CN102128924A - Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof - Google Patents
Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof Download PDFInfo
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- CN102128924A CN102128924A CN2010100226688A CN201010022668A CN102128924A CN 102128924 A CN102128924 A CN 102128924A CN 2010100226688 A CN2010100226688 A CN 2010100226688A CN 201010022668 A CN201010022668 A CN 201010022668A CN 102128924 A CN102128924 A CN 102128924A
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Abstract
The invention provides an agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and a preparation method thereof. The agent kit is characterized by comprising emulsion particles, transferrin antibody and buffer liquid; according to appropriate paths for covalent bond between the transferrin antibody and the emulsion particles, a turbidity reaction system between 340-700 nm detection is established below the low-concentration transferrin level and the transferrin antibody bonded with the emulsion particles. The preparation method comprises the following steps of: activation of emulsion particles; bonding of the transferrin antibody and the emulsion particles; and storing. The agent kit can be used for detecting the urinary transferrin level with the concentration to be 0-12 mg/L in the urine; the accuracy can trace back to CRM470, and the precision is smaller than 10% so as to satisfy the clinic requirements.
Description
Technical field
The present invention relates to a kind of latex enhance immunity turbidimetry for Determination urine transferrins kit, be specifically related to a kind of latex particle enhancement techniques of using and improve the sensitivity that transferrins concentration detects, can be used for the detection of low concentration transferrins in the urine.
Background technology
Glomerular filtration membrane filters protein two kinds of barriers, i.e. volume barrier and electrostatic barrier.The size that filtration membrane filters the aperture has determined the volume barrier; The generation of electrostatic barrier is because glomerular filtration membrane is rich in electronegative constituent, makes electronegative protein be difficult to filter, and transferrins belongs to this proteinoid.In case existing proof renal glomerular disease, electronegative composition sialic acid and heparin sulfate content reduce in the glomerular filtration membrane, and this moment, electrostatic barrier was impaired makes electronegative protein be easy to leach, thereby enter capsula glomeruli and can drain from urine.
At present the method for clinical detection transferrins commonly used is an immunoturbidimetry, and the method is quick with it, and is easy to be reliable, the characteristics that can use on various automatic biochemistry analyzer and in wide clinical application.But its sensing range is usually at 2-15g/L, can only be used for detecting the concentration (term of reference: 2-3.6g/L), and be difficult to detect transferrins concentration level (urine transferrins term of reference: 0-0.2mg/L) in the urine of serum transferrins.If can develop the transferrins that the immunoturbidimetry kit that can use is used for detecting the urine low strength range on automatic biochemistry analyzer, will provide strong experimental basis for clinical early detection ephrosis.
Summary of the invention
The present invention is exactly in order to address the above problem, method by the enhancing of research latex particle, improve the sensitivity that detects, thereby but solve the clinical problem that does not have conventional sense urine transferrins means, a kind of latex enhance immunity turbidimetry for Determination urine transferrins kit is provided.This invention is applicable to that the clinical conventional sense urine transferrins that carries out detects.
The present invention studies and selects suitable latex particle, and transferrins antibody and the covalently bound suitable pathways of latex particle, set up under the low concentration transferrins level and form the detectable turbidity reaction system of 340nm-700nm in conjunction with the transferrins antibody response of latex particle, comprise the suitable damping fluid that reaction is essential, pH and ionic strength etc.
The technical matters that will solve required for the present invention can be achieved through the following technical solutions:
A kind of latex enhance immunity turbidimetry for Determination urine transferrins kit is characterized in that, described kit comprise latex particle, transferrins antibody and damping fluid; The covalently bound suitable pathways of transferrins antibody and latex particle is set up under the low concentration transferrins level and is formed on the turbidity reaction system that 340nm-700nm detects in conjunction with the transferrins antibody response of latex particle.
A kind of preparation method of latex enhance immunity turbidimetry for Determination urine transferrins kit is characterized in that, may further comprise the steps:
(1), the activation of latex particle
1. get the anti-human transferrin antibody of 1000 μ l rabbits and add 2000 μ l 20mM phosphate buffers, pH7.50, mixing is standby;
2. get 600 μ l 10%20-40nm latex particles, centrifugal 4 ℃ of 21000rpm 90min discard filtrate;
3. add 1000 μ l 10mM phosphate buffers, pH6.00 ultrasonic cleaning agent dissolved particles, twice of eccentric cleaning;
4. use 3000 μ l 10mM phosphate buffers, pH6.00 dissolves latex particle, and making the latex particle activation concentration is 2%; Add the 0.036g N-hydroxy-succinamide, make reaction density reach 12mg/ml; 0.060g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride makes reaction density reach 20mg/ml;
5. room temperature vibration 15min;
6. 4 ℃ of high speed centrifugation 60min discard filtrate;
7. with 3000 μ l 2mM hydrochloric acid ice baths dissolving latex particle, 4 ℃ of high speed centrifugations clean 90min, are used for combination at once;
(2), the combination of transferrins antibody and latex particle
1. add the anti-human transferrin antibody of rabbit damping fluid, making latex particle is 2% in conjunction with concentration;
2. room temperature vibration 2hr;
3. 4 ℃ of high speed centrifugation 60min, separating filtrate is preserved filtrate;
4. use 3000 μ l 20mM phosphate buffers, pH7.50, the dissolving latex particle, high speed centrifugation cleans, and makes preservation solution, is used at once preserving;
(3), preserve
1. add 3000 μ l and preserve solution, pH7.50 dissolves latex particle, and making latex particle preserve concentration is 2%;
2. mixing, 4 ℃ of preservations are used to stablize.
Described high speed centrifugation is that 21000rpm is centrifugal.
Wherein, the frequency of described room temperature vibration is 2000 times/minute.
Wherein, described room temperature is meant that temperature is not higher than 30 ℃.
Beneficial effect of the present invention:
This kit can be used for detecting that concentration range is the urine transferrins level of 0-12mg/L in the urine, accuracy can be traceable to the abbreviation that CRM470[CRM is English Certified Reference Material (reference material of approval), CRM 470 is reference preparations of 14 (serum) analysis of protein things], precision＜10%, thus satisfy clinical needs.
Embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect is easy to understand,, further set forth the present invention below in conjunction with specific embodiment.
Embodiment 1
(1) anti-transferrins antibody sandwich latex particle
A kind of latex enhance immunity turbidimetry for Determination urine transferrins kit is characterized in that, described kit comprise latex particle, transferrins antibody and damping fluid; The covalently bound suitable pathways of transferrins antibody and latex particle is set up under the low concentration transferrins level and is formed the detectable turbidity reaction system of 340nm-700nm in conjunction with the transferrins antibody response of latex particle, may further comprise the steps:
1, the activation of latex particle
(1) get 1000 μ lQ0327 (the anti-human transferrin antibody of rabbit, Denmark Dako company produces) and add 2000 μ l 20mM phosphate buffers, pH7.50 (numbering 20PS5), mixing is standby;
(2) get 600 μ l 10%K1-0005 latex particles (production of Merck company), centrifugal 4 ℃ of 21000rpm90min discard filtrate;
(3) add 1000 μ l 10mM phosphate buffers, pH6.00 (numbering 10PS) uses ultrasonic cleaning agent dissolved particles, twice of eccentric cleaning;
(4) with 3000 μ l 10mM phosphate buffers, pH6.00 (numbering 10PS) dissolving latex particle, making the latex particle activation concentration is 2%; Add the 0.036g N-hydroxy-succinamide, make reaction density reach 12mg/ml; 0.060g 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride makes reaction density reach 20mg/ml;
(5) 2000 times/minute hunting of frequency 15min of room temperature, room temperature is not higher than 30 ℃;
(6) centrifugal 4 ℃ of 21000rpm 60min discard filtrate;
(7) with 3000 μ l 2mM hydrochloric acid ice baths dissolving latex particle, 4 ℃ of 21000rpm 90min eccentric cleaning are used for combination at once;
2, the combination of transferrins antibody and latex particle
(1) add the anti-human transferrin antibody of rabbit damping fluid, making latex particle is 2% in conjunction with concentration;
(2) 2000 times/minute hunting of frequency 2hr of room temperature;
(3) centrifugal 4 ℃ of 21000rpm 60min, separating filtrate is preserved filtrate;
(4) with 3000 μ l 20mM phosphate buffers, pH7.50 (numbering 20PS5), the dissolving latex particle, eccentric cleaning is made preservation solution, is used at once preserving;
3, preserve
(1) add 3000 μ l and preserve solution, pH7.50 (numbering SB) dissolving latex particle, making latex particle preserve concentration is 2%;
(2) mixing, 4 ℃ of preservations are used to stablize.
(2) kit preparation
1. reagent 1--reaction buffer: 200mM Tris damping fluid 50ml, pH7.15 sees Table 1.
The prescription of table 1 reagent 1
| Tris | High?Pure | 1.2110g |
| PEG6000 | CP | 1.5000g |
| ProClin?300 | N/A | 25μl |
2. the preparation of reagent 2:
1) reagent dilute solution: 50mmol/l Tris 50ml, pH7.50 sees Table 2.
The prescription of table 2 reagent 2
| Tris | High?Pure | 0.3027g |
| Bovine serum albumin(BSA) | High-purity | 0.2900g |
| ProClin?300 | N/A | 25μl |
2) reagent 2: antibodies particle preservation solution and the antibody diluent of getting 2% concentration are prepared according to 2:25 (2+23) ratio.Get the antibodies particle of 2% concentration and preserve solution 2ml+ antibody diluent 23ml.
Embodiment 2: urine transferrins immune turbidimetry detection kit is used the detection of step and analytical performance
1 testing conditions
1.1 instrument
The Olympus full automatic biochemical apparatus
Set 1.2 detect
Wavelength: 340nm
Detect consumption: 30+150+50
Reaction buffer: 200mmol/L, Tris pH value of solution 7.15 (numbering RB)
2. detect
2.1 assay method
A) mark test tube: standard pipe, sample hose, Quality Control pipe.
B) with distilled water spectrophotometer is returned to zero at the 340nm place.
C) add titer after the pre-dilution of 30 μ L respectively, sample, control liquid is to each corresponding test tube.
D) every pipe adds 150 μ L transferrins reagent 1, and 37 ℃ of pre-temperature are 3～5 minutes behind the mixing, measure absorbance A 1.
E) every pipe adds 30 μ L transferrins reagent 2, and 37 ℃ were reacted 5 minutes behind the mixing, measured absorbance A 2.
F) calculate Δ A (A2-A1), find corresponding concentration value according to curve.
2.2 result's calculating
With the albumen calibration solution in proportion (1/2.5,1/4,1/6,1/11,1/31) with physiological saline diluted protein calibration solution, by above-mentioned steps operation, drawing standard curve.With Δ A is the y axle, and each calibration solution concentration value is the x axle.Search the concentration value of sample from curve.
2.3 withinrun precision
Use a blood serum sample, use kit METHOD FOR CONTINUOUS DETERMINATION 20 times, calculate measurement result coefficient of variation CV (%) by formula (1), (2) and (3).
CV(％)＝S/x×100(％)………………………………………………………(3)
2.4 extreme difference between batch
Get three lot number kits, each lot number one cover reagent is measured with a blood serum sample, and every box reagent is surveyed 3 times.Calculate the average of every batch of reagent mensuration and the grand mean of three batches of reagent measurement results.Maximal value is relative extreme difference (%) between criticizing with the difference of minimum value and the number percent of grand mean in three batch means.With relative extreme difference (%) expression betweenrun precision.
2.5 accuracy
With treating the high and low two parts of quality-control products of check reagent three pipe replicate determinations, calculate the average of each concentration, should meet performance and the 3rd regulation that requires table 1 in 4.2.
2.6 minimum detectable level
Make sample with physiological saline, use kit METHOD FOR CONTINUOUS DETERMINATION 20 times, calculate the mean value of measuring and add 2SD.
2.7 sensing range
The a high value blood serum sample of equal proportion (by 20%, 40%, 60%, 80%, 100% preparation) dilution is answered replication 3 times at every.Calculate correlation coefficient r according to formula (4).
Formula
R---related coefficient;
In:
X
i---measure the concentration of pipe solution;
Y
i---3 replications with measure the corresponding absorbance average of pipe solution concentration;
i——1，2，3，……，n；
N---working sample number.
3 detect data
See Table 3.
Table 3 detects data
| TRF concentration (mg/L) | 2.02 | 3.86 | 10.48 | 21.9 | 51.7 | 125.2 |
| Absorbance (Abs) | 0.0157 | 0.0349 | 0.0639 | 0.0748 | 0.1007 | 0.1718 |
4 testing results
Testing result reaches re-set target substantially, and is as shown in table 4.
Table 4 transferrins kit performance table
| Sequence number | Project name | Index |
| 1 | Withinrun precision | CV≤1.3％ |
| 2 | Extreme difference between batch | ≤1.1％ |
| 3 | Accuracy | In the sign scope |
| 6 | Minimum detectable level | ≤0.04g/L |
| 8 | Sensing range | 0.2-12g/L in the scope, correlation coefficient r=0.9999 |
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the instructions just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (5)
1. a latex enhance immunity turbidimetry for Determination urine transferrins kit is characterized in that, described kit comprise latex particle, transferrins antibody and damping fluid; The covalently bound suitable pathways of transferrins antibody and latex particle is set up under the low concentration transferrins level and is formed on the turbidity reaction system that 340nm-700nm detects in conjunction with the transferrins antibody response of latex particle.
2. the preparation method of a latex enhance immunity turbidimetry for Determination urine transferrins kit is characterized in that, may further comprise the steps:
(1), the activation of latex particle
1. get the anti-human transferrin antibody of 1000 μ l rabbits and add 2000 μ l 20mM phosphate buffers, pH7.50, mixing is standby;
2. get 600 μ l 10%20-40nm latex particles, centrifugal 4 ℃ of 21000rpm 90min discard filtrate;
3. add 1000 μ l 10mM phosphate buffers, pH6.00 ultrasonic cleaning agent dissolved particles, twice of eccentric cleaning;
4. use 3000 μ l 10mM phosphate buffers, pH6.00 dissolves latex particle, and making the latex particle activation concentration is 2%; Add the 0.036g N-hydroxy-succinamide, make reaction density reach 12mg/ml; 0.060g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride makes reaction density reach 20mg/ml;
5. room temperature vibration 15min;
6. 4 ℃ of high speed centrifugation 60min discard filtrate;
7. with 3000 μ l 2mM hydrochloric acid ice baths dissolving latex particle, 4 ℃ of high speed centrifugations clean 90min, are used for combination at once;
(2), the combination of transferrins antibody and latex particle
1. add the anti-human transferrin antibody of rabbit damping fluid, making latex particle is 2% in conjunction with concentration;
2. room temperature vibration 2hr;
3. 4 ℃ of high speed centrifugation 60min, separating filtrate is preserved filtrate;
4. use 3000 μ l 20mM phosphate buffers, pH7.50, the dissolving latex particle, high speed centrifugation cleans, and makes preservation solution, is used at once preserving;
(3), preserve
1. add 3000 μ l and preserve solution, pH7.50 dissolves latex particle, and making latex particle preserve concentration is 2%;
2. mixing, 4 ℃ of preservations are used to stablize.
3. preparation method according to claim 2 is characterized in that, described high speed centrifugation is that 21000rpm is centrifugal.
4. preparation method according to claim 2 is characterized in that, wherein, the frequency of described room temperature vibration is 2000 times/minute.
5. preparation method according to claim 4 is characterized in that, wherein, described room temperature is meant that temperature is not higher than 30 ℃.
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| CN102628866A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Liquid double reagent kit for determining ferritin in serum by utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay |
| CN102645537A (en) * | 2012-04-26 | 2012-08-22 | 北京美康生物技术研究中心 | Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application |
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| CN103460046A (en) * | 2011-03-31 | 2013-12-18 | 积水医疗株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunonephelometry |
| CN106053838A (en) * | 2016-07-12 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining transferrin and preparation method thereof |
| CN108593940A (en) * | 2018-07-20 | 2018-09-28 | 迪瑞医疗科技股份有限公司 | A kind of urine transferrins detection kit |
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| CN101236210A (en) * | 2008-01-29 | 2008-08-06 | 王学生 | Rapid joint inspection reagent for hemoglobin -haptoglobin complex and occult blood in stool |
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| US10048268B2 (en) | 2011-03-31 | 2018-08-14 | Sekisui Medical Co., Ltd. | Latex particle for measurement reagent, coated latex particle, and measurement reagent for immunoturbidimetric method |
| CN103460046A (en) * | 2011-03-31 | 2013-12-18 | 积水医疗株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunonephelometry |
| CN103460046B (en) * | 2011-03-31 | 2016-08-03 | 积水医疗株式会社 | Measure reagent latex particle, sensitizing latex particle and immunoturbidimetry with measuring reagent |
| CN102628866A (en) * | 2011-12-30 | 2012-08-08 | 北京九强生物技术股份有限公司 | Liquid double reagent kit for determining ferritin in serum by utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay |
| CN102628866B (en) * | 2011-12-30 | 2014-07-16 | 北京九强生物技术股份有限公司 | Liquid double reagent kit for determining ferritin in serum by utilization of IGY ferritin antibody through latex enhanced turbidimetric immunoassay |
| CN102621325A (en) * | 2012-04-06 | 2012-08-01 | 上海蓝怡科技有限公司 | Kit for detecting concentration of docetaxel in blood |
| CN102621325B (en) * | 2012-04-06 | 2014-11-12 | 上海蓝怡科技有限公司 | Kit for detecting concentration of docetaxel in blood |
| CN102645537A (en) * | 2012-04-26 | 2012-08-22 | 北京美康生物技术研究中心 | Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application |
| CN102645537B (en) * | 2012-04-26 | 2014-07-30 | 北京美康生物技术研究中心 | Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
| CN106053838A (en) * | 2016-07-12 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining transferrin and preparation method thereof |
| CN108593940A (en) * | 2018-07-20 | 2018-09-28 | 迪瑞医疗科技股份有限公司 | A kind of urine transferrins detection kit |
| CN114047338A (en) * | 2021-11-10 | 2022-02-15 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
| CN114047338B (en) * | 2021-11-10 | 2024-03-12 | 上海捷门生物技术有限公司 | Urine transferrin detection kit and detection method thereof |
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Application publication date: 20110720 |