5～8.Mixing behind step (3) the adding crosslinking chemical, eccentric cleaning behind the incubation.Described crosslinking chemical is the carbodiimide class, and as N, N '-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), this type of crosslinking chemical are widely used in the condensation of amino and carboxyl.Carboxylic acid ion adds to protonated carbodiimide, forms highly active O-acylureas, and O-acylureas and amino group reaction produce protected peptide and urea derivative.Incubation behind the adding crosslinking chemical, temperature is 15～45 ℃, time is 15～60 minutes, can make carboxyl and carbodiimide fully react the formation stable intermediate products like this, eccentric cleaning can flush away not be participated in the crosslinking chemical and the urea derivative of reaction, this step is extremely important, directly has influence on next step cross-linking efficiency, and cleaning is clean even may cause not having the c reactive protein antibodies to microballoon.
The described acid-base modifier of step (4) is the organic acid of 2 to 6 C atoms, comprises acetate.The described sealer of step (4) is a non-ionics, comprises triton x-100, Tween 80.This class surfactant has excellent biological compatibility, and cheap.The purpose that adds sealer is a carboxylic group of not participating in reaction in order to seal, also can make c reactive protein antibody become monodisperse status to distribute at microsphere surface, and certain distance is arranged each other, and what be unlikely to arrange is closely excessive.The temperature of incubation is 15～45 ℃, and the time is can reach better effect in 1.5～20 hours.
Liquid and the described damping fluid of step (6) that step (5) is cleaned usefulness can be liquid of the same race, be nonionic both sexes damping fluid, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, the damping fluid ion concentration is 20～100mM, pH value is 4.5～8, and these all help the long-term stable preservation of immunoturbidimetry reagent.
Embodiment
The covalent cross-linking method of the carboxylic polystyrene microsphere of embodiment and c reactive protein antibody may further comprise the steps:
(1) carboxylic polystyrene microsphere of selection 200nm;
(2) be 30mM with the carboxylic polystyrene microsphere ion concentration, PH is 7.8 phosphate buffer eccentric cleaning 2 times, carries out resuspension with ultrasonic method;
(3) add mixing behind the carbodiimide hydrochloride, incubation then, 25 ℃ of the temperature of incubation, the time is 30 minutes, with phosphate buffer eccentric cleaning 2 times;
(4) adding acetate, to regulate pH value be 5, then adds c reactive protein antibody and Tween 80, incubation then, and 25 ℃ of the temperature of incubation, the time is 3 hours;
(5) clean carboxylic polystyrene microsphere with hydroxyethyl piperazine second thiosulfonic acid;
(6) adding ion concentration is that 50mM, pH value are 7.8 damping fluid hydroxyethyl piperazine second thiosulfonic acid, carries out resuspension with ultrasonic method, is stored in the damping fluid damping fluid.
Use the method, the covalent cross-linking rate of carboxylic polystyrene microsphere and c reactive protein antibody can reach 90%, can stablize and place 2 years.
Claims (20)
1. the covalent cross-linking method of carboxylic polystyrene microsphere and c reactive protein antibody is characterized in that may further comprise the steps:
(1) selects the carboxylic polystyrene microsphere of appropriate particle size as required;
(2) carboxylic polystyrene microsphere is used damping fluid eccentric cleaning, resuspension;
(3) mixing behind the adding crosslinking chemical, eccentric cleaning behind the incubation;
(4) add c reactive protein antibody and sealer, incubation behind the adding acid-base modifier;
(5) clean carboxylic polystyrene microsphere;
(6) add damping fluid, resuspension is stored in the damping fluid.
2. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 1 and c reactive protein antibody, it is characterized in that the particle diameter of the described carboxylic polystyrene microsphere of step (1) is 50～300nm.
3. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 1 and c reactive protein antibody, it is characterized in that the described damping fluid of step (2) is acetate buffer or phosphate buffer, the ion concentration of damping fluid is 20～80mM, and pH value is 7.5～8.
4. according to the covalent cross-linking method of claim 1,2 or 3 described carboxylic polystyrene microspheres and c reactive protein antibody, it is characterized in that the described crosslinking chemical of step (3) comprises the carbodiimide class, the concentration of crosslinking chemical was 1～20mg/mL after step (3) was finished, the temperature of described incubation is 15～45 ℃, and the time is 15～60 minutes.
5. according to the covalent cross-linking method of claim 1,2 or 3 described carboxylic polystyrene microspheres and c reactive protein antibody, it is characterized in that the described acid-base modifier of step (4) is the organic acid of 2 to 6 C atoms, comprise acetate.
6. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 4 and c reactive protein antibody, it is characterized in that the described acid-base modifier of step (4) is the organic acid of 2 to 6 C atoms, comprise acetate.
7. according to the covalent cross-linking method of claim 1,2 or 3 described carboxylic polystyrene microspheres and c reactive protein antibody, it is characterized in that the described sealer of step (4) is a non-ionics, comprise triton x-100, Tween 80.
8. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 4 and c reactive protein antibody, it is characterized in that the described sealer of step (4) is a non-ionics, comprise triton x-100, Tween 80.
9. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 5 and c reactive protein antibody, it is characterized in that the described sealer of step (4) is a non-ionics, comprise triton x-100, Tween 80.
10. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 6 and c reactive protein antibody, it is characterized in that the described sealer of step (4) is a non-ionics, comprise triton x-100, Tween 80.
11. according to the covalent cross-linking method of described carboxylic polystyrene microsphere of claim 10 and c reactive protein antibody, it is characterized in that the temperature of the described incubation of step (4) is 15～45 ℃, the time is 1.5～20 hours.
12. covalent cross-linking method according to claim 1,2 or 3 described carboxylic polystyrene microspheres and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
13. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 4 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
14. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 5 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
15. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 6 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
16. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 7 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
17. the covalent cross-linking method of described according to Claim 8 carboxylic polystyrene microsphere and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
18. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 9 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
19. covalent cross-linking method according to described carboxylic polystyrene microsphere of claim 10 and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
20.
The covalent cross-linking method of 11 described carboxylic polystyrene microspheres and c reactive protein antibody, it is characterized in that the described damping fluid of step (6) is nonionic both sexes damping fluids, comprise hydroxyethyl piperazine second thiosulfonic acid, 2-(N-morphine quinoline) ethyl sulfonic acid, described damping fluid ion concentration is 20～100mM, and pH value is 4.5～8.
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Cited By (6)
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| CN102432685A (en) * | 2011-11-01 | 2012-05-02 | 中国环境科学研究院 | Immune latex microspheres for detecting recombined mouse basic fibroblast growth factor and preparation method and application of immune latex microspheres |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN104215778A (en) * | 2014-09-26 | 2014-12-17 | 上海科华生物工程股份有限公司 | C-peptide monoclonal antibody cross-linked with magnetic particles, preparation method thereof and C-peptide test kit including same |
| CN106674403A (en) * | 2017-01-03 | 2017-05-17 | 宏葵生物（中国）有限公司 | Preparation method of polystyrene microspheres for enhancing immunoturbidimetry and application thereof |
| CN108761089A (en) * | 2018-06-29 | 2018-11-06 | 迈克生物股份有限公司 | Preparation method for the reagent for detecting β2-microglobulin |
| CN112979798A (en) * | 2019-12-18 | 2021-06-18 | 东莞市朋志生物科技有限公司 | An isolated binding protein comprising a C-reactive protein antigen binding domain |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432685A (en) * | 2011-11-01 | 2012-05-02 | 中国环境科学研究院 | Immune latex microspheres for detecting recombined mouse basic fibroblast growth factor and preparation method and application of immune latex microspheres |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN103073642B (en) * | 2012-08-29 | 2016-06-15 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation technology |
| CN104215778A (en) * | 2014-09-26 | 2014-12-17 | 上海科华生物工程股份有限公司 | C-peptide monoclonal antibody cross-linked with magnetic particles, preparation method thereof and C-peptide test kit including same |
| CN106674403A (en) * | 2017-01-03 | 2017-05-17 | 宏葵生物（中国）有限公司 | Preparation method of polystyrene microspheres for enhancing immunoturbidimetry and application thereof |
| CN108761089A (en) * | 2018-06-29 | 2018-11-06 | 迈克生物股份有限公司 | Preparation method for the reagent for detecting β2-microglobulin |
| CN112979798A (en) * | 2019-12-18 | 2021-06-18 | 东莞市朋志生物科技有限公司 | An isolated binding protein comprising a C-reactive protein antigen binding domain |
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Application publication date: 20101027 |