CN105044351A - Antistreptolysin O immunological turbidimetry reagent and preparation method thereof - Google Patents

Antistreptolysin O immunological turbidimetry reagent and preparation method thereof Download PDF

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Publication number
CN105044351A
CN105044351A CN201510376941.XA CN201510376941A CN105044351A CN 105044351 A CN105044351 A CN 105044351A CN 201510376941 A CN201510376941 A CN 201510376941A CN 105044351 A CN105044351 A CN 105044351A
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reagent
antistreptolysin
aso
liquid
immunoturbidimetry
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张春东
孙珊
黄宝福
谷万俊
韩东成
淳林
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NANJING PERLONG MEDICAL EQUIPMENT CO Ltd
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NANJING PERLONG MEDICAL EQUIPMENT CO Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • G01N33/56944Streptococcus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854Immunoglobulins

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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
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  • Hematology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses an antistreptolysin O immunological turbidimetry reagent. The reagent comprises a freeze-dried reagent R1 and R2, which are fixed in a same container. The freeze-dried reagent R1 and R2 are prepared by adding liquid reagent R1 and R2 into an excipient and a protective agent and then freeze-drying the mixture; wherein the liquid reagent R1 is a buffering solution containing PEG, the liquid reagent R2 is latex coupled with antistreptolysin O or colloidal gold coupled with antistreptolysin O. The operation of the provided reagent for detection is convenient, time is saved, at the same time, the reagent is convenient for transportation and preservation, moreover, the reagent can be transported in cold areas, and the reagent stability is improved.

Description

A kind of antistreptolysin O (ASO) immunoturbidimetry reagent and preparation method thereof
Technical field
Immunoturbidimetry field of the present invention, is specifically related to the technique of a kind of antistreptolysin O (ASO) immunoturbidimetry reagent and preparation thereof.
Background technology
A kind of metabolic product energy lysed erythrocyte that hemolytic streptococcus produces, so this product is named as " O " hemolysin (SLO), after human infection A group hemolytic streptococcus, will produce specific antibody.Streptolysin antibody (ASO) is the type of special immunoglobulin be present in serum.
Streptolysin antibody (ASO) mensuration is one of important indicator diagnosing A race hemolytic streptococcus (A chain) to infect.Body, because of after the infection A group of streptococcus such as pharyngitis, tonsillitis, scarlet fever, erysipelas, pyoderma, rheumatic fever, can produce streptolysin O antibody, i.e. " Anti-StreptolysinO (ASO) ".This kind of antibody horizontal significantly increase with active rheumatic fever or acute nephritis pathology closely related, sustainable existence or level decline, then point out disease to be nonmobile phase or convalescence, clinical dynamic monitoring and significance.The patient with rheumatoid arthritis streptolysin antibody (ASO) of about 80% is positive simultaneously.
The antistreptolysin O (ASO) detection kit overwhelming majority for turbidimetry detection on current domestic and international market is liquid double reagent; Because freeze thawing can affect the performance of the R2 in reagent, make product inactivation, also there are no the reagent of freeze-drying, therefore winter northern area reagent transport or preserve produce larger problem; Specialty section office of increasing hospital or basic hospital all wish that manufacturer provides POCT reagent and instrument clinically simultaneously, can obtain detection data as early as possible like this, carry out diagnosis and treatment in time to patient; Wherein instant POCT reagent is subject to the favor of numerous doctors especially, but instant immunoturbidimetry reagent is owing to taking up an area larger in its space, laboratory cannot provide enough refrigerating spaces to preserve reagent, and therefore urgent hope can solve transport and the preservation problem of immunoturbidimetry reagent clinically.
Simultaneously due to the mutual interference between reagent, use double reagent clinically always, do not solve the problem of single reagent; Single reagent will make operation steps simpler, reduce the time.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the invention provides a kind of antistreptolysin O (ASO) immunoturbidimetry reagent and preparation method thereof.
Technical scheme: for solving the problems of the technologies described above, a kind of antistreptolysin O (ASO) immunoturbidimetry reagent of the present invention, comprise and be fixed on freeze-drying R1 reagent in same container and R2 reagent, freeze-drying R1 reagent and R2 reagent add excipient by liquid R1 reagent and R2 reagent and protective agent freeze-drying forms; Wherein liquid R1 reagent is the damping fluid containing PEG, and liquid R2 reagent is that coupling has the latex of streptolysin O or coupling to have the collaurum of streptolysin O.
Further, described excipient and protective agent are is any one or combination in any in organic compound in trehalose, sucrose, fructose, sweet mellow wine, PEG, PVP, glycerine, ethylene glycol, amino acid.
Further, described container is the cuvette that can be directly used in detection.
Further, the addition of described excipient and protective agent is mass percent concentration is 0.1% ~ 20%.
Further, described liquid R1 reagent and R2 ratio of reagents are arbitrary proportion.
Further, described liquid R1 reagent and R2 reagent add indicator.
The preparation method of above-mentioned antistreptolysin O (ASO) Immunoturbidimetric kit, its preparation process is:
1) in liquid R1 and R2 reagent, excipient and protective agent is added respectively;
2) put into-80 DEG C of refrigerator freezings after being mixed by above-mentioned liquid R1 and R2 reagent to spend the night, then open frozen vacuum dryer freeze-drying in freezing 20 hours, be loaded in container.
The another kind of preparation method of above-mentioned antistreptolysin O (ASO) immunoturbidimetry reagent, its preparation process is:
1) in liquid R1 and R2 reagent, excipient and protective agent is added respectively;
2) just above-mentioned liquid R1 and R2 reagent freezing shaping take out freezing respectively, opens frozen vacuum dryer, freezing 20 hours;
3) last packing is fixed in container.
Beneficial effect: the present invention in terms of existing technologies, possesses following advantage:
1) the invention solves the transportation problem of immunoturbidimetry reagent, reagent freeze-drying is shaped, although it is the ordinary skill in the art that freeze-drying is preserved albumen and antibody, but after freeze-drying can the activity of protected protein and antibody unaffected, this does not provide corresponding technical scheme in prior art, because the albumen in the present invention or antibody are coupled on nano rubber latex microballoon or collaurum, nano rubber latex microballoon or collaurum can not freezingly be these is that those skilled in the art are confessed, properties of product are affected because of breakdown of emulsion being there is after it is freezing, and the present invention thoroughly solve nano rubber latex microballoon or collaurum freezing after can there is breakdown of emulsion and affect the technical matters of properties of product.
2) R1 and R2 reagent is the invention enables at room temperature to preserve, coexist a container and mutual interference does not occur, operation steps and running time is decreased in clinical examination, simultaneously reagent is due to through freeze drying, be conducive to product to preserve, liquid reagent needs to preserve at refrigerator 2-8 degree, storage life is generally 1 year, at room temperature preserve through checking reagent of the present invention and do not change for more than 6 months, storing under cryogenic conditions (lower than 0 degree) simultaneously or transport can not affect properties of product in one month.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 detects linear graph.
Fig. 2 is that embodiment 2 detects linear graph.
Fig. 3 is that embodiment 3 detects linear graph.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.
Embodiment 1:
A preparation method for antistreptolysin O (ASO) Immunoturbidimetric kit, its preparation process is:
1) in liquid R1 and R2 reagent, 20% sucrose is added respectively, 0.1% glycocoll; Wherein, R1 reagent is the damping fluid of PEG, and R2 reagent is the latex of coupling people antistreptolysin O (ASO) polyclonal antibody;
2) put into freeze overnight after being mixed by above-mentioned liquid R1 and R2 reagent, then open frozen vacuum dryer freeze-drying in freezing 20 hours, rearmounted-20 DEG C of preservations of sealer are loaded in container for 1 month.
Then above-mentioned antistreptolysin O (ASO) Immunoturbidimetric kit detected, its detecting step is as follows:
1, open instrument preheating 15 minutes, before measuring sample, reagent calibration card is positioned in the appointed area of specific protein analyser, after the scale operation description operation of instrument, then carries out pattern detection (now card can be removed).
2, the place of reagent from storage is taken out;
3, open sealed membrane, get 500ul deionized water, be placed in and detect cup, softly rock and make dispensing reagents even, then add 5ul sample, again softly rock mixing.The time of mixing, within 5 seconds, puts into the specific protein detector after calibration;
4, instrument detects antistreptolysin O (ASO) testing result automatically.
Its testing result is as follows:
(1) repeatability
Under repeated condition, with Landau quality-control product 329LPC and 330LPC test kit, repeated test at least 10 times (n>=10), the mean value of computation and measurement value respectively with standard deviation (s).The coefficient of variation (CV) is calculated, the precision (coefficient of variation, CV)≤10.0% of acquired results by formula (1).
C V = s / x ‾ × 100 % ... ( 1 )
Precision 329LPC 330LPC
1 366.21 494.93
2 367.85 498.06
3 385.64 531.08
4 354.17 497.25
5 391.07 543.46
6 345.12 516.11
7 381.23 540.49
8 369.12 493.38
9 394.56 515.72
10 371.26 566.84
n=10 10 10
Mean 372.62 519.73
SD 15.78 25.07
CV 4.23% 4.82%
(2) range of linearity
With close to the range of linearity upper limit enriched sample and close to or equal the low concentration sample (5% physiological saline) of range of linearity lower limit, high level (H) sample, low value (L) sample are pressed each self-configuring mixing of 1L, 0.75L+0.25H, 0.50L+0.50H, 0.25L+0.75H, 1H relation, forms the equidistant sample of 5 concentration levels.Test kit respectively, each dilute concentration tests 3 times, obtains the average (y of measurement result respectively i).Using statistics Epidemiological Analysis software (as Excel, SPSS) carries out linear regression analysis, with dilute concentration (x i) be independent variable, with measurement result average (y i) be dependent variable, obtain equation of linear regression and related coefficient (R).By dilute concentration (x i) substitute into equation of linear regression, calculate y iestimated value and the absolute deviation of yi and estimated value and relative deviation.
As shown in Figure 1, testing result shows, the reagent after freeze-drying stores one month at-20 DEG C, and its properties of product do not decline.
Embodiment 2:
A preparation method for antistreptolysin O (ASO) Immunoturbidimetric kit, its preparation process is:
1) in liquid R1 reagent, add 10% trehalose, add 2% sweet mellow wine and 0.1%PVP mixed liquor in liquid R2, wherein liquid R1 reagent is the damping fluid of PEG, and R2 reagent is the collaurum that coupling has streptolysin O;
2) by the freezing shaping take out freezing respectively of above-mentioned liquid R1 and R2 reagent, open frozen vacuum dryer, freezing 20 hours envelopes, film is placed on room temperature and places 6 months;
3) last packing is fixed in container.
Then above-mentioned antistreptolysin O (ASO) Immunoturbidimetric kit detected, its detecting step is as follows:
1, open instrument preheating 15 minutes, before measuring sample, reagent calibration card is positioned in the appointed area of specific protein analyser, after the scale operation description operation of instrument, then carries out pattern detection (now card can be removed).
2, by reagent (wherein R2 reagent is the latex of how anti-coupling, and R1 adds 1% trehalose, and R2 adds 2% sweet mellow wine and glycocoll, and both are frozen into bead respectively, freeze drying 10 hours, and sealer is placed on room temperature and places 6 months), take out from the place of storage;
3, open sealed membrane, get 600ul deionized water, be placed in and detect cup, softly rock and make dispensing reagents even, then add 2ul sample, again softly rock mixing, the time of mixing, within 5 seconds, puts into the specific protein detector after calibration;
4, instrument detects antistreptolysin O (ASO) testing result automatically.
Its testing result is as follows:
(1) repeatability
Under repeated condition, with Landau quality-control product 329LPC and 330LPC test kit, repeated test at least 10 times (n>=10), the mean value of computation and measurement value respectively with standard deviation (s).The coefficient of variation (CV) is calculated, the precision (coefficient of variation, CV)≤10.0% of acquired results by formula (1).
C V = s / x ‾ × 100 % ... ( 1 )
Precision 329LPC 330LPC
1 386.32 486.34
2 362.34 504.13
3 387.21 531.08
4 374.74 497.25
5 365.18 542.9
6 359.56 526.8
7 358.45 553.12
8 387.26 493.38
9 375.26 498.49
10 376.36 542.36
n=10 10 10
Mean 373.27 517.59
SD 11.39 24.28
CV 3.05% 4.69%
(2) range of linearity
With close to the range of linearity upper limit enriched sample and close to or equal the low concentration sample (5% physiological saline) of range of linearity lower limit, high level (H) sample, low value (L) sample are pressed each self-configuring mixing of 1L, 0.75L+0.25H, 0.50L+0.50H, 0.25L+0.75H, 1H relation, forms the equidistant sample of 5 concentration levels.Test kit respectively, each dilute concentration tests 3 times, obtains the average (y of measurement result respectively i).Using statistics Epidemiological Analysis software (as Excel, SPSS) carries out linear regression analysis, with dilute concentration (x i) be independent variable, with measurement result average (y i) be dependent variable, obtain equation of linear regression and related coefficient (R).By dilute concentration (x i) substitute into equation of linear regression, calculate y iestimated value and the absolute deviation of yi and estimated value and relative deviation.
Shown in Fig. 2, testing result shows, the reagent after freeze-drying at room temperature stores six months, and its properties of product do not decline.
Embodiment 3:
A preparation method for antistreptolysin O (ASO) Immunoturbidimetric kit, its preparation process is:
1) mix at liquid R1 reagent and liquid R2, wherein liquid R1 reagent is the damping fluid of PEG, and R2 reagent is the collaurum that coupling has streptolysin O;
2) be shaped and take out freezing in after instillation in above-mentioned liquid R1 and R2 reagent being added 10% glycerine and fructose mixed liquor, opening frozen vacuum dryer, freezing 10 hours envelopes, film is placed in 37 degree of refrigerators and places 15 days;
3) last packing is fixed in container.
Then above-mentioned antistreptolysin O (ASO) Immunoturbidimetric kit detected, its detecting step is as follows:
1. open instrument preheating 15 minutes, before measuring sample, reagent calibration card is positioned in the appointed area of specific protein analyser, after the scale operation description operation of instrument, then carries out pattern detection (now card can be removed).
2. the place of reagent from storage is taken out;
3. open sealed membrane, get 495ul deionized water, be placed in and detect cup, softly rock and make dispensing reagents even, then add 2.5ul sample, again softly rock mixing.The time of mixing, within 5 seconds, puts into the specific protein detector after calibration;
4. instrument detects antistreptolysin O (ASO) testing result automatically.
Its testing result is as follows:
One) repeatability
Under repeated condition, with Landau quality-control product 329LPC and 330LPC test kit, repeated test at least 10 times (n>=10), the mean value of computation and measurement value respectively with standard deviation (s).The coefficient of variation (CV) is calculated, the precision (coefficient of variation, CV)≤10.0% of acquired results by formula (1).
C V = s / x ‾ × 100 % ... ( 1 )
Precision 329LPC 330LPC
1 386.24 486.3
2 362.37 504.13
3 387.21 531.08
4 374.09 497.25
5 365.18 542.9
6 359.56 526.8
7 358.45 553.12
8 387.26 493.38
9 375.26 498.49
10 376.36 542.36
n=10 10 10
Mean 373.20 517.58
SD 11.37 24.29
CV 3.05% 4.69%
(2) range of linearity
With close to the range of linearity upper limit enriched sample and close to or equal the low concentration sample (5% physiological saline) of range of linearity lower limit, high level (H) sample, low value (L) sample are pressed each self-configuring mixing of 1L, 0.75L+0.25H, 0.50L+0.50H, 0.25L+0.75H, 1H relation, forms the equidistant sample of 5 concentration levels.Test kit respectively, each dilute concentration tests 3 times, obtains the average (y of measurement result respectively i).Using statistics Epidemiological Analysis software (as Excel, SPSS) carries out linear regression analysis, with dilute concentration (x i) be independent variable, with measurement result average (y i) be dependent variable, obtain equation of linear regression and related coefficient (R).By dilute concentration (x i) substitute into equation of linear regression, calculate y iestimated value and the absolute deviation of yi and estimated value and relative deviation.
As shown in Figure 3, testing result shows, the reagent after freeze-drying stores 15 days at 37 DEG C, and its properties of product do not decline.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (8)

1. an antistreptolysin O (ASO) immunoturbidimetry reagent, is characterized in that: comprise and be fixed on freeze-drying R1 reagent in same container and R2 reagent, and freeze-drying R1 reagent and R2 reagent add excipient by liquid R1 reagent and R2 reagent and protective agent freeze-drying forms; Wherein liquid R1 reagent is the damping fluid containing PEG, and liquid R2 reagent is that coupling has the latex of streptolysin O or coupling to have the collaurum of streptolysin O.
2. antistreptolysin O (ASO) immunoturbidimetry reagent according to claim 1, is characterized in that: it is any one or combination in any in organic compound in trehalose, sucrose, fructose, sweet mellow wine, PEG, PVP, glycerine, ethylene glycol, amino acid that described excipient and protective agent are.
3. antistreptolysin O (ASO) immunoturbidimetry reagent according to claim 1, is characterized in that: described container is the cuvette that can be directly used in detection.
4. antistreptolysin O (ASO) immunoturbidimetry reagent according to claim 1, is characterized in that: the addition of described excipient and protective agent is mass percent concentration is 0.1% ~ 20%.
5. antistreptolysin O (ASO) immunoturbidimetry reagent according to claim 1, is characterized in that: described liquid R1 reagent and R2 ratio of reagents are arbitrary proportion.
6. the antistreptolysin O (ASO) immunoturbidimetry reagent according to claim 1 or 6, is characterized in that: described liquid R1 reagent and R2 reagent add indicator.
7. a preparation method for the antistreptolysin O (ASO) immunoturbidimetry reagent according to any one of claim 1 ~ 7, is characterized in that: its preparation process is:
1) in liquid R1 and R2 reagent, excipient and protective agent is added respectively;
2) above-mentioned liquid R1 and R2 reagent is mixed rear freeze-drying, be loaded in container.
8. a preparation method for the antistreptolysin O (ASO) immunoturbidimetry reagent according to any one of claim 1 ~ 7, is characterized in that: its preparation process is:
1) in liquid R1 and R2 reagent, excipient and protective agent is added respectively;
2) by the freezing shaping take out freezing respectively of above-mentioned liquid R1 and R2 reagent;
3) last packing is fixed in container.
CN201510376941.XA 2015-07-01 2015-07-01 Antistreptolysin O immunological turbidimetry reagent and preparation method thereof Pending CN105044351A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105527434A (en) * 2015-12-31 2016-04-27 辽宁迈迪生物科技有限公司 A kit used for detecting N1,N<12>-diacetylspermine (DAS)
CN106771148A (en) * 2016-12-28 2017-05-31 广州华弘生物科技有限公司 A kind of immune globulin M detection reagent box and detection method
CN106855571A (en) * 2016-12-29 2017-06-16 广州华弘生物科技有限公司 A kind of antistreptolysin O (ASO) quick diagnosis reagent kit and its application method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105527434A (en) * 2015-12-31 2016-04-27 辽宁迈迪生物科技有限公司 A kit used for detecting N1,N<12>-diacetylspermine (DAS)
CN105527434B (en) * 2015-12-31 2017-12-19 辽宁迈迪生物科技有限公司 One kind is used for the kit of diacetyl spermine (DAS) detection
CN106771148A (en) * 2016-12-28 2017-05-31 广州华弘生物科技有限公司 A kind of immune globulin M detection reagent box and detection method
CN106855571A (en) * 2016-12-29 2017-06-16 广州华弘生物科技有限公司 A kind of antistreptolysin O (ASO) quick diagnosis reagent kit and its application method

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