CN103675299A - Kit and method for detecting concentration of fibronectin in urine - Google Patents
Kit and method for detecting concentration of fibronectin in urine Download PDFInfo
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- CN103675299A CN103675299A CN201310724601.2A CN201310724601A CN103675299A CN 103675299 A CN103675299 A CN 103675299A CN 201310724601 A CN201310724601 A CN 201310724601A CN 103675299 A CN103675299 A CN 103675299A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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Abstract
The invention provides a kit for detecting the concentration of fibronectin in urine, which comprises two reagents. A reagent I comprises phosphate buffer; a reagent II is suspension formed by cross-linking a rabbit anti-human fibronectin antiserum, a goat anti-human fibronectin antiserum, a rat anti-human fibronectin antiserum or a rat anti-human fibronectin monoclonal antibody on latex particles. The invention solves the problems of complex steps, low accuracy and poor repeatability when a radial immunodiffusion method and an ELISA (Enzyme Linked Immunosorbent Assay) double-antibody sandwich technology are adopted to measure the concentration of the fibronectin in the prior art. The invention also provides a method for detecting the concentration of the fibronectin in human urine by adopting the kit. The kit and the method which are provided by the invention are used for measuring the concentration of the fibronectin in the urine, adopt simple steps, have high accuracy and good repeatability and are used for an automatic analysis meter.
Description
Technical field
The invention belongs to bioengineering field, relate in particular to a kind of detection kit, particularly a kind of employing strengthens latex immunoturbidimetry and detects kit and the method thereof that fibronectin in human urine claims again fibronectin (FN) concentration.
Background technology
Fibronectin claims again fibronectin (Fibronectin is called for short FN), two similar polypeptied chain subunits, consists of, and the molecular weight of subunit is 220-250KD.Available technology adopting SRID and ELISA double-antibody sandwich technology are measured fibronectin (FN) concentration in urine, and its shortcoming is complex operation, working strength is large, the retention time is long, generally need 8-48 hour, its quantitative degree of accuracy poor.
Because the concentration of fibronectin in urine (FN) is low, so the report of so far there are no both at home and abroad useful kit and method.
Summary of the invention
For the above-mentioned defect existing in prior art, technical matters to be solved by this invention is to provide a kind of kit and method thereof for detection of fibronectin concentration in urine, the described this kit for detection of fibronectin concentration in urine and method thereof will solve available technology adopting SRID and ELISA double-antibody sandwich technology is measured Urinary fibronectin (FN) concentration process complexity, the technical matters that accuracy is not high.
The invention provides a kind of kit for detection of fibronectin concentration in urine, comprise two kinds of reagent, I reagent is by sodium hydrogen phosphate, potassium dihydrogen phosphate, PEG6000-8000, disodium ethylene diamine tetraacetate (EDTA-NA
2) and Triton X-100 (TX-100) composition, in described I reagent, the mass percent concentration of described sodium hydrogen phosphate is 28.6g/L, the mass percent concentration of described potassium dihydrogen phosphate is 2.7g/L, the mass percent concentration of described PEG6000-8000 is 50g/L, described EDTA-NA
2mass percent concentration be 1g/L, the volumetric concentration of described TX-100 is 0.2ml/L, and II reagent is that the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or mouse-anti people fibronectin monoclonal antibody are cross-linked the suspension forming on latex particle.
Further, described II reagent is to utilize carbodiimide reaction that the mouse-anti people fibronectin monoclonal antibody IgG after the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or purifying is covalently bound on latex particle.
Concrete, the diameter of latex particle is 60-160nm, material is polystyrene.
Concrete, the purity of above-mentioned Triton X-100 is 100%.
Further, the anti-human fibronectin antiserum of described rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin is sero-fast tires as 1:64.
The present invention also provides the method for fibronectin (FN) concentration in a kind of detection human urine, adopt above-mentioned kit, comprise a step that adopts Biochemical Analyzer to measure urine sample, at described one, urine sample is adopted in the step that Biochemical Analyzer measures, urine samples is added after the first reagent R1, hatch 5min for 37 ℃, read to survey the 1st sample A1, add the second reagent R2, hatch 5min for 37 ℃, read to survey the 2nd sample A2, sample A=sample A2-sample A1; Also comprise a step that adopts Biochemical Analyzer to measure urine calibration object, in a described step that urine calibration employing Biochemical Analyzer is measured, urine calibration object is added after the first reagent R1, hatch 5min for 37 ℃, read to survey the 1st calibration A1, add the second reagent R2, hatch 5min for 37 ℃, read to survey the 2nd calibration A2, calibration A=calibration A2-calibration A1;
Result is calculated:
The parameter of said determination is (strengthening latex immunoturbidimetry): 37 ℃ of temperature, predominant wavelength 546nm or 600nm, commplementary wave length 700nm, urine sample or calibration object 3-4 μ l, R
1: 240 μ l, R
2: 60 μ l, the reaction time is 10 minutes.
The anti-human fibronectin antiserum of rabbit of the present invention or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or mouse-anti people fibronectin monoclonal antibody can be bought in market, also can prepare by conventional immunization method, chemical reagent is bought by market.
The present invention is at the surface-crosslinked monoclonal of polymer latex microballoon or polyclone fibronectin list (FN) antibody, after the crosslinked microballoon that has antibody is combined with antigen, can flock together rapidly at short notice, increased and in solution, reacted turbidity, be that penetrability reduces, absorbance increases the concentration of fibronectin list (FN) in reaction human urine.
Kit of the present invention and method are to adopt the measuring principle that strengthens latex immunoturbidimetry, utilize fibronectin (FN) antigen and specific antibody (the anti-human fibronectin of rabbit (FN) antiserum) to combine, form insolubilized immune complexes, reactant liquor is produced muddy, its turbidity height, be the concentration that penetrability reduces, absorbance increases fibronectin (FN) in reflection human urine sample, the dose-effect curve that the concentration of Urinary fibronectin (FN) can be done by calibration object is calculated.
Adopt the concentration of fibronectin (FN) in the urine that kit of the present invention and method record to can be used as scientific research and teaching and clinical use.
The present invention compares with prior art, and its technical progress is significant.It is simple and convenient that kit of the present invention and method are used for measuring the process of urine fibronectin (FN) concentration, and accuracy is high.Meanwhile, the present invention can also be for the tumour early screening of urinary system.
Accompanying drawing explanation
Fig. 1 is by urine fibronectin (FN) concentration done of calibration object and the dose-effect curve figure of absorbance.
Embodiment
Embodiment 1
The invention provides a kind of kit for detection of fibronectin concentration in urine, comprise two kinds of reagent, I reagent is by sodium hydrogen phosphate, potassium dihydrogen phosphate, PEG6000-8000, disodium ethylene diamine tetraacetate (EDTA-NA
2) and Triton X-100 (TX-100) composition, in described I reagent, the mass percent concentration of described sodium hydrogen phosphate is 28.6g/L, and the mass percent concentration of described potassium dihydrogen phosphate is 2.7g/L, the mass percent concentration of described PEG6000 is 50g/L, described EDTA-NA
2mass percent concentration be 1g/L, the volumetric concentration of described TX-100 is 0.2ml/L, and II reagent is that the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or mouse-anti people fibronectin monoclonal antibody are cross-linked the suspension forming on latex particle.
Further, described II reagent is to utilize carbodiimide reaction that the mouse-anti people fibronectin monoclonal antibody IgG after the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or purifying is covalently bound on latex particle.
Concrete, the diameter of latex particle is 60-160nm, material is polystyrene.
Concrete, the purity of above-mentioned Triton X-100 is 100%.
Further, the anti-human fibronectin antiserum of described rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin is sero-fast tires as 1:64.
Embodiment 2
2.1 applicable instruments
Semi-automatic, automatic clinical chemistry analyzer.
2.2 analytical approach
Immunity turbidimetry.
2.3 performance requirement
2.3.1 reagent outward appearance
R1: achromaticity and clarification transparency liquid;
R2: milky suspension.
2.3.2 reagent blank absorbance (A)
Absorbance (A): R
1+ R
2≤ 2600A(is 37 ℃ of temperature; Predominant wavelength 546nm, 600nm(commplementary wave length 700nm)).
2.3.3 precision
2.3.3.1 withinrun precision
CV≤10%。
2.3.4 betweenrun precision
Relative extreme difference≤10%.
2.3.5 accuracy
Inaccuracy: in ± 10% scope.
2.3.6 sensitivity for analysis
Absorbance (A) >2600A.
2.3.7 linear
Within the scope of 20mg/L-100mg/L, related coefficient (γ) >=0.9900.
2.3.8 stability
Reagent is stored to effective former and later two months in the end of term 2 ℃ of-8 ℃ of lucifuges, detects, and its quality meets the regulation of item.
Embodiment 3 experimental techniques
3.1 testing conditions
3.1.1 Biochemical Analyzer (hereinafter to be referred as instrument)
Hitachi's 7060 automatic biochemistry analyzers.
3.1.2 operating ambient temperature
Room temperature 15-32 ℃.
Indoor humidity 45-85%RH.
3.1.3 main location parameter and operation steps
37 ℃ of temperature; Predominant wavelength 600nm(commplementary wave length 700nm), urine sample or urine calibration object 3 μ l; R
1: 240 μ l; R
2: 60 μ l, 10 minutes reaction time.
Method type: 2 end-point methods.
After urine sample or urine calibration object reagent adding R1, hatch 5min for 37 ℃, read to survey the 1st point (A1), add reagent R2, hatch 5min for 37 ℃, read to survey the 2nd point (A2), sample A=A2-A1.
Result is calculated:
3.1.4 calibration object
In this standard, calibration object used is the standard items that our company produces.
3.2 reagent outward appearances
Visual observation agent box under light, R1: achromaticity and clarification transparency liquid; R2: suspension.
3.3 reagent blank absorbances
Get R
1240 μ l, adding distil water 3 μ l, put 37 ℃ and hatch after 5min, add R
260 μ l, put 37 ℃ and hatch after 5min, on 7060 automatic biochemistry analyzers, use 600nm wavelength, measure the regulation that its absorbance should meet 1.3.2 item in embodiment 1.
3.4 precision
3.4.1 withinrun precision test method
Under instrument normal running conditions, to use with a collection of reagent, follow-on test (about 100mg/L) sample 20 times, calculates the mean value of its measured value
and standard deviation (SD), then calculate as follows the value of the coefficient of variation (CV%), its result should meet the regulation of 1.3.3.1 item in embodiment 1.
n
SD={∑（Xi-X）
2/（N-1）}
1/2
I=1
In formula: SD-standard deviation;
CV-coefficient of variation;
The measured value that Xi-the is i time;
N-measurement number of times.
3.4.2 betweenrun precision test method
Under instrument normal running conditions, get three lot number reagent, each lot number is got a set of.Measure respectively (about 200mg/L) sample each 3 times.Then calculate the average of every batch of measurement result
grand mean with three batches of reagent measurement results
the relative extreme difference (%) of obtaining the mensuration average of three lot number reagent according to following formula, its result should meet the regulation of 1.3.4 item in embodiment 1.
In formula:
for
middle maximal value,
for
middle minimum value.
3.5 accuracy
Under instrument normal running conditions, the standard items that reagent is produced with our company, after calibration, are measured the sample of concentration known, replication 10 times, and the relative deviation of the average of its measurement result should meet the regulation of 1.3.5 item in embodiment 1.
RE%=absolute deviation/TV * 100%
Absolute deviation=testing result average-standard items target value
In formula: the target value of TV-bioassay standard product
3.6 sensitivity for analysis
R
1reagent 240 μ l, add 20mg/L sample 3 μ l, put 37 ℃ and hatch after 5min, add R
260 μ l, put 37 ℃ and hatch after 5min, and on 7060 automatic biochemistry analyzers, with 600nm, predominant wavelength (commplementary wave length 700nm), surveys absorbance, and its result should meet the regulation of 1.3.6 item in embodiment 1.
3.7 linear test
Sample or calibration object: select by 1 of embodiment 3 preparations
#, 2
#, 3
#, 4
#, and 5
#each test concentrations sample or calibration object.
Determination step: under instrument normal running conditions, after calibration, measure above five concentration samples or calibration object with reagent, each concentration samples or calibration object are measured respectively 3 times, and getting average is measured value Yi.
Calculate: calculate as follows related coefficient γ, its result should meet the regulation of 1.3.7 item in embodiment 1.
In formula: γ-related coefficient
Xi-i
#the theoretical value of sample
Yi-i
#the measured value of sample
The numbering of i-variable concentrations test sample book
5.8 stability
Be taken at and under regulation storage requirement, be saved to the reagent in two months before and after effective end of term and detect, its result should meet the regulation of 1.3.8 item in embodiment 1.
The preparation of embodiment 4 reagent
Getting the urine sample that a content is about 100mg/L, is sample 5
#, by sample 5
#according to the form below method is mixed with 5 test sample books with physiological saline or pure water.(this sample matching while using)
| ? | 1 # | 2 # | 3 # | 4 # | 5 # |
| Calibration object | — | 2μl | 4μl | 6μl | 3μl |
| Physiological saline | 3μl | 8μl | 6μl | 4μl | — |
| Sampling amount after dilution | — | 3μl | 3μl | 3μl | — |
| Concentration (mg/L) | 0 | 20 | 40 | 60 | 100 |
After sample or calibration object reagent adding R1, hatch 5min for 37 ℃, read to survey the 1st point (A1), add reagent R2, hatch 5min for 37 ℃, read to survey the 2nd point (A2), sample A=A2-A1.
Result is calculated:
By the mensuration of above-mentioned sample, obtained the dose-effect curve figure of (FN) concentration of the Urine fibronectin shown in Fig. 1 and absorbance.(Fig. 1 is the curve of doing according to the OD value of predominant wavelength 600nm, the use only for reference of commplementary wave length 700nm.）
Pass through Fig. 1, measured 100 routine normal persons' urine, fibronectin (FN) normal reference value in urine: below 20mg/L, illustrate that the concentration of fibronectin (FN) in the urine that adopts kit of the present invention and method mensuration is still accurately with reliably.
Fibronectin (FN) concentration (mg/L) in 100 routine normal person's human urines
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Numerical value | 6mg | 2mg | 3mg | 11mg | 7mg | 8mg | 2mg | 1mg | 5mg | 12mg |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Numerical value | 13mg | 7mg | 4mg | 5mg | 12mg | 16mg | 3mg | 5mg | 12mg | 14mg |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Numerical value | 19mg | 15mg | 11mg | 15mg | 17mg | 16mg | 12mg | 9mg | 8mg | 13mg |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| Numerical value | 12mg | 7mg | 5mg | 6mg | 11mg | 12mg | 17mg | 19mg | 15mg | 15mg |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| Numerical value | 7mg | 8mg | 3mg | 8mg | 7mg | 9mg | 11mg | 13mg | 4mg | 7mg |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| Numerical value | 15mg | 6mg | 8mg | 11mg | 12mg | 15mg | 8mg | 7mg | 11mg | 12mg |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| Numerical value | 4mg | 3mg | 6mg | 7mg | 9mg | 13mg | 15mg | 11mg | 8mg | 9mg |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| Numerical value | 6mg | 5mg | 12mg | 8mg | 11mg | 9mg | 15mg | 10mg | 8mg | 9mg |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| Numerical value | 13mg | 12mg | 9mg | 5mg | 4mg | 6mg | 8mg | 11mg | 13mg | 9mg |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| Numerical value | 15mg | 11mg | 7mg | 8mg | 11mg | 6mg | 15mg | 13mg | 12mg | 7mg |
Claims (4)
1. for detection of a kit for fibronectin concentration in urine, comprise two kinds of reagent, it is characterized in that: I reagent is by sodium hydrogen phosphate, potassium dihydrogen phosphate, PEG6000-8000, EDTA-NA
2form with TX-100, in described I reagent, the mass percent concentration of described sodium hydrogen phosphate is 28.6g/L, and the mass percent concentration of described potassium dihydrogen phosphate is 2.7g/L, the mass percent concentration of described PEG6000-8000 is 50g/L, described EDTA-NA
2mass percent concentration be 1g/L, the volumetric concentration of described TX-100 is 0.2ml/L, and II reagent is that the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or mouse-anti people fibronectin monoclonal antibody are cross-linked the suspension forming on latex particle.
2. a kind of kit for detection of fibronectin concentration in urine as claimed in claim 1, is characterized in that: described II reagent is to utilize carbodiimide reaction that the mouse-anti people fibronectin monoclonal antibody IgG after the anti-human fibronectin antiserum of rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin antiserum or purifying is covalently bound on latex particle.
3. a kind of kit for detection of fibronectin concentration in urine as claimed in claim 1, is characterized in that: the anti-human fibronectin antiserum of described rabbit or goat-anti people fibronectin antiserum or mouse-anti people fibronectin is sero-fast tires as 1:64.
4. the kit of employing claim 1 detects the method for fibronectin concentration in urine, it is characterized in that: comprise a step that adopts Biochemical Analyzer to measure urine sample, at described one, urine sample is adopted in the step that Biochemical Analyzer measures, urine samples is added after the first reagent R1, hatch 5min for 37 ℃, read to survey the 1st sample A1, add the second reagent R2, hatch 5min for 37 ℃, read to survey the 2nd sample A2, sample A=sample A2-sample A1; Also comprise a step that adopts Biochemical Analyzer to measure calibration object, at described one, calibration object is adopted in the step that Biochemical Analyzer measures, calibration object is added after the first reagent R1, hatch 5min for 37 ℃, read to survey the 1st calibration A1, add the second reagent R2, hatch 5min for 37 ℃, read to survey the 2nd calibration A2, calibration A=calibration A2-calibration A1;
The parameter of said determination is: 37 ℃ of temperature, predominant wavelength 546nm or 600nm, commplementary wave length 700nm, sample or calibration object 3-4 μ l, R
1: 240 μ l, R
2: 60 μ l, the reaction time is 10 minutes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106370862A (en) * | 2016-08-30 | 2017-02-01 | 山东博科生物产业有限公司 | Stable and sensitive fibronectin detection reagent |
| CN106645694A (en) * | 2016-09-24 | 2017-05-10 | 济南博鑫生物技术有限公司 | Fn (fibronectin) detection kit |
| WO2018032125A1 (en) * | 2016-08-16 | 2018-02-22 | 刘中令 | Urine fibronectin concentration assay kit and method |
| CN108780095A (en) * | 2016-03-31 | 2018-11-09 | 积水医疗株式会社 | Utilize the cancer embryo fibronectin detection method of simple immunoassays |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108780095A (en) * | 2016-03-31 | 2018-11-09 | 积水医疗株式会社 | Utilize the cancer embryo fibronectin detection method of simple immunoassays |
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| CN106370862A (en) * | 2016-08-30 | 2017-02-01 | 山东博科生物产业有限公司 | Stable and sensitive fibronectin detection reagent |
| CN106370862B (en) * | 2016-08-30 | 2018-07-20 | 山东博科诊断科技有限公司 | A kind of stabilization, sensitive fibronectin detection reagent |
| CN106645694A (en) * | 2016-09-24 | 2017-05-10 | 济南博鑫生物技术有限公司 | Fn (fibronectin) detection kit |
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