CN109613260A - A kind of detection kit and its detection method of complete homogeneous determination microdose urine protein - Google Patents
A kind of detection kit and its detection method of complete homogeneous determination microdose urine protein Download PDFInfo
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Abstract
The invention belongs to biomedical inspection fields, and in particular to a kind of detection kit and its method of homogeneous detection technique measurement microdose urine protein.Kit involved in the present invention, (reagent R1 and R2) epitope different with albumin combines the monoclonal antibody of the different labels of two kinds had, forms the immunocomplex formation of antibody-antigen-antibody；After addition antioxidant (reagent R3) mixes gently, it is added detection substrate (reagent R4).Horseradish peroxidase enzyme catalytic peroxide generates hydroxidion, and hydroxidion time present in system is very short, and due to the presence of antioxidant in system, can guarantee that portion propagates hydroxidion in the molecule.Acridine derivatives generate optical signal under the action of strong oxidizer.Luminous intensity and the content of albumin are directly proportional.The detection technique is compared with other detection techniques based on double antibodies sandwich, which, which has, does not need sessile antibody, is not needed to wash, is reacted more abundant, rapid advantage.
Description
Technical field
The invention belongs to biomedical inspection fields, and in particular to a kind of using homogeneous detection technique measurement urine Microalbunin
White kit and preparation method thereof.
Background technique
Albumin molecule amount is 69KD, is a kind of high molecular weight protein with negative electrical charge in blood plasma, accounts for Total plasma protein
60% or so of content.Under normal circumstances, since glomerular capillary basement membrane has filtering function, glomerulus in addition
Reabsorption, only minimal amount of albumin can be discharged in vitro by urine.But if there is certain lesions cause kidney
The charge selective barrier of bead basilar memebrane is damaged or filtration hole increases, then may cause the increase of albumin excretion amount.Diabetes
Nephrosis is one of serious microvascular complication of diabetes, shows diabetic nephropathy according to American Diabetes Association (ADA) statistics
The probability occurred in diabetes patient's (including 1 type and 2 types) is 20%~30%.If not taking any intervening measure, 20%
~40% will progress to dominant nephrosis with the type 2 diabetic patient of microalbuminuria, and about 20% has been diagnosed as glycosuria
The patient of sick nephrosis will progress to end-stage renal disease in 20 years.
In order to make this criterion of albumin in urine, albumin in urine is indicated using protein secretion rate in the world
Discharge rate.Content < 20mg~30mg/24h of albumin in healthy human urine, when albumin in human urine content 20~
When 30mg/24h, referred to as microalbuminuria；It is referred to as a large amount of white when albumin content is more than 300mg/24h in human urine
Albuminuria.Microdose urine protein is an early signal of nephrosis, the change and serum urea nitrogen, creatinine much higher than routine urinalysis
It increases.If not paying attention to the detection of microdose urine protein, the lasting regular period is urinated in microdose urine protein and is constantly promoted, is urinated
Conventional determining will appear protein positive, and the pathological lesion of kidneys of patients is irreversible at this time.Only Microalbunin is urinated in detection
The white stage is treated, and positive mode is taken to be intervened, and is just avoided that the generation of nephrosis, so for diabetic,
The detection of microdose urine protein has very important significance.
The method of common detection microdose urine protein domestic at present has: immunoturbidimetry, colloidal gold method, Enzyme-linked Immunosorbent Assay
Method, board-like chemoluminescence method and Magnetism particulate immuno chemistry luminescence method.It is now immunoturbidimetry using more method.Immunoturbidimetry and glue
Body gold method have the shortcomings that it is obvious, due to the limitation of method, detection inaccuracy in the low concentration range, albumin in urine
Content, which must reach a certain concentration (>=30mg/L), just can guarantee its accuracy.Enzyme linked immunosorbent assay, board-like chemoluminescence method
With the methods of Magnetism particulate immuno chemistry luminescence method there is also apparent defect, it is required to immobilized antibody, in entire detection process
It needs to wash, a large amount of medical waste liquids can be generated, and due to being heterogeneous reaction, detection process is cumbersome, and detection time is long.
Summary of the invention
In consideration of it, it is necessary to, repetitions long, complicated for operation for detection time in existing urine microalbumin detection method
Property and poor reproducibility, medical waste liquid yield it is big, be unsuitable for emergency treatment and clinical patient diagnoses in time the defects of needs, provide one
The kit and detection method of kind detection microdose urine protein content.
The present invention is achieved by the following technical solutions:
A kind of detection kit of complete homogeneous determination microdose urine protein, including reagent R1, reagent R2, reagent R3, examination
Agent R4, microdose urine protein calibration object and quality-control product.
Further, the reagent R1 includes following components:
No. 1 antibody of anti-human serum albumin of horseradish peroxidase-labeled, concentration are 0.5~3 μ g/mL；Buffer is 5
The MES solution of~20mM, pH are 6.0~7.0, the CaCl of bovine serum albumin(BSA), 5~10mM containing 0.5~1wt%2, 50~
The MgSO of 100mM4, the enzyme stabilizers of 0.01~0.05wt%, 0.5~1% NaCl, 0.05~0.2% KCl, 0.5~
The Krovin300M of the mannitol of 1wt%, the PEG20000 of 0.5~2wt%, 0.01~0.1wt%.
Further, include following final concentration component in the reagent R2:
No. 2 antibody of anti-human serum albumin of Acridine derivatives label, concentration are 1~5 μ g/mL；The buffer is 5
The PBS solution of~50mM, pH are 7.0~8.0, contain 0.5~1wt% bovine serum albumin(BSA), 0.5%~1wt%
The Krovin 300M of PEG20000 and 0.01~0.1wt%.
Further, include following final concentration component in the reagent R3:
The buffer of reagent be 5~50mM MES, pH value be 5.5~6.5, containing 0.001~0.02% vitamin C,
0.0001~0.0075% glutathione and 0.05~0.01% sodium thiosulfate.
It further, include urea peroxide 5~20% in the reagent R4, the buffer of reagent is the Tris- of 5~30mM
HCl solution, pH is 7.5~8.5.
Wherein, the microdose urine protein calibration object and quality-control product are made of humanized's seralbumin and matrix；It is described
Calibration object is mixed with quality-control product matrix by urine and buffer with 1:19；The PBS solution that the buffering is 10~50mM,
In containing concentration be 0.5~1% BSA, 0.05~0.1% Krovin 300M, human urine matrix 5%；When detection, work school
Quasi- product be include one group of microdose urine protein that humanized's microdose urine protein concentration is respectively 0,0.2,1,5,10,20 μ g/mL
Calibration object.
Further, in the R1 reagent, No. 1 antibody of anti-human serum albumin of horseradish peroxidase-labeled is to pass through
Coupling reagent obtains No. 1 antibody coupling of horseradish peroxidase and anti-human serum albumin；In the R2 reagent, acridine spreads out
No. 2 antibody of anti-human serum albumin of biomarker are by Acridine derivatives and anti-human serum albumin 2 through overactivation
Number antibody coupling obtains.
Further, the coupling reagent is respectively selected from succinimide -4- (N- maleimide) thiacyclohexane -1-1 hydroxyl
Acid esters, N- succinimido-S- acetylthioacetate, 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimine hydrochloric acid
It is salt, n-hydroxysuccinimide, one or more in glutaraldehyde.
Further, the Acridine derivatives through overactivation include N- succinimide -9,10- acridan ester,
One of N- succinimide -9,10- acridan thioesters, N- succinimide -9,10- acridan sulfanilamide (SN).
The preparation method of kit of the invention, includes the following steps:
S1: the preparation of reagent R1
S11: the preparation of No. 1 antibody of anti-human serum albumin with horseradish peroxidase label:
Horseradish peroxidase is activated using succinimide -4- (N- maleimide) thiacyclohexane -1-1 carboxylic esters, it is raw
At maleimide-horseradish peroxidase derivative；It is activated using N- succinimido-S- acetylthioacetate anti-
Body generates N- succinimide-antibody derivatives of anti-human serum albumin 1；The two is mixed, N- succinimide-
- SH in No. 1 antibody derivatives of anti-human serum albumin, will be single in conjunction with maleimide-horseradish peroxidase derivative
Clonal antibody A and horseradish peroxidase connect to obtain horseradish peroxidase-labeled anti-human serum albumin 1 are anti-
Body.
S12: reagent R1 buffer preparation
By 10g sodium chloride, 1g potassium chloride, 10g magnesium sulfate, 0.2g enzyme stabilizers, 5g PEG20000,1g Krovin
300M, 1g calcium chloride, 2g MES, 5g mannitol are dissolved in 900mL ultrapure water, adjust pH to 6.0~7.0 with NaOH, with super
Pure water is settled to 1000mL.
S13: by No. 1 antibody of anti-human serum albumin of horseradish peroxidase-labeled made from step (1) and step (2)
Buffer mixing obtained, obtains reagent R1.
S2: the preparation of reagent R2
S21: the preparation of No. 2 antibody of anti-human serum albumin with Acridine derivatives label:
Acridine derivatives (such as: N- succinimide -9,10- acridan ester) through overactivation are white with anti-human serum
No. 2 antibody of albumen mix, and the activated group-NHS in the Acridine derivatives through overactivation can be with primary in monoclonal antibody
Amine (- NH2) is connected, and forms No. 2 antibody of anti-human serum albumin of Acridine derivatives label.
S22: reagent R2 buffer preparation
By 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 5g bovine serum albumin(BSA),
5g PEG20000 and 1g Krovin 300M is dissolved in 900mL ultrapure water, is adjusted pH to 7.0~8.0, is settled to 1000mL.
S23: No. 2 antibody of anti-human serum albumin of the label of Acridine derivatives made from step (1) and step (2) are made
The buffer mixing obtained, obtains reagent R2.
S3: the preparation of reagent R3
By 0.2g vitamin C, 0.05g glutathione, 0.2g sodium thiosulfate, 1g MES are dissolved in 900mL ultrapure water,
PH to 5.5~6.5 is adjusted, is settled to 1000mL, 2~8 DEG C save backup.
S4: the preparation of reagent R4
50~200g urea peroxide, 1~3g Tris are dissolved in 800mL ultrapure water, pH to 7.5~8.5 is adjusted, it is fixed
Hold to 1000mL, 2~8 DEG C save backup.
S5: the preparation of calibration object
S51: calibration object dilution preparation
By 8g sodium chloride, 0.2g potassium chloride, 4.32g disodium hydrogen phosphate, 0.72g potassium dihydrogen phosphate, 5g bovine serum albumin(BSA)
It is dissolved in 900mL ultrapure water with 1g Krovin 300M, adjusts pH to 7.0~8.0, be settled to 1000mL.
S52: the preparation of working calibration product
Urine sample is collected according to correct way, after 2~8 DEG C of 2~3h of placement, after urine sample centrifugal filtration, adds source of people thereto
Property seralbumin, detects it with other contrast agent boxes, to its preliminary definite value；It is according to its definite value as a result, micro- with urinating
Urine sample of the amount albumin content lower than 1 μ g/mL is diluted.4 μ g/mL, 20 μ g/mL, 100 μ g/mL, 200 μ g/ are prepared respectively
The intermediate calibration object of mL and 400 μ g/mL obtain 0 μ g/L, 0.2 μ g/mL, 1 μ g/mL, 5 μ with 20 times of calibration object diluted
G/mL, 10 μ g/mL, 20 μ g/mL working calibration product.
Kit semi-finished product are prepared according to above-mentioned formula, can just be assembled into microdose urine protein after verifying is qualified
Detection kit.
A kind of detection method of complete homogeneous determination microdose urine protein, step include:
(1), the peak area of sample and calibration object is obtained respectively；
(2), using the microdose urine protein concentration of calibration object as X-coordinate, using peak area as Y-coordinate, it is anti-to make dosage-
Curve is answered, the concentration of microdose urine protein in sample is calculated according to the curve.
Further, step (1) operation are as follows:
A, then calibration object/sample to be examined is added in the reagent adding R1 in reaction cup, reagent R2 is then added, in 37 DEG C of items
15min is incubated under part；
B, reagent R3 is added after being incubated for 15min, R4 is added after mixing；Immediately continuous detection a period of time (usually 1~
3S), every minor tick 0.05S, calculates its peak area.
Further, sample/calibration object in the step (1), reagent R1, reagent R2, reagent R3, reagent R4 ratio be
1:8:8:1:15
The invention has the advantages that:
The present invention is intended to provide a kind of detection kit and its method of complete homogeneous determination microdose urine protein, the reagent
Box is white using Acridine derivatives label No. 1 antibody of anti-human serum albumin, antigen, horseradish peroxidase-labeled anti-human serum
No. 2 antibody of albumen form antibody-antigen-antibody compound, are not necessarily to washing process, after reagent R3 standing 0.5min is added, are added
Continuously detection a period of time (usually 1~3S), every minor tick 0.05S calculate its peak area to R4 immediately, microdose urine protein
Content is positively correlated with its peak area.Its testing principle is that the hydrogen peroxide in horseradish peroxidase oxidising agent R2 can produce
Free radical, free radical aoxidizes the Acridine derivatives on anti-human microdose urine protein antibody immediately, due to the half-life period of free radical
It is very short, it middle in the molecule can only spread, therefore only forming antibody-antigen-antibody compound could react reflective.
Innovation of the invention is:
1, the kit is combined using homogeneous detection technique with chemiluminescence, using single step reaction mode, makes to examine
Performance (such as: precision, sensitivity, accuracy) is surveyed to greatly improve.
2, the kit is different from traditional detection based on double antibodies sandwich technology, and traditional urine based on double antibodies sandwich technology is micro-
It measures albumin detection method (such as ELISA, board-like chemiluminescence, magnetic microparticle chemiluminescence), needs to wash in this detection process
It washs, but the entire reaction process of the kit does not need to be washed, the medical waste water that entire detection process can be made to generate
Greatly reduce；
3, the kit can be such that the entire reaction process time greatly shortens due to the uniqueness of its testing principle, and same
The kit of type compares, and the reaction time can shorten to 15min or so, and other kit reaction time 20min with
On, it can greatly improve in the unit time, the detection flux of microdose urine protein.
Detailed description of the invention
Fig. 1 is to the canonical plotting in kit of the present invention.
Fig. 2 is the comparative diagram of kit of the present invention Yu commercial reagent box A clinical assays result.
Specific embodiment
Technical solution problem to be solved, the technical solution of use and reach beneficial in order to better illustrate the present invention
Effect is further described now in conjunction with specific embodiment.It is worth noting that technical solution of the present invention is including but not limited to following
Embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
Embodiment
1, sample collection
Urine sample, after the urine sample of collection is placed at room temperature for 2h, centrifuging and taking supernatant are collected using the clinical correct method for collecting urine sample
It is detected.Urine sample after centrifugation need to be detected in 4h, if sample cannot detect completion in 4h, need to be set sample
It is saved in 2~8 DEG C；If you need to save for a long time, needs to be frozen to -20 DEG C, avoid multigelation.Room is returned to before use
Temperature gently shakes mixing.
2, experimental method
Prepared reagent is placed in detecting instrument, calibration object, quality-control product and urine sample are placed in instrument sample rack
On, prepare detection.
3, instrument parameter is arranged
This reagent according to external diagnosis reagent analyze Performance Evaluation guideline, performance evaluation is carried out to it, can reach as
Lower index:
Standard curve is linear: R >=0.9900 (as shown in Figure 1)；
Most bottom detection limit :≤0.1 μ g/mL.
To sample duplicate measurements 20 times of 0 μ g/mL of this kit, detection data is shown in Table 1, it is known that examines at the most bottom of this kit
Survey≤0.1 μ g/mL of limit.
The most bottom of table 1. detection limit testing result
For the accuracy for assessing detection reagent, 2 clinical samples are taken at random, and cooperation is added recovery experiment.Due to urine
Microalbumin takes standard substance GBW (E) 090619 to be diluted to 50mg/L without national standard and international standard substance, adds body
Product is the 5% of original volume, is added recovery experiment.The results are shown in Table 2, as can be seen from the results, TIANZHU XINGNAO Capsul be 90%~
110%.
Table 2. adds recovery experiment result (accuracy validation)
With commercialization Quality Control, batch interior essence of the kit is evaluated in the microalbumin height Quality Control of DAKO company urine production
Density, determination data is as shown in table 3, by measurement result it is found that the reagent and withinrun precision≤5% of the invention.
The evaluation of 3. withinrun precision of table
With commercialization Quality Control, 3 batches of the kit are evaluated in the microalbumin height Quality Control of DAKO company urine production
Betweenrun precision, determination data is as shown in table 4, by measurement result it is found that the reagent and betweenrun precision≤7% of the invention.
The evaluation of 4. betweenrun precision of table
Clinical Urinary sample is taken at random, measuring its albumin content is 15.7 μ g/mL, and it is pure to add human serum albumins thereto
Substance prepares high level urine sample, and theoretical concentration is respectively 1000 μ g/mL (sample 1), 2000 μ g/mL (sample 2), 4000 μ g/mL
(sample 3) is diluted evaluation, as a result such as table 5.The result shows that the kit maximum dilution multiple is more than 60 times.
The evaluation of 5. maximum dilution multiple of table
120 parts of Clinical Urinary samples and commercially available microdose urine protein detection kit A are compared, reagent more of the present invention and with
The correlation of commercially available microdose urine protein reagent and testing result.The results are shown in Table 6, and reagent of the invention is shown in attached drawing 2
The linear equation that box is compareed with commercially available microdose urine protein detection kit A are as follows: Y=1.00416X-1.00951, R=
0.996383。
6. kits of table are compareed with commercially available microdose urine protein detection kit A
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of detection kit of complete homogeneous determination microdose urine protein, which is characterized in that the kit includes reagent
R1, reagent R2, reagent R3, reagent R4, microdose urine protein calibration object and quality-control product；The reagent R1 includes horseradish peroxidase
0.5~3 μ g/mL of anti-human serum albumin monoclonal antibody of enzyme label；The reagent R2 includes what Acridine derivatives marked
1~5 μ g/mL of anti-human serum albumin monoclonal antibody；The reagent R4 includes urea peroxide 5~20%.
2. detection kit according to claim 1, which is characterized in that further include buffer in the reagent R1；It is described
Buffer be 5~20mM MES solution, pH be 6.0~7.0, wherein the bovine serum albumin(BSA) containing 0.5~1wt%, 5~
The CaCl of 10mM2, 50~100mM MgSO4, 0.01~0.05wt% enzyme stabilizers, 0.5~1% NaCl, 0.05~
0.2% KCl, the mannitol of 0.5~1wt%, the PEG20000 of 0.5~2wt%, 0.01~0.1wt%Krovin 300M.
3. detection kit according to claim 1, which is characterized in that further include buffer in the reagent R2；It is described
Buffer be 5~50mM PBS solution, pH be 7.0~8.0, wherein containing 0.5~1wt% bovine serum albumin(BSA), 0.5%~
PEG20000 and 0.01~0.1wt%Krovin 300M of 1wt%.
4. detection kit according to claim 1, which is characterized in that the MES in the reagent R3 including 5~50mM is slow
Fliud flushing, pH value are 5.5~6.5；In the buffer containing 0.001~0.02% vitamin C, 0.0001~0.0075%
Glutathione and 0.05~0.01% sodium thiosulfate.
5. detection kit according to claim 1, which is characterized in that further include buffer in the reagent R4；It is described
Buffer is the Tris-HCl solution of 5~30mM, and pH is 7.5~8.5.
6. detection kit according to claim 1, which is characterized in that the calibration object and quality-control product are by humanized's serum
Albumin and matrix composition；The calibration object is mixed with quality-control product matrix by urine and buffer with 1:19；The buffering
For the PBS solution of 10~50mM, wherein being 0.5~1% BSA, 0.05~0.1% Krovin 300M, human urine containing concentration
Matrix 5%.
7. detection kit according to claim 1, which is characterized in that the horseradish peroxidase mark in the R1 reagent
The anti-human serum albumin antibody of note is to be resisted horseradish peroxidase and anti-human serum albumin monoclonal by coupling reagent
Body coupling obtains；The anti-human serum albumin monoclonal antibody of Acridine derivatives label in the R2 reagent is to pass through coupling
Reagent obtains the Acridine derivatives through overactivation with the coupling of anti-human serum albumin monoclonal antibody.
8. detection kit according to claim 7, which is characterized in that the Acridine derivatives through overactivation include
N- succinimide -9,10- acridan ester, N- succinimide -9,10- acridan thioesters, succinimide -9 N-,
One of 10- acridan sulfanilamide (SN).
9. the preparation method of detection kit described in claim 1-8 any one, which is characterized in that comprising steps of
S1: the preparation of reagent R1: being made the anti-human serum albumin antibody and buffer of horseradish peroxidase-labeled respectively, will
The two mixing；
S2: the anti-human serum albumin monoclonal antibody and buffering of Acridine derivatives label the preparation of reagent R2: are made respectively
Liquid mixes the two；
S3: the preparation of reagent R3: vitamin C, glutathione, sodium thiosulfate, MES are dissolved in ultrapure water, adjust pH to
5.5~6.5, it is settled to 1000mL, 2~8 DEG C save backup；
S4: the preparation of reagent R4: urea peroxide, Tris-HCl are dissolved in ultrapure water, are adjusted pH to 7.5~8.5, are settled to
1000mL, 2~8 DEG C save backup；
S5: the preparation of calibration object.
10. a kind of detection method of complete homogeneous determination microdose urine protein, which is characterized in that comprising steps of
(1), the peak area of sample and calibration object is obtained respectively；
(2), using the microdose urine protein concentration of calibration object as X-coordinate, using peak area as Y-coordinate, dose-response song is made
Line calculates the concentration of microdose urine protein in sample according to the curve.
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| CN102333886A (en) * | 2009-02-27 | 2012-01-25 | 贝克曼考尔特公司 | Solution phase homogeneous assays |
| CN108196043A (en) * | 2017-11-28 | 2018-06-22 | 泰州泽成生物技术有限公司 | Kit of microdose urine protein content and preparation method thereof in a kind of detection serum |
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2018
- 2018-12-21 CN CN201811575007.0A patent/CN109613260A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102333886A (en) * | 2009-02-27 | 2012-01-25 | 贝克曼考尔特公司 | Solution phase homogeneous assays |
| CN108196043A (en) * | 2017-11-28 | 2018-06-22 | 泰州泽成生物技术有限公司 | Kit of microdose urine protein content and preparation method thereof in a kind of detection serum |
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Application publication date: 20190412 |