CN101046473A - Method of improving stability of antigen or antibody particle combined with latex - Google Patents
Method of improving stability of antigen or antibody particle combined with latex Download PDFInfo
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- CN101046473A CN101046473A CN 200610025351 CN200610025351A CN101046473A CN 101046473 A CN101046473 A CN 101046473A CN 200610025351 CN200610025351 CN 200610025351 CN 200610025351 A CN200610025351 A CN 200610025351A CN 101046473 A CN101046473 A CN 101046473A
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Abstract
The method of improving stability of antigen or antibody particle combined with latex is used for clinical reagent compounding. The present invention features that the reagent solution contains blocking agent in 0.1-2 % and stabilizer in 2-20 %. The blocking agent is selected from blocking proteins, including albumin, gel protein, etc; and the stabilizer is glycerin, cane sugar and other matter with special regulated density of 1.01-1.09 g/ml. The present invention can ensure the stability of biological body fluid diagnosing reagent for latex reinforcing process and has wide clinical application foreground.
Description
Technical field:
The invention belongs to biological technical field.Be specifically related to a kind of antigen of latex combination or method of antibody particle stability improved.
Background technology
It is micron-sized particle that singer in 1956 and plotz bring into use diameter, detect some and be present in micro-measured object in the body fluid by carry out slide agglutination qualitatively attached to the antibody in the antigen on the latex particle or antibody and the serum or antigen, mainly comprise rheumatoid disease, prealbumin, progesterone etc.Along with the development of particle synthetic technology, particle diameter is brought up to nanoscale by micron order, and particle surface can carry out some particular processing as required simultaneously, produce reactive group, nano level particle is applied to developing the diagnostic reagent of a series of quantitative measurements in the immunoturbidimetry mensuration, comprise rheumatoid disease, anti-O, Lp (a), the urine microglobulin, prealbumin, c reactive protein, progesterone, β2Wei Qiudanbai, troponin, glycosylated hemoglobin etc.Along with the raising that clinical detection requires, extensively adopt latex to strengthen reagent both at home and abroad trace of albumin is carried out quantitative measurement.
Latex enhancing method is a kind of new methods for clinical diagnosis, by with antigen or antibody with carry out antigen-antibody reaction with corresponding antibody or antigen after latex particle carries out covalent cross-linking, strengthened the sensitivity of common immunization significantly, at least improve an order of magnitude than common immunoturbidimetry, can reach the level of radioimmunoassay (RIA), but there is not radiocontamination, simple to operate, can measure with common Biochemical Analyzer, meet the requirement of modern clinical examination, realized the quantitative measurement of some low content materials.Owing to used latex particle, and on particle, combine albumen, the latex that forms strengthens particle volume and is generally 0.05～0.5um, but, they are to exist with suspended state in solution, can not be medium-term and long-term stable at solution, so must solve the antigen of glue latex combination or the stability of antibody particle.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of antigen of latex combination or method of antibody particle stability improved is provided, it is in steady state (SS) when the analyte concentration that biological fluid samples is carried out in quantitative measurement and the analyzing samples, can make reagent for clinical diagnosis measurement result repeatability better, and the reagent bottom of regular period can not occur depositing and small amount of precipitate occur, problems such as blank rising.
The objective of the invention is to be achieved through the following technical solutions: the invention provides a kind of antigen of latex combination or method of antibody particle stability improved, be used for the preparation of reagent for clinical diagnosis, it is characterized in that: add 0.1～2% sealer and 2～20% stabilizing agent in this reagent solution.
In mentioned reagent, sealer refers to multiple closed protein, comprises albumin, gel protein etc.; Stabilizing agent refers to the material of special adjusting density, comprises glycerine, sucrose etc., and adjusting density is 1.01～1.09g/ml.
In the mentioned reagent, described sealer optium concentration is 0.3%～1.2%.
In the mentioned reagent, described stabilizing agent optium concentration is 6～10%.
In the mentioned reagent, the optimum density that described stabilizing agent is regulated is 1.03～1.07g/ml.
The present invention is based on following principle the present invention is mainly used in and uses latex to strengthen reagent for clinical diagnosis stable of method, its essence is exactly by regulating the density of reagent, and use closed protein to guarantee that the electrically charged unanimity of particle institute is even, make the long-term suspension that the latex ion can be stable.Known is not fully by protein blocking in conjunction with the latex particle behind the albumen, need to use a certain amount of strong to the very little water capacity of reaction influence, electrically charged many albumen guarantees that latex strengthens particle sealing and homogeneous fully, closed protein used herein comprises albumin, gel proteins etc., content is generally 0.1～2%.Regulate material glycerine and sucrose that density is mainly used relative inertness, content is generally 2～20%, because the density of latex particle itself is generally 1.05g/ml, be 1.03～1.07g/ml so need to regulate density the best, guarantee that easier steady in a long-term suspension of particle that has the homogeneous electric charge exists.The latex that utilizes the present invention to prepare strengthens reagent after 9～12 months, still can meet clinical requirement, so have a wide range of applications 4 ℃ of storages on clinical examination.
The varieties of reagent that the present invention is applied to clinical diagnosis mainly contains: rheumatoid disease, anti-O, Lp (a), the urine microglobulin, prealbumin, c reactive protein (comprising common enhancing and super quick), progesterone, β2Wei Qiudanbai, troponin, glycosylated hemoglobin etc., but be not limited to these reagent, reagent normally seals with albumen, the sealer that also comprises some non-albumen, closed protein comprises albumin, gel protein etc., but be not limited to that these are several.Regulating the density material generally is the material of relative inertness, glycerine normally, and sucrose etc., but be not limited to that these are several.
When implementing this patent, composition according to detectable, the material of sealer that reasonable combination is suitable and appropriate adjusting density according to the different damping fluids that want detected object uses, is selected the sealer of variable concentrations and is regulated the density material with the requirement difference of reaction.
Embodiment
Embodiment one:
Prepare stable this reagent of latex enhancing method rheumatoid disease reagent (RF) and divide A, B two parts, elder generation is with 4 parts of reagent A and sample mix during use, hatched 300 seconds for 37 ℃, again a reagent B is added, react after 300 seconds, the absorbance variation when calculating under the 570nm (perhaps 600nm also can) terminal point and just having added reagent B, the absorbance of contrast calibration object changes, and calculates the content of rheumatoid factor in the sample.This calculating can be carried out on automatic analyzer automatically.Agent prescription is as follows:
Latex strengthens the preparation of particle:
The human IgG that is ready to handle, 0.124um is with the particle (concentration 10%mg/ml) of activated group, and prepares the MES damping fluid (2-(N-Morpholino) ethanesulfonic acid 106.7g/L) of 500mM, deionized water.It is as follows that preparation latex strengthens particle:
Add the solution 1ml that contains 10% latex particle
The MES solution 1ml of 500mM
Human IgG 14mg
EDAC(N-(3-dimethylaminopropyl)N’-ethylcarbodiimidehydrochloride)
100mg
Deionized water adds to 10ml mixing centrifugal supernatant that goes after 3 hours, adds following damping fluid to 40ml
EDTA Na2 0.0298g
BSA 0.4mg
Sucrose 3.2mg
PBS pH of buffer=8.0 100mM (Na
2HPO
412H
2O 33.9155g/l, KH
2PO
40.721275g/lNaCl 8.8g/l) to 40ml.
Stir and to obtain reagent B.
Reagent A: PBS pH of buffer=8.0 100mM
EDTA Na2 2mM
PEG8000 2％
BSA 1％
Reagent B:PBS pH of buffer=8.0 100mM
EDTA Na2 2mM
BSA 1％
Sucrose 8%
Latex strengthens particle 0.25%
Mentioned reagent A and B are each concentration of component of obtain solution, can convert the amount of each component according to concrete solution amount.
The reagent for preparing is placed 4 ℃ and is stored 1,3,6, after Dec, measures with a standard quality controlled serum, its degree of variation＜5%
Embodiment two:
Prepare stable this reagent of latex enhancing method c reactive protein reagent (CRP) and divide A, B two parts, elder generation is with 4 parts of reagent A and sample mix during use, hatched 300 seconds for 37 ℃, again a reagent B is added, react after 300 seconds, the absorbance variation when calculating under the 570nm (perhaps 600nm also can) terminal point and just having added reagent B, the absorbance of contrast calibration object changes, and calculates the content of c reactive protein in the sample.This calculating can be carried out on automatic analyzer automatically.Agent prescription is as follows:
Latex strengthens the preparation of particle:
The c reactive protein antibody that is ready to handle, 0.201um is with the particle (concentration 10%) of activated group, and prepares the MES damping fluid of 500mM, deionized water.It is as follows that preparation latex strengthens particle:
Add the solution 1ml that contains 10% latex particle
The MES solution 1ml of 500mM
C reactive protein antibody 21mg
EDAC 100mg
Deionized water adds to 10ml mixing centrifugal supernatant that goes after 3 hours, adds following damping fluid to 40ml
EDTA Na2 0.0298g
BSA 0.36mg
Sucrose 3.6mg
PBS pH of buffer=8.0 100mM (with example one) is to 40ml
Stir and to obtain reagent B.
Reagent A: PBS pH of buffer=8.0 100mM
EDTA Na2 2mM
PEG6000 2.4％
BSA 1％
Reagent B:PBS pH of buffer=8.0 100mM
EDTA Na2 2mM
BSA 0.9％
Sucrose 9%
Latex strengthens particle 0.25%
Mentioned reagent A and B are each concentration of component of obtain solution, can convert the amount of each component according to concrete solution amount.
The reagent for preparing is placed 4 ℃ and is stored 1,3,6, after Dec, measures with a standard quality controlled serum, its degree of variation＜5%.
Example three
Utilize latex that the present invention prepares to strengthen the latex of preserving under reagent and the regular solution condition and strengthen particle and compare research (this place latex strengthen particle contrast use be the RF latex enhancing particle for preparing in the example one), the result is as follows:
Closed protein is regulated density
Solution 1:0.1%BSA does not have
Solution 2:0.3%BSA does not have
Solution 3:1%BSA does not have
Solution 4:2%BSA does not have
Solution 5:0.1%BSA sucrose 5%
Solution 6:0.1%BSA sucrose 8%
Solution 7:0.1%BSA sucrose 12%
Solution 8 0.3% closed protein sucrose 8%
Solution 9:1% closed protein sucrose 8%
Solution 10:2% closed protein sucrose 8%
Use spectrophotometer during measurement, blank be reagent A and reagent B by mixing in 4: 1, add make a living absorbance when managing salt solution of sample.The initial absorbance of 0 timing is decided to be 100% (initial absorbance Abs is 0.7211) here, the calibration object absorbance as 100% (the target value of Xuan Zeing is the calibration object of 150U/L here, and initial absorbance changes delta A is 1.0278)
Table 1: the number percent (%) of the blank absorbency of actual measurement and relative target value is not if add sealing albumen, and latex particle all precipitates and layering after one week, and 0.1% closed protein is used in institute's following table comparative study at least.
| After 1 month | After 3 months | After 6 months | After 12 months | |
| Solution 1 blank Abs | 110 | 129 | 154 | 182 |
| Relative target value Δ A | 91 | 78 | 61 | 39 |
| Solution 2 blank Abs | 108 | 121 | 142 | 164 |
| Relative target value Δ A | 93 | 84 | 70 | 56 |
| Solution 3 blank Abs | 106 | 111 | 124 | 138 |
| Relative target value Δ A | 95 | 90 | 83 | 72 |
| Solution 4 blank Abs | 113 | 118 | 125 | 136 |
| Relative target value Δ A | 89 | 86 | 82 | 73 |
| Solution 5 blank Abs | 107 | 123 | 145 | 167 |
| Relative target value Δ A | 94 | 83 | 68 | 54 |
| Solution 6 blank Abs | 104 | 109 | 118 | 129 |
| Relative target value Δ A | 97 | 92 | 86 | 78 |
| Solution 7 blank Abs | 105 | 111 | 119 | 130 |
| Relative target value Δ A | 96 | 90 | 85 | 78 |
| Solution 8 blank Abs | 103 | 106 | 112 | 117 |
| Relative target value Δ A | 98 | 95 | 89 | 86 |
| Solution 9 blank Abs | 101 | 103 | 106 | 109 |
| Relative target value Δ A | 100 | 98 | 95 | 92 |
| Solution 10 blank Abs | 105 | 107 | 111 | 115 |
| Relative target value Δ A | 96 | 94 | 90 | 88 |
Last table result shows, do not regulate under the situation of density only adding 0.1% closed protein, and it is very unstable that latex strengthens particle, measure blank absorbency simultaneously and can see that tangible blank absorbency increases, this be since latex particle that cohesion takes place is caused.Solution 2～4th adds the gradient of sealing albumen, can see that about 1% is best, and significantly blank is than higher later on to be higher than 2%, and when being lower than 0.2%, effect neither be clearly.Solution 5～7th, the result after the adjusting density, though it is fine equally latex to be strengthened particle-stabilised effect after being higher than 8%, but consider from the practical application angle, the optimum concentration that only needs to select to be effective gets final product, just regulate density and get final product more a little influence stability hardly to being fit to the particle-stabilised density of latex enhancing.By closed protein sealing with adjust that density is relative does not carry out stable latex particle stability and all be significantly improved.Solution 8～10th has carried out regulating the contrast of density and closed protein simultaneously, when using 1% sealing, contains in the solution of 8% sucrose, and blank absorbency has only increased by 9% after Dec, and the absorbance of calibration object changes and only descended 8% simultaneously.Can be in the practical application according to the different-diameter that uses, the particle of density, and the albumen that comprises different amounts on the particle are adjusted best closed protein concentration and are regulated optimum density.
Thereby, utilize the latex of the technology of the present invention preparation strengthen reagent 4 ℃ store for 9～Dec after, still can meet the requirement of clinical examination.
Claims (7)
1, a kind of antigen of latex combination or method of antibody particle stability improved is characterized in that adding 0.1～2% sealer and 2～20% stabilizing agent in the diagnosing reagent solution of preparation.
2, a kind of antigen of latex combination or method of antibody particle stability improved is characterized in that adding 0.3～1.2% sealer and 6～10% stabilizing agent in the diagnosing reagent solution of preparation.
3, a kind of antigen of latex combination or method of antibody particle stability improved according to claim 1, the adjusting density that it is characterized in that adding the diagnosing reagent solution after sealer and the stabilizing agent is 1.01～1.09%.
4, a kind of antigen of latex combination or method of antibody particle stability improved according to claim 1, the adjusting density that it is characterized in that adding the diagnosing reagent solution after sealer and the stabilizing agent is 1.03～1.07%.
5, a kind of antigen of latex combination or method of antibody particle stability improved according to claim 1 is characterized in that wherein said sealer is albumin or gel protein.
6, a kind of antigen of latex combination or method of antibody particle stability improved according to claim 1 is characterized in that wherein said stabilizing agent is glycerine or sucrose.
7, the application of the method for the antigen of the described improvement latex of a kind of claim 1 combination or antibody particle stability in rheumatoid disease, anti-O, LP (α), urine microglobulin, prealbumin, c reactive protein, progesterone, β2Wei Qiudanbai, troponin or the preparation of glycosylated hemoglobin diagnostic reagent liquid.
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|---|---|---|---|
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|---|---|---|---|
| CN 200610025351 CN101046473A (en) | 2006-03-31 | 2006-03-31 | Method of improving stability of antigen or antibody particle combined with latex |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893619A (en) * | 2010-02-10 | 2010-11-24 | 上海蓝怡科技有限公司 | Method for improving stability of latex suspension liquid |
| CN102749460A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Troponin detection kit and preparing method thereof |
| CN102788881A (en) * | 2012-08-16 | 2012-11-21 | 北京恩济和生物科技有限公司 | Prealbumin detection reagent kit and preparation method thereof |
| CN102914656A (en) * | 2012-07-23 | 2013-02-06 | 四川省新成生物科技有限责任公司 | Detection kit for saccharifying serum albumin by using indirect immunifaction and measuring method |
| CN105137090A (en) * | 2015-09-30 | 2015-12-09 | 山东博科生物产业有限公司 | High-accuracy prealbumin immunoturbidimetry detection kit |
| CN106093418A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring Troponin I and preparation method thereof |
| CN106442974A (en) * | 2016-10-03 | 2017-02-22 | 王贤俊 | Method for detecting AGEs content in human serum |
| CN106568944A (en) * | 2016-10-03 | 2017-04-19 | 王贤俊 | Latex immunoturbidimetric kit for detecting AGEs |
| CN107490677A (en) * | 2017-07-21 | 2017-12-19 | 王贤俊 | The cross-linking composition liquid and its cross-linking method of a kind of carboxylated latex microballoon and glycosylated hemoglobin antibody |
| CN107525936A (en) * | 2017-07-21 | 2017-12-29 | 王贤俊 | The composition of troponin antibodies stability in a kind of lifting latex immunoturbidimetry |
| CN107561292A (en) * | 2017-09-30 | 2018-01-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of progesterone detection kit and its application method |
| CN107589267A (en) * | 2017-08-25 | 2018-01-16 | 柏荣诊断产品（上海）有限公司 | A kind of blood urine is the same as the microglobulins of the gamut β 2 measure kit surveyed |
| CN107607706A (en) * | 2017-03-31 | 2018-01-19 | 迈克生物股份有限公司 | Suppress the super quick C reactive proteins latex enhancing immune of rheumatoid factor interference than turbid kit |
| CN107727871A (en) * | 2017-09-30 | 2018-02-23 | 安徽伊普诺康生物技术股份有限公司 | A kind of preparation method of progesterone detection kit |
| CN108680754A (en) * | 2018-05-04 | 2018-10-19 | 湖北科技学院 | PA detection kit |
| CN110806475A (en) * | 2019-12-05 | 2020-02-18 | 广东菲鹏生物有限公司 | Protective agent of latex microsphere and application and product thereof |
| CN111398606A (en) * | 2020-03-31 | 2020-07-10 | 深圳市锦瑞生物科技有限公司 | Kit and detection system |
| CN116298324A (en) * | 2023-05-25 | 2023-06-23 | 武汉大学人民医院（湖北省人民医院） | Method, device, equipment and readable storage medium for detecting beta 2-microglobulin |
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| CN101893619A (en) * | 2010-02-10 | 2010-11-24 | 上海蓝怡科技有限公司 | Method for improving stability of latex suspension liquid |
| CN101893619B (en) * | 2010-02-10 | 2013-11-13 | 上海蓝怡科技有限公司 | Method for improving stability of latex suspension liquid |
| CN102914656A (en) * | 2012-07-23 | 2013-02-06 | 四川省新成生物科技有限责任公司 | Detection kit for saccharifying serum albumin by using indirect immunifaction and measuring method |
| CN102914656B (en) * | 2012-07-23 | 2014-10-29 | 四川新健康成生物股份有限公司 | Detection kit for saccharifying serum albumin by using indirect immunifaction |
| CN102749460A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Troponin detection kit and preparing method thereof |
| CN102788881A (en) * | 2012-08-16 | 2012-11-21 | 北京恩济和生物科技有限公司 | Prealbumin detection reagent kit and preparation method thereof |
| CN105137090A (en) * | 2015-09-30 | 2015-12-09 | 山东博科生物产业有限公司 | High-accuracy prealbumin immunoturbidimetry detection kit |
| CN106093418A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring Troponin I and preparation method thereof |
| CN106442974B (en) * | 2016-10-03 | 2019-06-18 | 王贤俊 | A method of for detecting AGEs content in human serum |
| CN106568944A (en) * | 2016-10-03 | 2017-04-19 | 王贤俊 | Latex immunoturbidimetric kit for detecting AGEs |
| CN106442974A (en) * | 2016-10-03 | 2017-02-22 | 王贤俊 | Method for detecting AGEs content in human serum |
| CN107656065A (en) * | 2017-03-31 | 2018-02-02 | 迈克生物股份有限公司 | Suppress the RBP ELISA latex enhancing immune of rheumatoid factor interference than turbid reagent |
| CN107607706A (en) * | 2017-03-31 | 2018-01-19 | 迈克生物股份有限公司 | Suppress the super quick C reactive proteins latex enhancing immune of rheumatoid factor interference than turbid kit |
| CN107607706B (en) * | 2017-03-31 | 2020-04-07 | 迈克生物股份有限公司 | Hypersensitivity C-reactive protein latex enhanced immunoturbidimetry kit for inhibiting rheumatoid factor interference |
| CN107656065B (en) * | 2017-03-31 | 2020-06-12 | 迈克生物股份有限公司 | Retinol binding protein latex enhanced immunoturbidimetry reagent for inhibiting rheumatoid factor interference |
| CN107525936A (en) * | 2017-07-21 | 2017-12-29 | 王贤俊 | The composition of troponin antibodies stability in a kind of lifting latex immunoturbidimetry |
| CN107490677A (en) * | 2017-07-21 | 2017-12-19 | 王贤俊 | The cross-linking composition liquid and its cross-linking method of a kind of carboxylated latex microballoon and glycosylated hemoglobin antibody |
| CN107525936B (en) * | 2017-07-21 | 2020-02-07 | 王贤俊 | Composition for improving stability of troponin antibody in latex immunoturbidimetry |
| CN107589267A (en) * | 2017-08-25 | 2018-01-16 | 柏荣诊断产品（上海）有限公司 | A kind of blood urine is the same as the microglobulins of the gamut β 2 measure kit surveyed |
| CN107589267B (en) * | 2017-08-25 | 2019-08-02 | 柏荣诊断产品（上海）有限公司 | A kind of same gamut β2-microglobulin assay kit surveyed of blood urine |
| CN107561292A (en) * | 2017-09-30 | 2018-01-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of progesterone detection kit and its application method |
| CN107727871A (en) * | 2017-09-30 | 2018-02-23 | 安徽伊普诺康生物技术股份有限公司 | A kind of preparation method of progesterone detection kit |
| CN108680754A (en) * | 2018-05-04 | 2018-10-19 | 湖北科技学院 | PA detection kit |
| CN108680754B (en) * | 2018-05-04 | 2021-02-26 | 湖北科技学院 | Prealbumin determination kit |
| CN110806475A (en) * | 2019-12-05 | 2020-02-18 | 广东菲鹏生物有限公司 | Protective agent of latex microsphere and application and product thereof |
| CN111398606A (en) * | 2020-03-31 | 2020-07-10 | 深圳市锦瑞生物科技有限公司 | Kit and detection system |
| CN116298324A (en) * | 2023-05-25 | 2023-06-23 | 武汉大学人民医院（湖北省人民医院） | Method, device, equipment and readable storage medium for detecting beta 2-microglobulin |
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