CN104931706B - A kind of antistreptolysin O detection kit and its preparation - Google Patents
A kind of antistreptolysin O detection kit and its preparation Download PDFInfo
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Abstract
The present invention provides a kind of antistreptolysin O (ASO) detection kit, the kit is based on latex enhancing immune turbidimetry, it is liquid double reagent, including reagent R1 and R2, the reagent R1 is reactant, reagent R2 is the solution containing ASO latex particles, its feature is the crosslinking of applied chemistry method, hemolysin " O " and Carboxylated Polystyrene latex covalent cross-linking are made by water-soluble carbodiimide (EDC) and N HOSu NHSs (NHS), ASO emulsion reagents are formed.This reagent sensitivity is high, high specificity, and preparation of reagents is simple, and worth further genralrlization is used.
Description
Technical field：
The invention belongs to biological technical field, it is related to a kind of antistreptolysin O (ASO) detection kit and its system
It is standby.
Background technology：
Streptococcus hemotoxin " O " is one of metabolite of hemolytic streptococcus, is a kind of protein containing-SH bases,
Energy lysed erythrocyte, is easily oxidized, and haematolysis ability can be temporarily lost with air contact, is formed-SS bases but can be made by reducing agent
It rejuvenates, and again with haemocylolysis, hemotoxin " O " has very strong antigenicity.After people is infected by Streptococcus hemolyticus
The patient of about 85-90% after infection 2-3 weeks to after recovering several months or 1 year can find streptococcus lysotoxin OAb (referred to as
Anti- OAb), if there are corresponding antibodies in tested serum, combined with hemotoxin " O ", haemolysis is occurred without, represent body in the recent period once
Receive hemolytic streptococcal infection or receive hemolytic streptococcus infringement repeatedly.Therefore according to streptococcus hemotoxin OAb
Potency height can aid in the diagnosis of the streptococcal allergic disease such as rheumatic fever, acute glomerulonephritis.
The measure (i.e. anti-ASO) of antistreptolysin O has haemolysis to suppress two kinds of method and emulsion method, and haemolysis suppresses method behaviour
Make method cumbersome, influence factor is more, because the haemocylolysis of streptolysin " O " is easily inactivated by cholesterol, in all serum
Cholesterol content increased perosn, its anti-" O " may occur in which false positive, for example：Take on a full stomach blood, serum be contaminated by bacterial, serum
Middle CHF is raised, the various primary cell membrane courages with Secondary cases hypercholesterolemia, various haemolysis and inflammation damnification are consolidated
Alcohol is released into blood flow etc..Emulsion method is easy, and influence factor is few, haemolysis, piarhemia, hyperbilirubinemia, hypercholesterolemia, class wind
The result that wet factor positive and sample are tested by bacterium slight pollution all without influence.It is widely used in this way.
Emulsion method principle：If there is the ASO of high titre in tested serum, the latter's aggegation can be caused when being reacted with ASO latex,
After being neutralized with 25 bonding units/ml hemolysins " O " in advance, the units of ASO ＜ 250 in aggegation person's explanation serum are occurred without, be cloudy
Property, still there are the units of ASO ＞ 250 in aggegation person's explanation serum, it is positive.
The content of the invention：
It is an object of the present invention to largely prepare ASO emulsion reagents, obtained ASO emulsion reagents are used to prepare anti-chain
Pneumoniae pneumolysin " O " detection kit.
Technical scheme：Applied chemistry method is crosslinked, by water-soluble carbodiimide (EDC) and N- hydroxyl ambers
Amber acid imide (NHS) makes hemolysin " O " and Carboxylated Polystyrene latex covalent cross-linking, prepares ASO emulsion reagents, using what is obtained
ASO emulsion reagents, prepare antistreptolysin O detection kit.
In the present invention, including ASO emulsion reagents preparation and the preparation of antistreptolysin O detection kit.Tool
Gymnastics is made as follows：
The preparation of ASO emulsion reagents：
1. the preparation of relevant buffers
Activation buffer：800ml deionized water dissolving 9.76gN- morpholino b acids (MES) is taken, 0.1%NaOH adjusts PH extremely
6.1, it is settled to 1L, 15psi moist heat sterilizations 20min with deionized water.
Mark buffer solution：Take 800ml deionized water dissolving 9.13g potassium phosphate,monobasics and 1.36g potassium dihydrogen phosphates, 0.1%
Hydrochloric acid adjusts PH to 7.4, and 1L, 15psi moist heat sterilizations 20min are settled to deionized water.
Preserve buffer solution：800ml deionized water dissolving 9.13g potassium phosphate,monobasics, 1.36g potassium dihydrogen phosphates, 1.0g
Tween 20,0.1% hydrochloric acid adjusts PH to 7.4, and 1L, 15psi moist heat sterilizations 20min are settled to deionized water.
EDC activation buffers：20mg EDC are taken, is dissolved in 10ml activation buffers, be made EDC activation buffers.
NHS activation buffers：40mg NHS are taken, is dissolved in 10ml activation buffers, be made NHS activation buffers.
The preparation of 2.ASO emulsion reagents
Raw material：Carboxylated Polystyrene latex particle size is 100-150nm, and concentration is 10%, takes 1ml, is dissolved in 3-7ml activation slow
It is diluted in fliud flushing.1ml EDC activation buffers, 1ml NHS activation buffers, room temperature are added in the microspheres solution for having diluted
20-25 DEG C of concussion 10min, 37 DEG C of water bath with thermostatic control 20min.2-8 DEG C of centrifugation (12000rpm, 30min), removes supernatant, with mark
After note buffer solution washed once, 7ml is diluted to mark buffer solution.50mg hemolysins " O " (commercially available) is added while stirring, with mark
Note buffer solution complements to 10ml, room temperature 20-25 DEG C of concussion 3hour, 37 DEG C of water bath with thermostatic control 3hour.Centrifugation (12000rpm,
30min), supernatant is removed, and is washed twice with mark buffer solution buffer solution.After washing 1% (weight is made into preservation buffer solution
Volume ratio) suspension, with 0.1% (w/v) sodium azide solution anti-corrosion, as ASO emulsion reagents.
The preparation of antistreptolysin O detection kit：
This detection kit includes reagent R1 and R2, and specific composition is as follows：
With detection kit R1：400ml, R2：Prepared as a example by the specification of 100ml.
The preparation of reagent R1：320ml deionized waters are taken, 0.73g Tris are added, stirred 4-5 minutes at room temperature, added
0.74g disodium salt disodiums, are stirred 4-5 minutes at room temperature, are eventually adding 2.34g sodium chloride, and 4-5 points is stirred at room temperature
Clock, 400ml is settled to deionized water, is stirred at room temperature 4-5 minutes and is well mixed.
The preparation of reagent R2：80ml deionized waters are taken, 0.58g sodium chloride is added, stirred 4-5 minutes at room temperature, added
40ml ASO emulsion reagents, are stirred 4-5 minutes at room temperature, and 100ml is settled to deionized water, are stirred at room temperature 4-5 minutes and are mixed
Close uniform.
The Cleaning Principle of this detection kit is that hemolysin " O " is crosslinking in present latex particulate, anti-with ASO in test serum
There is antigen-antibody reaction in body, cause microparticle agglutination, form certain turbidity, under 540nm wavelength, by same treatment
Calibration object is compareed, and quantitative determination goes out the content of ASO antibody in sample.
Brief description of the drawings：
Fig. 1：Using BS-420 automatic clinical chemistry analyzers, the reagent to 5 gradient concentrations is measured, and to measured value
Carry out correlation analysis.What wherein X-axis was represented is diluted concentration, and what Y-axis was represented is measured value average.Coefficient correlation：r2=
0.9977, linear equation is：Y=1.054x-9.443.
Specific embodiment：
Embodiment
The use of the antistreptolysin O detection kit of embodiment 1.
1) detecting instrument：With 540nm wavelength, 1cm optical paths, 37 DEG C of thermostats Biochemical Analyzer.
2) test serum：Not haemolysis serum, 2-8 DEG C can stablize one week, and 6 avoid fat turbid.
3) program is specifically detected：
4) result of calculation：The concentration (IU/ml) of ASO=Δ AT/ Δs AS × calibration solution concentration in sample
In formula：Δ AT take blank tube absorbance as the sample cell absorbance for compareing；
Δ AS take blank tube absorbance as the calibration pipe absorbance for compareing；
5) reference range：ASO：≤200IU/ml.
6) precision：CV≤8% in batch；Relative extreme difference≤10% between batch.
7) degree of accuracy：Measured value should be in the range of ± 10% with quality controlled serum target value relative deviation.
8) range of linearity：0-800IU/ml.
9) sensitivity for analysis：0.0010-0.0025A.
The preparation of embodiment 2.ASO emulsion reagents and preparation (1) ASO glue of antistreptolysin O detection kit
The newborn specific composition of reagent is formulated as follows：
Carboxylated Polystyrene latex particle size is 100nm, and concentration is 10%, takes 1ml, and being dissolved in 3ml activation buffers is carried out
Dilution.1ml EDC activation buffers, 1ml NHS activation buffers, 20-25 DEG C of shake of room temperature are added in the microspheres solution for having diluted
Swing 10min, 37 DEG C of water bath with thermostatic control 20min.2-8 DEG C of centrifugation (12000rpm, 30min), removes supernatant, is washed with mark buffer solution
After washing once, 7ml is diluted to mark buffer solution.50mg hemolysins " O " (commercially available) is added while stirring, is mended with mark buffer solution
Foot to 10ml, 20-25 DEG C of room temperature shakes 3hour, 37 DEG C of water bath with thermostatic control 3hour.Centrifugation (12000rpm, 30min), removes supernatant
Liquid, and washed twice with mark buffer solution buffer solution.1% (w/v) suspension is made into preservation buffer solution after washing, with
0.1% (w/v) sodium azide solution anti-corrosion, as ASO emulsion reagents.
The preparation of antistreptolysin O detection kit
This detection kit includes reagent R1 and R2, and specific composition is as follows：
With detection kit R1：400ml, R2：Prepared as a example by the specification of 100ml.
The preparation of reagent R1：320ml deionized waters are taken, 0.73g Tris are added, stirred 4 minutes at room temperature, added
0.74g disodium salt disodiums, stir 4 minutes at room temperature, are eventually adding 2.34g sodium chloride, stir 4 minutes at room temperature, use
Deionized water is settled to 400ml, stirs at room temperature 4 minutes and is well mixed.
The preparation of reagent R2：80ml deionized waters are taken, 0.58g sodium chloride is added, stirred 4 minutes at room temperature, added
40mlASO emulsion reagents, are stirred 4 minutes at room temperature, and 100ml is settled to deionized water, mixing in 4 minutes are stirred at room temperature equal
It is even.
The performance indications of antistreptolysin O detection kit：
1) precision is determined：Continuous drawing is measured for 20 times in same sample, calculate the mean of measured value, standard deviation and
The coefficient of variation,
The precision testing result of table 1
Coefficient of variation CV is generally used for weighing a precision for assay method, and CV values are smaller, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.CV values are less than 3% in table 1, show that the inventive method has excellent precision.
2) accuracy determination：Same quality-control product, replication 3 times is used to average, should with quality-control product target value relative deviation
In the range of ± 15%.
The degree of accuracy testing result of table 2
Relative deviation CB=-1.64% in table 2, in the range of ± 15%, shows that the inventive method has excellent standard
Exactness.
3) linear determination：Using deionized water by reagent dilutions into 5 gradient concentrations, each gradient concentration is detected 3 times, taken
Average value, regression analysis is made to measured value and desired value, calculates r values and relative deviation (result is shown in Fig. 1, unit IU/ml).
The linear correlation detection result of table 3
| Diluted concentration | 120 | 200 | 400 | 600 | 800 |
| Measured value | 122 | 191 | 406 | 595 | 829 |
| 122 | 203 | 430 | 592 | 843 | |
| 114 | 204 | 432 | 614 | 866 | |
| Average | 119 | 200 | 422 | 600 | 846 |
| Absolute deviation | -1 | 0 | 22 | 0 | 46 |
| Relative deviation | - 0.83% | 0.00% | 5.50%% | 0.00% | 5.75% |
Coefficient correlation is obtained by table 3：r2=0.9977, linear equation is：Y=1.054x-9.443, as a result shows this
Reagent correlation is good, with good specificity and accuracy.
4) sensitivity for analysis is defined as：To 20 measure of zero calibration object, its 2 times of average deviation is taken, it is bent in standard
Corresponding concentration is sensitivity for analysis, sensitivity for analysis on line：1IU/ml：0.0018.
The preparation of embodiment 3.ASO emulsion reagents and the preparation (2) of antistreptolysin O detection kit
The specific composition of ASO emulsion reagents is formulated as follows：
Carboxylated Polystyrene latex particle size is 150nm, and concentration is 10%, takes 1ml, and being dissolved in 7ml activation buffers is carried out
Dilution.1mlEDC activation buffers, 1mlNHS activation buffers, 20-25 DEG C of concussion of room temperature are added in the microspheres solution for having diluted
10min, 37 DEG C of water bath with thermostatic control 20min.2-8 DEG C of centrifugation (12000rpm, 30min), removes supernatant, is washed with mark buffer solution
After once, 7ml is diluted to mark buffer solution.50mg hemolysins " O " (commercially available) is added while stirring, is supplied with mark buffer solution
To 10ml, room temperature 20-25 DEG C of concussion 3hour, 37 DEG C of water bath with thermostatic control 3hour.Centrifugation (12000rpm, 30min), removes supernatant
Liquid, and washed twice with mark buffer solution buffer solution.1% (w/v) suspension is made into preservation buffer solution after washing, with
0.1% (w/v) sodium azide solution anti-corrosion, as ASO emulsion reagents.
The preparation of antistreptolysin O detection kit
This detection kit includes reagent R1 and R2, and specific composition is as follows：
With detection kit R1：400ml, R2：Prepared as a example by the specification of 100ml.
The preparation of reagent R1：320ml deionized waters are taken, 0.73g Tris are added, stirred 5 minutes at room temperature, added
0.74g disodium salt disodiums, stir 5 minutes at room temperature, are eventually adding 2.34g sodium chloride, stir 5 minutes at room temperature, use
Deionized water is settled to 400ml, stirs at room temperature 5 minutes and is well mixed.
The preparation of reagent R2：80ml deionized waters are taken, 0.58g sodium chloride is added, stirred 5 minutes at room temperature, added
40ml ASO emulsion reagents, are stirred 5 minutes at room temperature, and 100ml is settled to deionized water, mixing in 5 minutes are stirred at room temperature equal
It is even.
The performance indications of antistreptolysin O detection kit：
Result shows precision, the degree of accuracy, the difference between linear correlation and sensitivity for analysis and the data of reagent described in example 2
Different in the range of nominal error (data summary).
Claims (1)
1. a kind of antistreptolysin O detection kit, it is characterised in that：The kit is based on latex enhancing immune ratio
Turbid method, is liquid double reagent, including reagent R1 and R2, and the reagent R1 is reactant, and reagent R2 is to contain streptococcus haemolysis
The solution of plain " O " latex particle, reagent R1 and the specific compositions of reagent R2 are as follows：
Reagent R1：
Disodium salt disodium 5.0mmol/L
Sodium chloride 100mmol/L
Tris buffer solutions 15mmol/L
Reagent R2：
Sodium chloride 100mmol/L
Antistreptolysin O emulsion reagent 400ml/L
The antistreptolysin O emulsion reagent is prepared by the following method：
（1）The preparation of relevant buffers
Activation buffer：Take 800ml deionized water dissolving 9.76gN- morpholino b acids, 0.1%NaOH adjusts pH to 6.1, spend from
Sub- water is settled to 1L, 15psi moist heat sterilizations 20min；
Mark buffer solution：800ml deionized water dissolving 9.13g potassium phosphate,monobasics and 1.36g potassium dihydrogen phosphates are taken, 0.1% hydrochloric acid is adjusted
PH to 7.4,1L, 15psi moist heat sterilizations 20min are settled to deionized water；
Preserve buffer solution：800ml deionized water dissolving 9.13g potassium phosphate,monobasics, 1.36g potassium dihydrogen phosphates, 1.0g Tween
20,0.1% hydrochloric acid adjusts pH to 7.4, and 1L, 15psi moist heat sterilizations 20min are settled to deionized water；
EDC activation buffers：20mg EDC are taken, is dissolved in 10ml activation buffers, be made EDC activation buffers；
NHS activation buffers：40mg NHS are taken, is dissolved in 10ml activation buffers, be made NHS activation buffers；
（2）The preparation of antistreptolysin O emulsion reagent
Raw material：Carboxylated Polystyrene latex particle size is 100-150nm, and concentration is 10%, takes 1ml, is dissolved in 3-7ml activation buffers
In be diluted, in the microspheres solution for having diluted add 1ml EDC activation buffers, 1ml NHS activation buffers, 20-25 DEG C
Concussion 10min, 37 DEG C of water bath with thermostatic control 20min, at 2-8 DEG C, are centrifuged 30min under the conditions of 12000rpm, supernatant is removed, with mark
After note buffer solution washed once, 7ml is diluted to mark buffer solution, 50mg antistreptolysin Os are added while stirring, used
Mark buffer solution complements to 10ml, and 20-25 DEG C shakes 3 hours, 37 DEG C of waters bath with thermostatic control 3 hours, is centrifuged under 12000rpm
30min, removes supernatant, and is washed twice with mark buffer solution buffer solution, and 1% weighing body is made into preservation buffer solution after washing
The suspension of product ratio, with 0.1% w/v sodium azide solution anti-corrosion, as antistreptolysin O emulsion reagent.
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| CN110146706A (en) * | 2019-05-28 | 2019-08-20 | 中生北控生物科技股份有限公司 | Antistreptolysin O latex immunoturbidimetry detection kit and preparation method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0475786B1 (en) * | 1990-09-13 | 1996-07-17 | Wako Pure Chemical Industries Ltd | Method for determination of antistreptolysin O |
| US5856202A (en) * | 1994-11-15 | 1999-01-05 | Asahi Kasei Kogyo Kabushiki Kaisha | Method for determining antistreptolysin O antibody |
| CN103304645A (en) * | 2012-03-16 | 2013-09-18 | 深圳迈瑞生物医疗电子股份有限公司 | Recombinant streptococcolysin O protein, nucleotide sequence, preparation method and application thereof |
| CN104374911A (en) * | 2014-12-05 | 2015-02-25 | 重庆乾德生物技术有限公司 | Detection kit for quantitative detection of rheumatoid factors and anti streptolysin O |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161716B (en) * | 2010-12-30 | 2014-03-05 | 北京九强生物技术股份有限公司 | Method and reagent for latex sensitization |
| CN102964435B (en) * | 2011-08-31 | 2014-12-03 | 北京利德曼生化股份有限公司研发中心 | Truncated-form streptococcus hemolyticus bacteriolysin O and detection kit using same |
| CN103308698B (en) * | 2013-06-17 | 2015-04-29 | 北京北检·新创源生物技术有限公司 | Method for covalently coupling amino-containing molecules to microspheres |
| 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 |
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- 2015-05-02 CN CN201510227792.0A patent/CN104931706B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0475786B1 (en) * | 1990-09-13 | 1996-07-17 | Wako Pure Chemical Industries Ltd | Method for determination of antistreptolysin O |
| US5856202A (en) * | 1994-11-15 | 1999-01-05 | Asahi Kasei Kogyo Kabushiki Kaisha | Method for determining antistreptolysin O antibody |
| CN103304645A (en) * | 2012-03-16 | 2013-09-18 | 深圳迈瑞生物医疗电子股份有限公司 | Recombinant streptococcolysin O protein, nucleotide sequence, preparation method and application thereof |
| CN104374911A (en) * | 2014-12-05 | 2015-02-25 | 重庆乾德生物技术有限公司 | Detection kit for quantitative detection of rheumatoid factors and anti streptolysin O |
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