CN107167586B - A kind of antistreptolysin O (ASO) detection kit and preparation method thereof - Google Patents
A kind of antistreptolysin O (ASO) detection kit and preparation method thereof Download PDFInfo
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- CN107167586B CN107167586B CN201710547376.8A CN201710547376A CN107167586B CN 107167586 B CN107167586 B CN 107167586B CN 201710547376 A CN201710547376 A CN 201710547376A CN 107167586 B CN107167586 B CN 107167586B
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- 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
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- G—PHYSICS
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- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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Abstract
The invention discloses a kind of antistreptolysin O (ASO) detection kits, solve the step of prior art is required to centrifugation or ultrafiltration, time-consuming and laborious, are unfavorable for industrialized problem.The present invention includes reagent two, the reagent two be using latex, buffer, EDC and HEPES hybrid reaction after, add NaOH adjust pH to 7.00~8.00, finally add manufactured finished product after SLO, sealer I and sealer II；The concentration of each substance in finished product are as follows: latex 58.00ml/L~62.00ml/L；Buffer 140.00ml/L；EDC 0.0525g/L~0.0924g/L；HEPES 5.9578g/L；SLO 74.00mg/L~78.00mg/L；Sealer I 20.00ml/L；Sealer II 10.00ml/L.The invention avoids the impurity eliminations processes such as the centrifugation of conventional latex sensitization process, ultrafiltration, substantially increase production efficiency, have saved time cost and human cost.
Description
Technical field
The present invention relates to a kind of kits and preparation method thereof, and in particular to a kind of antistreptolysin O (ASO) detection reagent
Box and preparation method thereof.
Background technique
Immunoturbidimetry (Turbidimetric inhibition immuno assay) is that antigen-antibody combines dynamic to survey
Determine method.The basic principle is that: when antigen and antibody react and suitable (the general provision antibody of ratio in special dilution system
It is excessive) when, under the action of the poly- agent of rush of the soluble immune complex of formation in dilution system (polyethylene glycol etc.), from liquid phase
It is precipitated, forms particle, reaction solution is made turbidity occur.When antibody concentration is fixed, the amount of the immune complex of formation is with sample
The increase of middle amount of antigen and increase, the turbidity of reaction solution is consequently increased.By the turbidity and series of standards that measure reaction solution
Product control, can calculate the content of antigen in sample.
Latex enhancing immune turbidimetry is that the corresponding antibody/antigen of test substance is coated on diameter as nano rubber latex
On particle, antigen-antibody conjugate is made to form reticular structure, after light passes through, the Strength Changes of transmitted light and scattering light are more aobvious
It writes, to improve the sensibility of test.
The patent of Beijing Strong Biotechnologies, Inc. discloses the method for being related to latex sensitization and reagent (application
Number 201010615283.2), specifically, the method for the latex sensitization invented is to first pass through chemical bond for latex nothing to do with egg
White combination reuses glutaraldehyde by antigen or antibody linked on unrelated protein, to make latex sensitization.The preparation of reagent two
Journey, which needs to be centrifuged twice, changes liquid, is unfavorable for preparation of industrialization.
The patent of Ningbo Meikang Biotechnology Co., Ltd. discloses a kind of detection of antistreptolysin O (ASO) antibody
Kit (application number 201410804871.9).Its described albumen latex particle composites is that combined protein is marked in polystyrene
The compound formed on latex particle；The combined protein is by streptolysin O and inert protein by being chemically crosslinked.
The preparation process of reagent two needs a ultrafiltration, and liquid is changed in centrifugation twice, time-consuming and laborious.
Medical University Of Tianjin's invention is related to a kind of side of coupling protein matter on carboxylated polystyrene latex particles with spacerarm
Method (application number 201210005525.5).Its is described to use when separating the latex microsphere for being coupled antibody with remaining antibody
Tangential flow filtration technology, overcome antibody in centrifugal separation process it is easy to fall off and inactivation the shortcomings that.
To sum up, in preparation method in the prior art in order to reach removal excess component, change buffer, change the mesh such as pH value
Be required to carry out centrifugation or ultrafiltration step, cause existing preparation method time-consuming and laborious, be unfavorable for industrializing.Moreover, existing skill
Inert protein or unrelated protein are introduced in the file that art is recorded, the process of the introducing inert protein or unrelated protein is cumbersome, and
Success rate is low, low efficiency and time-consuming.
Summary of the invention
The technical problems to be solved by the present invention are: the step of prior art is required to centrifugation or ultrafiltration, time-consuming and laborious,
It is unfavorable for industrialized problem, and it is an object of the present invention to provide a kind of antistreptolysin O (ASO) detection kit and preparation method thereof,
By the optimization of the range that feeds intake of each component, it can effectively reach the step of removing excess component without centrifugation, ultrafiltration etc., make sensitization
Latex afterwards participates in reacting directly as reagent two, substantially increases the preparation efficiency of conventional latex enhancing immunoturbidimetry reagent,
Avoid the purification process such as the centrifugation taken time and effort, ultrafiltration.
The present invention is achieved through the following technical solutions:
A kind of antistreptolysin O (ASO) detection kit, including reagent two, the reagent two be using latex, buffer,
After EDC and HEPES hybrid reaction, adds NaOH and adjust pH to 7.00~8.00, finally add SLO, sealer I and closing
Manufactured finished product after agent II；The concentration of each substance in finished product are as follows:
The present invention is avoided that the introducing of inert protein or unrelated protein, greatly simplifies behaviour by the optimization of said components
Make step.Also, the present invention is based on multiple experiments, and then determine the range that feeds intake of each component in reagent two, press institute of the present invention
The range that feeds intake established is fed intake, and can be removed the purpose of centrifugation or ultrafiltration removal excess component from, is highly suitable for industrialization
Production, improves production efficiency, and effect is more significant.
Further, the buffer is MES-NaOH solution, and the sealer I is Tween-20 solution, the sealer
II is BSA solution.Further, the concentration of the MES-NaOH solution is 25~50mM, pH value 6.00；The Tween-20
The concentration of solution is 20%；The concentration of the BSA solution is 10%.
The value range of the present invention through the invention is preferably provided with, and can be effectively improved while simplifying preparation step
Accuracy in detection.
Preferably, also include in the BSA solution 3% NaN3。NaN3Effect be anti-corrosion, after being added into reagent two
Final concentration becomes 0.03%, still there is antisepsis.
Further, the latex is spaced arm latex.Streptolysin O molecular weight is about 69kd, and molecular weight is smaller,
After sensitization to latex, steric hindrance is formed, covers by the antibody recognition site in sample to be tested, is unfavorable for antigenic determinant
Exposure, is not easy to identify that reaction efficiency is low by the antibody in sample to be tested.The latex of spaced arm can be used in the present invention simultaneously
Conventional latex is replaced, which can be effectively reduced the steric hindrance problem of antigen-antibody combination, be conducive to antigen decision
The exposure of cluster occurs specific agglutination, reaches preferable latex intensified to be identified rapidly by the antibody in sample to be tested
The effect of immunoturbidimetry measurement.
Percent concentrations of the present invention refer both to w/v concentration, i.e. mass/volume concentration, unit g/ml.Said components
Middle EDC is carbodiimide, and HEPES is 4- hydroxyethyl piperazineethanesulfonic acid, and SLO is streptolysin O, and MES is 2- morpholine second sulphur
Acid, BSA are bovine serum albumin(BSA).
To guarantee the accuracy weighed, the mode of EDC addition are as follows: it is molten that EDC is configured to the EDC that concentration is 0.7~1.2%
Liquid, the dosage of EDC solution are 7.50ml/L~7.70ml/L.Meanwhile the concentration of heretofore described NaOH solution is preferably provided with
It is 20%.
A kind of preparation method of antistreptolysin O (ASO) detection kit, wherein the preparation method of reagent two includes:
Latex, buffer, EDC, HEPES, SLO, sealer I and sealer II are taken respectively；EDC solution is prepared, is prepared
NaOH solution；
Latex is added in buffer, EDC solution is added and is activated, add HEPES, adjusted with NaOH solution
PH, is added SLO and is chemically crosslinked, and sealer I and sealer II is then added and is closed, finished product is eventually fabricated.
Further, the time that the EDC solution is activated is 15min.The time of the chemical crosslinking is 2h.The envelope
The time closed is 0.5h.
Compared with prior art, the present invention having the following advantages and benefits:
1, control process of the present invention is very precisely stringent, avoids the impurity eliminations such as centrifugation, the ultrafiltration of conventional latex sensitization process
Process substantially increases the preparation efficiency of conventional latex enhancing immunoturbidimetry reagent, substantially increases production efficiency；
2, superior performance of the present invention, preparation process is very simple, easy, and time cost and human cost is greatly saved,
The shortcomings that overcoming the prior art.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, the present invention is made
Further to be described in detail, exemplary embodiment of the invention and its explanation for explaining only the invention, are not intended as to this
The restriction of invention.
Embodiment 1
A kind of antistreptolysin O (ASO) detection kit, including reagent one and reagent two, in the present embodiment, the reagent two
In each substance concentration are as follows:
The latex of the reagent two is preferably spaced arm latex in the present embodiment.
Buffer is preferably the MES-NaOH solution that concentration is 25~50mM, and preparation method is: making after being dissolved using MES
At the solution of 25~50mM concentration, pH to 6.0 is then adjusted using NaOH, MES-NaOH solution is made.Sealer I is preferably
The Tween-20 solution that concentration is 20%.
Sealer II is preferably the BSA solution that concentration is 10%, in the BSA solution also containing 3% NaN3；Sealer II
Preparation process are as follows: weigh the NaN of the BSA and 3g of 10g3Afterwards, the two is dissolved into water, and BSA is made to 100ml in constant volume
Solution.
By taking 100ml as an example, the reagent two of the present embodiment specific the preparation method is as follows:
NaOH is configured to the NaOH solution that concentration is 20%, EDC is configured to the EDC solution that concentration is 0.9%；Respectively
It is molten to measure latex 6ml, MES-NaOH solution 14ml, EDC solution 0.76ml, HEPES 0.5958g, SLO 7.6mg, Tween-20
Liquid 2ml and BSA solution 1ml；HEPES is dissolved.
Latex is added in buffer, add EDC solution carry out carboxyl activation, activation time 15min, then plus
Enter HEPES, using NaOH solution adjust pH to 7.60, be added SLO be chemically crosslinked, the chemical crosslinking time be 2h, then plus
Enter sealer I and sealer II is closed, off-period 0.5h, finished product is made in last constant volume to 100ml.
The reagent one of the present embodiment is the reagent one in existing antistreptolysin O (ASO) detection kit, in the present embodiment
The composition of one of reagent one is provided, concrete composition composition is as follows:
By taking 1000ml as an example, the reagent one of the present embodiment specific the preparation method is as follows:
About 800ml purified water is measured, HEPES 23.83g is weighed and dissolve, pH value is adjusted to 7.00 with 20%NaOH, claims
Take and dissolve 9.00~15.00g of NaCl, measure and dissolve Tween-20 10.0ml, weigh and dissolve PEG6000 25.00~
33.00g measures and dissolves preservative 0.30ml, measures and dissolve 10%BSA 10ml, and last constant volume to 1000ml is made into
Product.
Embodiment 2
The present embodiment the difference from embodiment 1 is that, the amount of the preparation of reagent two in the present embodiment is different, the present embodiment with
For 500ml, the reagent two of the present embodiment specific the preparation method is as follows:
Latex 30ml, buffer 70ml, EDC 3.8ml, HEPES 2.9789g, SLO 38mg, sealer I are measured respectively
10ml and sealer II 5ml；EDC is configured to the EDC solution that concentration is 0.7~1.2%, HEPES is dissolved, it will
NaOH is configured to the NaOH solution that concentration is 20%；
Latex is added in buffer, add EDC solution carry out carboxyl activation, activation time 15min, then plus
Enter HEPES, using NaOH solution adjust pH to 7.40, be added SLO be chemically crosslinked, the chemical crosslinking time be 2h, then plus
Enter sealer I and sealer II is closed, off-period 0.5h, finished product is made in last constant volume to 500ml.
Embodiment 3
The present embodiment the difference from embodiment 1 is that, two preparation method of reagent is different in the present embodiment, and the present embodiment increases
The step of centrifugation, the present embodiment are specifically provided that
Latex 6ml, buffer 14ml, EDC 0.76ml, HEPES 0.5958g, SLO 7.6mg, sealer I are measured respectively
2ml and sealer II 1ml；；EDC is configured to the EDC solution that concentration is 0.7~1.2%；It is molten using NaOH after HEPES dissolution
Liquid adjusts pH value to 7.50, obtains and redissolves liquid；
Latex is added in buffer, add EDC solution carry out carboxyl activation, activation time 15min,
18000r/min is centrifuged 15min, adds the redissolution liquid that pH value is 7.50 and is redissolved, and SLO is added and is chemically crosslinked, chemistry
Crosslinking time is 2h, and 18000r/min is centrifuged 15min, adds the redissolution liquid that pH value is 7.50 and is redissolved, then with then adding
Enter sealer I and sealer II is closed, off-period 0.5h, finished product is made in last constant volume to 100ml.
It is detected using kit made of above-described embodiment 1- embodiment 3, calibration data is as shown in table 1, third party
Quality Control test result is as shown in table 2, and the testing result for doing clinical trial using 40 samples is as shown in table 3.
Table 1
Table 2
Table 3
By the data result of above-mentioned table 1- table 3 can prove that the present invention without the impurity eliminations process such as centrifugation, ultrafiltration, according to
So it can effectively make testing result very accurate.
Embodiment 4
The present embodiment the difference from embodiment 1 is that, the additive amount of each component is different in reagent two in the present embodiment, this reality
The concentration of each component in example is applied lower than the present invention, the present embodiment is specifically provided that
Latex 5.6ml, buffer 14ml, EDC solution 0.73ml, HEPES 0.5958g, SLO 7.2mg, sealer I
2ml and sealer II 1ml；The pH value that NaOH adjusts solution is 6.90.
Embodiment 5
The present embodiment the difference from embodiment 1 is that, the additive amount of each component is different in reagent two in the present embodiment, this reality
Apply specifically being provided that for example
Latex 5.9ml, buffer 14ml, EDC solution 0.755ml, HEPES 0.5958g, SLO 7.5mg, sealer I
2ml and sealer II 1ml；The pH value that NaOH adjusts solution is 7.10.
Embodiment 6
The present embodiment the difference from embodiment 1 is that, the additive amount of each component is different in reagent two in the present embodiment, this reality
Apply specifically being provided that for example
Latex 6.1ml, buffer 14ml, EDC solution 0.765ml, HEPES 0.5958g, SLO 7.7mg, sealer I
2ml and sealer II 1ml；The pH value that NaOH adjusts solution is 7.90.
Embodiment 7
The present embodiment the difference from embodiment 1 is that, the additive amount of each component is different in reagent two in the present embodiment, this reality
The concentration for applying each component in example is higher than the present invention, and the present embodiment is specifically provided that
Latex 6.4ml, buffer 14ml, EDC solution 0.79ml, HEPES 0.5958g, SLO 8.0mg, sealer I
2ml and sealer II 1ml；The pH value that NaOH adjusts solution is 8.10.
It is detected using kit made of above-described embodiment 2 and embodiment 4- embodiment 7, third party's Quality Control test knot
Fruit is as shown in table 4.
Table 4
The test data of 4-7, can be in letter it is found that being preferably provided with for value range through the invention through the foregoing embodiment
While changing preparation step, accuracy in detection is effectively improved, effect is more significant.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (9)
1. a kind of antistreptolysin O (ASO) detection kit, including reagent two, which is characterized in that the reagent two be using latex,
It after buffer, EDC and HEPES hybrid reaction, adds NaOH solution and adjusts pH to 7.00~8.00, finally add SLO, envelope
Close manufactured finished product after agent I and sealer II；The concentration of each substance in finished product are as follows:
The buffer is MES-NaOH solution, and the sealer I is Tween-20 solution, and the sealer II is BSA solution.
2. a kind of antistreptolysin O (ASO) detection kit according to claim 1, which is characterized in that the MES-
The concentration of NaOH solution is 25~50mM, pH value 6.00；The concentration of the Tween-20 solution is 20%；The BSA solution
Concentration be 10%.
3. a kind of antistreptolysin O (ASO) detection kit according to claim 2, which is characterized in that the BSA solution
In also include 3% NaN3。
4. a kind of antistreptolysin O (ASO) detection kit according to claim 1, which is characterized in that the latex is
Spaced arm latex.
5. a kind of antistreptolysin O (ASO) detection kit according to claim 1, which is characterized in that the NaOH is molten
The concentration of liquid is 20%；EDC is configured to the EDC solution that concentration is 0.7~1.2%, the dosage of EDC solution be 7.50ml/L~
7.70ml/L。
6. the preparation method of described in any item a kind of antistreptolysin O (ASO) detection kits according to claim 1~5,
It is characterized in that, the preparation method of reagent two includes:
Latex, buffer, EDC, HEPES, SLO, sealer I and sealer II are taken respectively；It prepares EDC solution respectively and NaOH is molten
Liquid；
By latex be added buffer in, add EDC solution and activated, add HEPES, with NaOH solution adjust pH to
7.00~8.00, SLO is added and is chemically crosslinked, sealer I and sealer II is then added and is closed, is eventually fabricated into
Product.
7. a kind of preparation method of antistreptolysin O (ASO) detection kit according to claim 6, which is characterized in that
The time that the EDC solution is activated is 15min.
8. a kind of preparation method of antistreptolysin O (ASO) detection kit according to claim 6, which is characterized in that
The time of the chemical crosslinking is 2h.
9. a kind of preparation method of antistreptolysin O (ASO) detection kit according to claim 6, which is characterized in that
The closed time is 0.5h.
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| CN108982827A (en) * | 2018-06-26 | 2018-12-11 | 浙江卓运生物科技有限公司 | A kind of latex immunoturbidimetry reagent |
| 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 |
| CN108761090A (en) * | 2018-06-29 | 2018-11-06 | 迈克生物股份有限公司 | Preparation method for the reagent for detecting c reactive protein |
| CN113311154B (en) * | 2021-05-28 | 2022-05-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
| CN114113578B (en) * | 2021-11-30 | 2024-08-16 | 湖南永和阳光生物科技股份有限公司 | Method for coupling fluorescent microsphere with antibody and application thereof |
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| JPS63298062A (en) * | 1987-05-28 | 1988-12-05 | Hitachi Chem Co Ltd | Determination of anti-streptolysin o antibody |
| AU619231B2 (en) * | 1987-12-21 | 1992-01-23 | Abbott Laboratories | Chromatographic binding assay devices and methods |
| CN101302526A (en) * | 2008-06-27 | 2008-11-12 | 四川省迈克科技有限责任公司 | Recombinant soluble streptococcus hemolyticus haemolysin O gene, recombinant protein and preparation thereof |
| CN102590524B (en) * | 2011-12-30 | 2016-01-06 | 北京九强生物技术股份有限公司 | Neutrophil gelatinase-associated lipocalin detection kit |
| CN104198723A (en) * | 2014-08-11 | 2014-12-10 | 南京普朗医疗设备有限公司 | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm |
| CN104215754B (en) * | 2014-08-14 | 2016-05-11 | 上海睿康生物科技有限公司 | The eukaryotic expression of SLO albumen and containing the kit of eukaryotic expression SLO albumen |
| CN104897904A (en) * | 2015-05-29 | 2015-09-09 | 中生北控生物科技股份有限公司 | Method for marking protein by carboxyl microsphere |
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