CN102650638B - II Collagen Type VI catabolite detection kit and preparation method thereof in urine - Google Patents
II Collagen Type VI catabolite detection kit and preparation method thereof in urine Download PDFInfo
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Abstract
The invention discloses II Collagen Type VI catabolite detection kit and preparation method thereof in a kind of urine, this kit mainly includes: biotin labeled CTX-II antigen: the concentration of CTX-II antigen is 4.5-5.5 μ g/ml; CTX-II monoclonal antibody solution, this CTX-II monoclonal antibody titre is 1: 950-1: 1050; The amount ratio of described biotin labeled CTX-II antigen and CTX-II monoclonal antibody solution is 1: 1.In described urine, II Collagen Type VI catabolite detection kit has all extraordinary advantage of specificity, sensitivity and precision.
Description
Technical field
The invention belongs to medical instruments field, relate to II Collagen Type VI catabolite detection kit and preparation thereof in a kind of urine specifically.
Background technology
The damage of cartilage structure integrality is the major histological performance of osteoarthritis and rheumatoid arthritis.II collagen type is the main organic principle of cartilage.After cartilage degradation, the fragment (CTX-II) of II collagen type discharges into the circulation system, enters in urine subsequently.CTX-II fragment in urine quantitatively can be detected by II Collagen Type VI catabolite detection kit in urine.
It is reported, in urine, II Collagen Type VI catabolite detects the development and other clinical and preclinical study that contribute to predicting osteoarthritis.
Summary of the invention
An object of the present invention is to provide II Collagen Type VI catabolite detection kit in a kind of urine.
The technical scheme realizing above-mentioned purpose is as follows:
II Collagen Type VI catabolite detection kit in a kind of urine, mainly includes:
(A) biotin labeled CTX-II antigen: the concentration of CTX-II antigen is 4.5-5.5 μ g/ml;
(B) CTX-II monoclonal antibody solution, this CTX-II monoclonal antibody titre is 1: 950-1: 1050;
The amount ratio of described biotin labeled CTX-II antigen and CTX-II monoclonal antibody solution is 1: 1.
Further, II Collagen Type VI catabolite detection kit in described urine, also includes peroxidase coupled antibody and bag by the microwell plate of chain bacterium avidin; The work titre of described enzyme coupled antibody is 1: 4550-5050.
Preferably, the concentration of described CTX-II antigen is 5 μ g/ml, and the titre of described CTX-II monoclonal antibody is 1: 1000.
Another object of the present invention is to provide the preparation method of II Collagen Type VI catabolite detection kit in a kind of urine.
The technical scheme realizing above-mentioned purpose is:
A preparation method for II Collagen Type VI catabolite detection kit in urine, mainly comprises the following steps:
1) biotin labeled CTX-II antigen is prepared; Get CTX-II synthetic peptide 25mg, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes, and obtains antigenic dilution; Get the biotin that concentration is 1.25mg/mL, antigenic dilution: biotin=1: 4 volume ratio mixing, adopt simple and easy Over-voltage protection biotin and CTX-II synthetic peptide to be cross-linked; Get the biotin labeled CTX-II antigen after preparation, add antiseptic and stabilizing agent; The concentration of described CTX-II antigen is 4.5-5.5 μ g/ml;
2) prepare CTX-II monoclonal antibody solution: get CTX-II monoclonal antibody, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, adding haematochrome is developer, and described CTX-II monoclonal antibody titre is 1: 950-1: 1050.
Preferably, the concentration of described haematochrome is 0.03mg/ml.
Being prepared as of described CTX-II monoclonal antibody: get healthy male rabbit, adopt injected s. c, immunity inoculation is carried out to position, rabbit 58 place, the CTX-II antigen 0.2mL of initial immunity injection 1mg/mL concentration, the 3rd week, the 4th week and within the 5th week, inject CTX-II antigen and the incomplete Freund's adjuvant equal-volume mixed solution 0.4mL of 1mg/mL concentration; Draw neck to put to death rabbit, collect blood, get supernatant, add antiseptic and stabilizing agent after the centrifugal 30min of 3000r/min, 4 DEG C of sealings are preserved.
Described urine II Collagen Type VI catabolite detection kit also includes peroxidase coupled antibody, being prepared as of this peroxidase coupled antibody: get mouse-anti rabbit igg freeze-dried powder 0.1g, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes; Getting the dilution of HRP solid (horseradish peroxidase) deionized water is 5mg/mL, adopts simple and easy Over-voltage protection HRP and mouse-anti rabbit igg to be cross-linked; Get the mouse-anti rabbit igg after preparation, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, adding cyanine is developer, and the concentration of cyanine is 0.08mg/ml, then adds antiseptic and stabilizing agent.The work titre of described enzyme coupled antibody is 1: 4550-5050, is preferably 1: 5000.
Kit of the present invention adopts euzymelinked immunosorbent assay (ELISA), based on a kind of monoclonal antibody to urine II Collagen Type VI fragment, or to being incorporated into the competitive binding of biotin labeling synthetic peptide of micropore inside surface of streptavidin bag quilt.
First, biotin labeled synthetic peptide is incorporated into the microwell plate inside surface being coated with streptavidin.After cleaning, add standard items, quality-control product and urine sample, then add monoclonal antibody solution.Again clean, and add the rabbit against murine immunoglobulin (Ig) of peroxidase conjugate.Again after cleaning, add chromogenic substrate, react by sulfuric acid color development stopping and measure absorbance.
Urine II Collagen Type VI catabolite detection kit of the present invention has all extraordinary advantage of specificity, sensitivity and precision.
Embodiment
Embodiment 1
(1) in the urine, described in the present embodiment, the main composition of II Collagen Type VI catabolite detection kit is as follows:
1. the microwell plate of streptavidin bag quilt
The bar shaped microwell plate (12 × 8 hole) that streptavidin is pre-coated, is placed in framework.
2. urinate CTX-II standard items 0
The TRIS buffer solution containing protein stabilizing agent, detergent and antiseptic that can directly use, 1 bottle (at least 3.0mL).
3. urinate CTX-II standard items 1-5
The synthetic peptide that can directly use is dissolved in TRIS buffer solution, and the actual concentrations of synthetic peptide is marked on each bottle, 5 bottles (at least 0.4mL/ bottle).
4. urinate CTX-II quality-control product
The synthetic peptide that can directly use, 2 bottles (at least 0.4mL).
5. biotin labeled urine CTX-II antigen
The biotin labeling synthetic peptide that can directly use 1 bottle (at least 12.0mL).
6. an antibody-solutions
The monoclonal antibody (being dissolved in the TRIS buffer solution containing protein stabilizing agent, antiseptic and orchil) 1 bottle (at least 12.0mL) that can directly use.
7. peroxidase coupled antibody
The rabbit against murine immunoglobulin (Ig) (being dissolved in the TRIS buffer solution containing detergent, antiseptic and blue dyes) 1 bottle (at least 12.0mL) of the peroxidase conjugate that can directly use.
8. substrate solution
Tetramethyl benzidine (TMB) substrate (being dissolved in acid solution) that can directly use.1 bottle (at least 12.0mL).
9. stop buffer
The 0.18M sulfuric acid solution that can directly use.1 bottle (at least 12.0mL).
10. cleaning solution (50 ×)
Concentrated cleaning buffer solution containing detergent and antiseptic.1 bottle (at least 20.0mL).
11. sealed membranes
The adhesive membrane of microwell plate is sealed when hatching.
Being prepared as follows of described kit:
One) standard items and quality-control product
1) originate:
CTX-II synthetic peptide freeze-dried powder is purchased from Nordic Bioscience Diagnostic A/S company.
2) quality-control product preparation:
CTX-II synthetic peptide freeze-dried powder is mixed with the TRIS damping fluid of 10mmol/L, in quality-control product 1, CTX-II concentration is about 1.50 μ g/L, in quality-control product 2, CTX-II concentration is about 4.50 μ g/L, and the Bronidox 5L adding the BSA and 0.018% of 1% is as antiseptic and stabilizing agent, respectively get 0.4mL packing, 4 DEG C of sealings are preserved.
3) standard items preparation:
The TRIS damping fluid of 3mL concentration 10mmol/L got by standard items 0, adds the Bronidox 5L of the BSA and 0.018% of 1% as antiseptic and stabilizing agent, and 4 DEG C of sealings are preserved.
Standard items 1-5 gets CTX-II synthetic peptide freeze-dried powder, and being diluted to CTX-II concentration with the TRIS damping fluid of 10mmol/L is respectively 0.75 μ g/L, 1.25 μ g/L, 2.50 μ g/L, 5.00 μ g/L and 10.00 μ g/L.Respectively get 0.4mL packing, 4 DEG C of sealings are preserved.
Two) biotin labeled CTX-II antigen
Get CTX-II synthetic peptide 25mg, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes, and obtains antigenic dilution; Get the biotin that concentration is 1.25mg/mL, antigenic dilution: biotin=1: 4 volume ratio mixing, adopt simple and easy Over-voltage protection biotin and CTX-II synthetic peptide to be cross-linked; Get the biotin labeled CTX-II antigen after preparation, add the Bronidox 5L of the BSA and 0.018% of 1% as antiseptic and stabilizing agent, 4 DEG C of sealings are preserved, and described CTX-II antigen concentration is 5 μ g/ml.
Three) primary antibodie solution
1) antibody preparation:
Get healthy male rabbit, adopt injected s. c, immunity inoculation is carried out to position, rabbit 5-8 place, the CTX-II antigen 0.2mL of initial immunity injection 1mg/mL concentration, the 3rd week, the 4th week and within the 5th week, inject CTX-II antigen and the incomplete Freund's adjuvant equal-volume mixed solution 0.4mL of 1mg/mL concentration.
Draw neck to put to death rabbit, collect blood, get supernatant after the centrifugal 30min of 3000r/min, add the solution 5L of the Bronidox containing the BSA of 1%, the Tween 20 of 0.1% and 0.0075% as antiseptic and stabilizing agent, 4 DEG C of sealings are preserved.
2) primary antibodie solution preparation:
Get the CTX-II monoclonal antibody after preparation, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, add haematochrome as developer, its concentration is 0.03mg/ml, get 12mL packing, 4 DEG C of sealings are preserved, and described CTX-II monoclonal antibody titre is 1: 1000.
The selection of primary antibodie concentration: dilutability measures: the microwell plate first the biotin labeling antigen of 0.1ml being added pre-coated chain enzyme avidin, a series of different dilutability (1: 1000 is added after washing, 1: 2000,1: 3000,1: 4000,1: 5000,1: 6000) primary antibodie is hatched, the peroxidase coupled antibody adding 0.1ml after washing is hatched, and adds substrate colour developing, add stop buffer cessation reaction after washing.In enzyme mark tintmeter, measure different dilution primary antibodie OD value, select that primary antibodie dilutability of OD value >=1.0 to be the working concentration of primary antibodie.Finally determine that the best effort concentration of primary antibodie is 1: 1000.
Four) peroxidase coupled antibody
General mouse-anti rabbit igg freeze-dried powder is purchased from Abcam company of Britain.Get mouse-anti rabbit igg freeze-dried powder 0.1g, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes.Getting the dilution of HRP solid deionized water is 5mg/mL, adopts simple and easy Over-voltage protection HRP and mouse-anti rabbit igg to be cross-linked.Get the mouse-anti rabbit igg after preparation, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, add cyanine as developer, the concentration of cyanine is 0.08mg/ml, add again the BSA of 1%, the Tween20 of 0.1% and 0.0075% Bronidox 5L as antiseptic and stabilizing agent, get 12mL packing, 4 DEG C of sealings are preserved.The work titre of described enzyme coupled antibody is 1: 5000.
Five) other
The microwell plate of pre-coated chain bacterium avidin, substrate solution, stop buffer and washing fluid provide by other commercial companies.Embodiment 2: use the kit described in embodiment 1 to detect II Collagen Type VI catabolite in urine
Before using, by all solution equilibrias to room temperature (18-22 DEG C).Determine to test required microwell plate quantity.Two parallel holes established by each sample.In addition, often wheel experiment needs 14 holes for standard items and Quality Control altogether.The microwell plate of right quantity is placed on plastic frame.Untapped microwell plate is sealed in Fresco Bag together with drying agent.Specifically comprise the following steps:
1) preincubate
Add 100 μ L biotin labeled urine CTX-II antigen to each hole, with sealed membrane sealing, hatch 30 ± 5 minutes under room temperature (18-22 DEG C), do not shake.
2) clean
Cleaning buffer solution (50 ×) is with the dilution proportion of 1 volume concentration buffer solution+50 volume distilled water.Manual cleaning microwell plate 5 times.Use automatic washer, usually cleaning 5 times.Guarantee each manual or automatically after cleaning by clean for the solution in micropore.
3) once hatch
In suitable hole, add 40 μ L urinate CTX-II standard items (0-5), quality-control product or urine sample to be measured, then add 100 μ L mono-antibody-solutions.Seal microwell plate with sealed membrane, in refrigerator, (2-8 DEG C) hatches 21 ± 3 hours, does not shake.
4) clean
See the 2nd step.
5) secondary is hatched
100 μ L peroxidase coupled antibody solution are added to each hole.Seal microwell plate with sealed membrane, hatch 60 ± 5 minutes under room temperature (18-22 DEG C), do not shake.
6) clean
See the 2nd step.
7) hatch with chromogenic substrate solution
Add 100 μ L substrate solutions to each hole, with sealed membrane sealing, under room temperature (18-22 DEG C), lucifuge hatches 15 ± 2 minutes, does not shake.
8) color development stopping reaction
100 μ L stop baths are added to each hole.
9) absorbance is measured
Be the absorbance with reference to measuring under 450nm with 650nm in 2 hours.
Sensing range
If the absorbance of sample to be tested is lower than standard 5, suggestion standard items 0 diluted sample also reanalyses.
Quality control
[reference value (term of reference)]
Mean value and the standard deviation of all kinds of crowd are exemplified below.
[explanation of assay]
Result treatment
Typical curve is built, by the concentration of urinating CTX-II in interpolation determination Quality Control and clinical samples with four parameter logistic curve fit.
Result is illustrated:
Correct with creatine concentration
Above-mentioned gained CTX-II value concentration of urinary creatinine corrects.
By the concentration (mmol/L) of UCr in clinical chemistry analyzer enzymic colorimetric determination sample, and correct with following formula:
CTX-II corrected value (ng/mmol)=1000 × urine CTX-II (μ g/L)/UCr (mmol/L)
Embodiment 3: kit quality analysis
One, batch difference: < 7.0% between
Between batch, difference is tested three urine samples determined by II Collagen Type VI catabolite detection kit in three batches of urine.
Detection limit: 0.20 μ g/L
Detection limit is 0.20ng/ml, the concentration of this value for urinating corresponding to CTX-II standard items 0 absorbance measurement mean value three standard deviations lower than 21.
Two, degree of accuracy≤12%
Carry out ten to urine sample to take turns analysis (diplopore) and determine degree of accuracy.
Three, dilute/linear 96%
In urine, the dilution recovery of II Collagen Type VI C peptide ending enzyme linked immune assay kit is 96%.4 sample urine CTX-II standard items 0 suitably dilute.In CTX-II concentration urine, II Collagen Type VI catabolite detection kit measures, recovery dilution factor correction.
DF: dilution gfactor; RC: the recovery
Four, disturb:
Add following compound to people's urine sample, do not detect that detecting analysis to II Collagen Type VI catabolite in urine produces interference.
The highest 30g/L of urea
The highest 10mg/L of creatinine
The highest 5mg/L of glucose
The highest 5mg/L of ascorbic acid
The highest 50mg/L of albumin.
Five, specificity:
In urine, II Collagen Type VI catabolite detection kit surveys antigenic determinant high conservative, and therefore test can be used for the urine sample of other species of major part, comprises non-human primates, ox, horse, pig, rabbit, rat and mouse.
Be more than illustrating for possible embodiments of the present invention, but this embodiment be not used to limit the scope of the claims of the present invention, allly do not depart from equivalence of the present invention and implement or change, all should be contained in the scope of the claims of the present invention.
Claims (2)
1. an II Collagen Type VI catabolite detection kit in urine, is characterized in that, mainly include:
(A) biotin labeled CTX-II antigen: the concentration of CTX-II antigen is 5 μ g/ml; The preparation method of biotin labeled CTX-II antigen is; Get CTX-II synthetic peptide 25mg, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes, and obtains antigenic dilution; Get the biotin that concentration is 1.25mg/mL, antigenic dilution: biotin=1:4 volume ratio mixing, adopts simple and easy Over-voltage protection biotin and CTX-II synthetic peptide to be cross-linked; Get the biotin labeled CTX-II antigen after preparation, add antiseptic and stabilizing agent;
(B) CTX-II Anti-TNF-α liquid solution, this CTX-II Anti-TNF-α body running titre is 1:1000; The amount ratio of described biotin labeled CTX-II antigen and CTX-II Anti-TNF-α liquid solution is 1:1;
Also include peroxidase coupled antibody and bag by the microwell plate of streptavidin; The work titre of described peroxidase coupled antibody is 1:5000;
Being prepared as of described peroxidase coupled antibody: get mouse-anti rabbit igg freeze-dried powder 0.1g, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes; Getting the dilution of HRP solid deionized water is 5mg/mL, adopts simple and easy Over-voltage protection HRP and mouse-anti rabbit igg to be cross-linked; Get the mouse-anti rabbit igg after preparation, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, adding cyanine is developer, and the concentration of developer is 0.08mg/ml, add antiseptic and stabilizing agent again, the work titre of described enzyme coupled antibody is 1:5000;
Being prepared as of described CTX-II polyclonal antibody: get healthy male rabbit, adopt injected s. c, immunity inoculation is carried out to position, rabbit 5-8 place, the CTX-II antigen 0.2mL of initial immunity injection 1mg/mL concentration, the 3rd week, the 4th week and within the 5th week, inject CTX-II antigen and the incomplete Freund's adjuvant equal-volume mixed solution 0.4mL of 1mg/mL concentration respectively; Draw neck to put to death rabbit, collect blood, get supernatant, add antiseptic and stabilizing agent after the centrifugal 30min of 3000r/min, 4 DEG C of sealings are preserved.
2. a preparation method for II Collagen Type VI catabolite detection kit in urinating, is characterized in that, mainly comprise the following steps:
1) biotin labeled CTX-II antigen is prepared; Get CTX-II synthetic peptide 25mg, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes, and obtains antigenic dilution; Get the biotin that concentration is 1.25mg/mL, antigenic dilution: biotin=1:4 volume ratio mixing, adopts simple and easy Over-voltage protection biotin and CTX-II synthetic peptide to be cross-linked; Get the biotin labeled CTX-II antigen after preparation, add antiseptic and stabilizing agent; The concentration of described CTX-II antigen is 5 μ g/ml;
2) prepare CTX-II Anti-TNF-α liquid solution: get CTX-II polyclonal antibody, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, adding haematochrome is developer, and described CTX-II polyclonal antibody titre is 1:1000; The concentration of haematochrome is 0.03mg/ml;
Also comprise and prepare peroxidase coupled antibody: get mouse-anti rabbit igg freeze-dried powder 0.1g, the TRIS damping fluid being 10mmol/L with 10mL concentration mixes; Getting the dilution of HRP solid deionized water is 5mg/mL, adopts simple and easy Over-voltage protection HRP and mouse-anti rabbit igg to be cross-linked; Get the mouse-anti rabbit igg after preparation, with the TRIS damping fluid of 10mmol/L and the EDTA mixing of 25mol/L, adding cyanine is developer, and the concentration of developer is 0.08mg/ml, add antiseptic and stabilizing agent again, the work titre of described peroxidase coupled antibody is 1:5000;
Also comprise preparation bag by the microwell plate of streptavidin;
Being prepared as of described CTX-II polyclonal antibody: get healthy male rabbit, adopt injected s. c, immunity inoculation is carried out to position, rabbit 5-8 place, the CTX-II antigen 0.2mL of initial immunity injection 1mg/mL concentration, the 3rd week, the 4th week and within the 5th week, inject CTX-II antigen and the incomplete Freund's adjuvant equal-volume mixed solution 0.4mL of 1mg/mL concentration respectively; Draw neck to put to death rabbit, collect blood, get supernatant, add antiseptic and stabilizing agent after the centrifugal 30min of 3000r/min, 4 DEG C of sealings are preserved.
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| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
| CN111868528B (en) * | 2018-04-03 | 2024-10-01 | 赛诺菲 | Lateral flow immunoassay strip device |
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| CN101236200A (en) * | 2008-02-06 | 2008-08-06 | 中国计量学院 | Chlorpromazine ELISA reagent kit and its detection method |
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| WO2002097121A1 (en) * | 2001-05-28 | 2002-12-05 | Nordic Bioscience A/S | Method for assaying osteoclast recruitment and resorption |
| CA2502926A1 (en) * | 2002-11-08 | 2004-05-27 | Linda J. Sandell | Uncoupled collagen synthesis and degradation assays |
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| CN1188235A (en) * | 1997-12-19 | 1998-07-22 | 北京化学试剂研究所 | Anti-human IV type collagen enzyme linked immunological quantitative determining kit and preparing method |
| CN1289924A (en) * | 2000-10-16 | 2001-04-04 | 北京市创伤骨科研究所 | II-type collegen enzyme-linked immunoassay analysis |
| CN101236200A (en) * | 2008-02-06 | 2008-08-06 | 中国计量学院 | Chlorpromazine ELISA reagent kit and its detection method |
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