CN108732358A - One kind is for measuring urine nuclear matrix protein immunofluorescent reagent item and its preparation method and application - Google Patents
One kind is for measuring urine nuclear matrix protein immunofluorescent reagent item and its preparation method and application Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a kind of immunofluorescent reagent items and its preparation method and application for measuring urine nuclear matrix protein.There are three regions for the fluorescence of the present invention reagent strip, respectively：Sample application zone, combined area, detection zone.The reagent strip is respectively from top to bottom water absorption pad, chromatographic film, bonding pad, sample pad；Tile the anti-NMP22 antibody complexes for having fluorescent material to mark on bonding pad, and anti-nuclear matrix albumen is coated on T lines in chromatographic film（NMP22）Monoclonal antibody can be used for detecting nuclear matrix protein in urine（NMP22）.It is used together with fluorescence analyser and quantitative detection may be implemented, it is simple to operate.It can be used for test in laboratory, it can also be used to which field quick detection, detection only need about 10min, operating time to be greatly reduced, which can be used for the early screening of carcinoma of urinary bladder and the auxiliary diagnosis of recurrence monitoring.
Description
Technical field
The invention discloses one kind for measuring urine nuclear matrix protein immunofluorescent reagent item and its preparation method and application,
The quantitative detection of concentration for the urine nuclear matrix protein (NMP22) in urine.This kit can be used for the early stage sieve of carcinoma of urinary bladder
Look into the auxiliary diagnosis with recurrence monitoring.Belong to field of immunological detection.
Technical background
Carcinoma of urinary bladder is the most common malignant tumour of China's urinary system.Bladder cancer recurrence rate up to 70%~80%,
10%~15% patient is dead due to tumour progression.Early detection, postoperative regular follow-up, detection recurrence are Therapy of Bladder Tumor
Key.
China carcinoma of urinary bladder age standardization incidence male is 3.8/10 ten thousand, and women is 1.8/10 ten thousand.In recent years, portion of China
The tumor incidence report of point city shows that bladder cancer morbidity has and increases trend.Carcinoma of urinary bladder male's incidence is 3-4 times of women.
Nuclear matrix protein (NMP22) is the important component of nuclear matrix, participates in constituting nucleus inner frame, is
It participates in maintaining a kind of tridimensional network albumen of cell kernel function, and synthesizes with DNA replication dna, RNA, hormone sensitive lipase gene and gene
The regulation and control of expression are related.NMP22 is caryomitosis device albumen (nuclear mitotic apparatus protein),
Nu-MAP a subunit).The main kinetic energy of Nu-MAP is that chromosome is correct, equably distributes during coordinating caryomitosis
To daughter cell.When canceration occurs for cell, inhereditary material distributes extremely abnormal, Nu-MAP synthesis in mitosis telophase in core
It increases sharply.NMP22 takes part in the adjusting of gene expression and coordinates NMP22 and is discharged into urine by apoptosis and reaches detectable
Level, it is closely related with urothelial tumor.The content of the NMP22 number higher than normal urothelium in carcinoma of urinary bladder epithelial cell
Ten times.
Research shows that：NMP22 joint cystoscopies, sensibility (recall rate) is up to 99% or more；NMP22 detection no matter
In early screening or recurrence monitoring, sensibility is much higher than Urine exfoliative cell, but specificity slightly below urine cell.
2012EAU (European Association of Urology Guidelines) carcinoma of urinary bladder guide is pointed out：
NMP22 has more sensibility than Urine exfoliative cell, and specificity is improved after screening case.Its high negative predictive value, can be
Using to postpone cystoscopy in monitoring.2015EAU carcinoma of urinary bladder guides are pointed out：NMP22 can be used for bedside diagnosis, and sensibility is
47~100%, specificity is 55~98%, and high cancer sensibility by stages is 75~83%.U.S. NCCN carcinoma of urinary bladder guides in 2016
It points out：Biopsy, pathological analysis etc. are based on for the management of patient, these information are aided with judgement recurrence may.It is contemplated that with FDA batches
Accurate NMP22 markers, are detected.
In consideration of it, establish it is a kind of effectively, quickly, the method for simple, sensitive quantitatively detection NMP22 is of great significance.
Invention content
The present invention provides one kind for measuring urine nuclear matrix protein immunofluorescent reagent item and its preparation method and application.
The kit is used together with fluorescence analyser and quantitative detection may be implemented, simple to operate.It can be used for test in laboratory,
It can be used for field quick detection, detection only needs about 10min, operating time to be greatly reduced, for the NMP22 albumen in urine
Concentration quantitative detection.This kit can be used for the early screening of carcinoma of urinary bladder and the auxiliary diagnosis of recurrence monitoring.
Present invention employs immunofluorescence chromatographic theories to be prepared for urine nuclear matrix protein immunofluorescent reagent item.The reagent strip
There are three regions, respectively：Sample application zone, combined area, detection zone.The structure of the reagent strip is by water absorption pad, chromatographic film, combination
Pad, the sequence close adhesion of sample pad from top to bottom are on bottom plate.The antibody complex Cy3-N-C1 tilings of fluorescent material label
There are detection line (T lines) and nature controlling line (C lines) in being drawn on bonding pad, in chromatographic film, anti-NMP22 monoclonal antibodies N-C2, C are coated on T lines
Sheep anti-mouse igg is coated on line.It drips and is flowed on bonding pad under the action of capillary chromatographs in the measuring samples liquid in sample pad,
NMP22 albumen in measuring samples forms compound after immune response occurs with the fluorescence antibody (Cy3-N-C1) on bonding pad,
It then moves to together on NC films, is combined and be enriched with the coated NMP22 monoclonal antibodies N-C2 of T lines and be retained at T lines when reaching T lines,
Remaining unreacted fluorescence antibody is combined and is enriched with the coated sheep anti-mouse igg of C lines and is retained at C lines, in immunofluorescence point
Detection obtains testing result in analyzer.T lines detect patient's NMP22 albumen concentration；C lines are unreacted fluorescence antibody and sheep anti mouse
IgG is combined, the Quality Control as this monitoring.
One kind is for measuring urine nuclear matrix protein immunofluorescent reagent item, it is characterised in that：In immunofluorescent reagent item, inhale
Water cushion, chromatographic film, bonding pad, sample pad are sequentially arranged；Sample pad corresponds to sample application zone, and bonding pad corresponds to combined area；Chromatography
Film corresponds to detection zone；Chromatographic film is equipped with detection line T lines and nature controlling line C line；NMP22 monoclonal antibodies N- is coated on detection line T lines
C2；Sheep anti-mouse igg is coated in nature controlling line C line；Tiling has the antibody complex Cy3-N-C1 that fluorescent material marks on bonding pad.
T lines, C lines dilution formula of liquid are：0.01M PBS, 5% sucrose, 1%BSA；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；Cy3-N-C1 dilutes formula of liquid：0.01M
PBS, 0.5%PVA, 5% sucrose, 1%BSA；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1%Tween-20,
1%BSA.
Sample pad, bonding pad, chromatographic film, water absorption pad are a reagent strip unit, adjacent reagent cells overlap region
Length is 2mm, and sequentially close adhesion cuts into 3mm on bottom plate after completing bonding for water absorption pad, chromatographic film, bonding pad, sample pad
Wide reagent strip.
Water absorption pad is blotting paper；Chromatographic film is nitrocellulose (NC) film；Bonding pad and sample pad are glass fibre element
Film.
The distance between detection line T lines and nature controlling line C line are 10mm.Coated NMP22 monoclonal antibodies N-C2 on detection line T lines,
A concentration of 0.5mg/mL, dosage are 3 μ l/cm.Coated sheep anti-mouse igg in nature controlling line C line, a concentration of 0.5mg/mL, dosage 3
μl/cm。
The antibody complex Cy3-N-C1 tilings (spray film) marked using the fluorescent material of a concentration of 1mg/mL are in bonding pad
On, the antibody complex Cy3-N-C1 dosages of fluorescent material label are 6 μ l/cm；The antibody complex Cy3- of fluorescent material label
In N-C1, fluorescent material is cyanine dyes Cy3.
A kind of preparation method for measuring urine nuclear matrix protein immunofluorescent reagent item includes the following steps：
1) sample pad is handled：The sample pad of 20mm wide is soaked in sample pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Sample pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,1%BSA；
2) bonding pad is handled：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；
3) prepared by the antibody complex Cy3-N-C1 of fluorescent material label：By anti-NMP22 antibody N-C1 0.15M NaCl
Solution room temperature is dialysed 4 hours, and then with fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, N-C1 and 10mg/mL Cy3 are pressed
According to 20:1 molar ratio mixed room temperature is protected from light 45 minutes, is then protected from light and was dialysed with 4 DEG C fresh of 0.15M NaCl solutions
Night, then it is protected from light dialysis 4 hours with 0.01M PBS solution room temperatures, then it is protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions,
It collects Cy3-N-C1 and preserves；
4) the antibody complex Cy3-N-C1 of fluorescent material label sprays film：Cy3-N-C1 is diluted with Cy3-N-C1 dilutions
For a concentration of 1mg/mL Cy3-N-C1 solution and spray film on bonding pad, the Cy3-N-C1 solution of 1mg/mL is on bonding pad
Dosage is 6 μ l/cm；Cy3-N-C1 dilutions：0.01M PBS, 0.5%PVA, 5% sucrose, 1%BSA；
5) T lines, the scribing line of C lines：It takes and draws T lines on the NC films of 30mm wide, the distance of C lines, T lines and C lines is 10mm；It is wrapped on T lines
There is anti-NMP22 monoclonal antibodies N-C2, adjusts a concentration of 0.5mg/mL of anti-NMP22 monoclonal antibodies N-C2 with T lines, C line dilutions, dosage is 3 μ
l/cm；It is coated with sheep anti-mouse igg on C lines, adjusts a concentration of 0.5mg/mL of sheep anti-mouse igg with T lines, C line dilutions, dosage is 3 μ
l/cm；T lines, C line dilutions：0.01M PBS, 5% sucrose, 1%BSA；
6) it assembles：By water absorption pad, chromatographic film, bonding pad, sample pad, sequentially close adhesion is on bottom plate, sample pad, combination
Pad, chromatographic film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is 2mm, after completing bonding
Cut into the reagent strip of 3mm wide.
The present invention also provides for measuring urine nuclear matrix protein immunofluorescent reagent item as the core base in detection human urine
The application of the reagent of the concentration of matter albumen.
Compared with the existing technology, beneficial effects of the present invention are：
1) it is used together for measuring urine nuclear matrix protein immunofluorescent reagent item and fluorescence analyser and may be implemented to quantify
Detection, it is simple to operate, do not need professional technician's participation.It can be used for test in laboratory, it can also be used to scene quickly inspection
It surveys, detection only needs about 10min.
2) it is glimmering as being immunized for measurement urine nuclear matrix protein independently to construct anti-NMP22 monoclonal antibodies N-C1 and N-C2 by the present invention
The core of light reagent strip structure, determines the accuracy and sensitivity of test strips.
Description of the drawings：
Fig. 1：One kind is for measuring urine nuclear matrix protein immunofluorescent reagent structural schematic diagram；
Fig. 2：A kind of canonical plotting for measuring urine nuclear matrix protein immunofluorescent reagent item；
Fig. 3：A kind of urine measuring patient urine and normal person for measuring urine nuclear matrix protein immunofluorescent reagent item
Middle NMP22 content balances scatter plot.
Specific implementation mode
Technical scheme of the present invention is described further with reference to embodiment.
Embodiment one：The preparation of the specific monoclonal antibody of anti-nuclear matrix albumen (NMP22)
1. animal immune：
The female Balb/c mouse of 6-8 week old are chosen as immunization, every mouse tail vein is taken a blood sample before initial immunity, just
200 μ l of immunogene after intraperitoneal injection emulsification when secondary immune (antigen adds Freund's complete adjuvant)；The 14th day after just exempting from, the 28th day with identical
Method carry out second and third time (antigen adds Freund's incomplete adjuvant) is immunized, after the third immunization the 5th day to every mouse into
The blood sampling of row eye socket takes serum to assess antibody titer, carries out within the 7th day booster immunization after the third immunization, and tail vein injection is immune
100 μ l of original take splenic lymphocytes and SP2/0 (5 on the 3rd day after booster immunization:1 mixed proportion) cell fusion is carried out, fusion is adopted
With PEG methods.After the completion of fusion based selective culture is cultivated with HAT.
2. subclone：
It is subcloned using limiting dilution assay.Feeder cells are laid in 96 orifice plates, by the hybridoma in positive hole,
Count, 3 concentration be diluted to HT culture mediums, spread 96 orifice plates, each concentration 2 arranges so that in per hole cell be 1/hole, 3/
Hole, 10/hole, be placed in 37 DEG C, cultivated 7-10 days in 5%CO2 incubators, the positive strongest hole of selection is carried out continuously 2-3 times
Subclone, the anti-NMP22 monoclonal antibodies N-C1 and N-C2 of positive strain carry out turn hole and expand culture.
3. prepared by monoclonal antibody
Monoclonal antibody preparation uses in vivo method i.e. mice celiac inoculation, takes the ascites of mouse peritoneal, the antibody of acquisition dense
Degree is high.The Balb/c female mices of 10 week old, mouse peritoneal is taken to inject the 500 sterilized atoleines of μ l.Hybridoma passes through
Cross collection centrifugation, intraperitoneal injection 2 × 106A cell/mouse.Mouse web portion obviously swells within about 1 week or so, uses syringe needle
Ascites is adopted from abdominal cavity.
4. antibody purification
Use the purifying that Protein G affinity columns carry out antibody.Ascites is diluted with isometric combination buffer,
Started column, and waited for that sample flowed completely out, column is crossed with appropriate combination buffer, and waited for that liquid is flowed completely out and cross column with appropriate eluent,
It simultaneously with the eluent in the small centrifuge tube of appropriate volume neutralizer and received is equipped with, is detected, is collected blue with Coomassie brilliant blue
The deeper centrifuge tube antibody of color is dialysed 3 days loaded in bag filter, and albumen concentration is detected after dialysis, is placed in -20 DEG C of preservations.
Embodiment two
One kind is for measuring urine nuclear matrix protein immunofluorescent reagent item, it is characterised in that：In immunofluorescent reagent item, inhale
Water cushion, chromatographic film, bonding pad, sample pad are sequentially arranged；Sample pad corresponds to sample application zone, and bonding pad corresponds to combined area；Chromatography
Film corresponds to detection zone；Chromatographic film is equipped with detection line T lines and nature controlling line C line；NMP22 monoclonal antibodies N- is coated on detection line T lines
C2；Sheep anti-mouse igg is coated in nature controlling line C line；Tiling has the antibody complex Cy3-N-C1 that fluorescent material marks on bonding pad.
T lines, C lines dilution formula of liquid are：0.01M PBS, 5% sucrose, 1%BSA；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；Cy3-N-C1 dilutes formula of liquid：0.01M
PBS, 0.5%PVA, 5% sucrose, 1%BSA；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1%Tween-20,
1%BSA.
Sample pad, bonding pad, chromatographic film, water absorption pad are a reagent strip unit, adjacent reagent cells overlap region
Length is 2mm, and sequentially close adhesion cuts into 3mm on bottom plate after completing bonding for water absorption pad, chromatographic film, bonding pad, sample pad
Wide reagent strip.
Water absorption pad is blotting paper；Chromatographic film is nitrocellulose (NC) film；Bonding pad and sample pad are glass fibre element
Film.
The distance between detection line T lines and nature controlling line C line are 10mm.Coated NMP22 monoclonal antibodies N-C2 on detection line T lines,
A concentration of 0.5mg/mL, dosage are 3 μ l/cm.Coated sheep anti-mouse igg in nature controlling line C line, a concentration of 0.5mg/mL, dosage 3
μl/cm。
The antibody complex Cy3-N-C1 tilings (spray film) marked using the fluorescent material of a concentration of 1mg/mL are in bonding pad
On, the antibody complex Cy3-N-C1 dosages of fluorescent material label are 6 μ l/cm；The antibody complex Cy3- of fluorescent material label
In N-C1, fluorescent material is cyanine dyes Cy3.
A kind of preparation method for measuring urine nuclear matrix protein immunofluorescent reagent item includes the following steps：
1) sample pad is handled：The sample pad of 20mm wide is soaked in sample pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Sample pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,1%BSA；
2) bonding pad is handled：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, soaked overnight under the conditions of 4 DEG C,
Taking-up is placed on drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Bonding pad prescription for the treatment of liquid is：0.1M
Tris-HCl, 0.1%Tween-20,0.5%PVA, 5% sucrose, 1%BSA；
3) prepared by the antibody complex Cy3-N-C1 of fluorescent material label：By anti-NMP22 antibody N-C1 0.15M NaCl
Solution room temperature is dialysed 4 hours, and then with fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, N-C1 and 10mg/mL Cy3 are pressed
According to 20:1 molar ratio mixed room temperature is protected from light 45 minutes, is then protected from light and was dialysed with 4 DEG C fresh of 0.15M NaCl solutions
Night, then it is protected from light dialysis 4 hours with 0.01M PBS solution room temperatures, then it is protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions,
It collects Cy3-N-C1 and preserves；
4) the antibody complex Cy3-N-C1 of fluorescent material label sprays film：Cy3-N-C1 is diluted with Cy3-N-C1 dilutions
For a concentration of 1mg/mL Cy3-N-C1 solution and spray film on bonding pad, the Cy3-N-C1 solution of 1mg/mL is on bonding pad
Dosage is 6 μ l/cm；Cy3-N-C1 dilutions：0.01M PBS, 0.5%PVA, 5% sucrose, 1%BSA；
5) T lines, the scribing line of C lines：It takes and draws T lines on the NC films of 30mm wide, the distance of C lines, T lines and C lines is 10mm；It is wrapped on T lines
There is anti-NMP22 monoclonal antibodies N-C2, adjusts a concentration of 0.5mg/mL of anti-NMP22 monoclonal antibodies N-C2 with T lines, C line dilutions, dosage is 3 μ
l/cm；It is coated with sheep anti-mouse igg on C lines, adjusts a concentration of 0.5mg/mL of sheep anti-mouse igg with T lines, C line dilutions, dosage is 3 μ
l/cm；T lines, C line dilutions：0.01M PBS, 5% sucrose, 1%BSA；
6) it assembles：By water absorption pad, chromatographic film, bonding pad, sample pad, sequentially close adhesion is on bottom plate, sample pad, combination
Pad, chromatographic film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is 2mm, after completing bonding
Cut into the reagent strip of 3mm wide.
Embodiment three：Standard curve for measuring urine nuclear matrix protein immunofluorescent reagent item
NMP22 antigenic solutions can be diluted into antigen to final concentration of 0ng/mL, 4ng/mL, 8ng/ with antigenic dilution
The serial standards solution of mL, 16ng/mL, 32ng/mL, 64ng/mL.
Antigenic dilution is to contain BSA, the 0.05mol/L phosphate buffers of sucrose, and pH=7.4 is every liter and contains 20g
BSA, 50g sucrose, 8g NaCl, 0.2g KCl, 0.24g KH2PO4, 1.44g Na2HPO4Aqueous solution.
According to the detecting step of urine nuclear matrix protein NMP22 immunofluorescent reagent assay methods, NMP22 series marks are obtained
The fluoroscopic examination intensity of quasi- product.According to the fluorescence intensity level value of standard items and a concentration of reference axis, suitable mathematical model is selected
Standard curve is established, standard curve using four parametric methods as shown in Fig. 2, be fitted its correlation coefficient r=0.9982.
Example IV：Repeatability for the sample for measuring urine nuclear matrix protein immunofluorescent reagent item
According to the detecting step of urine nuclear matrix protein NMP22 immunofluorescent reagent assay methods, with two concentration levels
Sample respectively repeat detection 10 times, calculate 10 times measurement concentration results average value M and standard deviation SD, obtained according to formula (2)
Coefficient of variation CV.
The formula of CV=SD/M × 100% (2)
In formula：
CV-the coefficient of variation；
The standard deviation of 10 measurement results of SD-；
The average value of 10 measurement results of M-.
The results are shown in Table 1 for repeatability, and coefficient of variation CV is respectively less than 10%, shows that the kit has when detecting sample
Preferable repeatability：
Table 1：Kit repeatability measures
Embodiment five：Pattern detection clinical application situation for measuring urine nuclear matrix protein immunofluorescent reagent item
According to the detecting step of urine nuclear matrix protein NMP22 immunofluorescent reagent assay methods, 30 bladders have been selected
Carninomatosis people, the urine specimen of 20 examinees, is detected, and obtains the concentration value of NMP22 in urine, draws scatter plot.It surveys
Determine shown in result figure 3, the concentration value of NMP22 in the patient of the kit measurement and the serum of normal person has notable difference.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein, it is characterised in that：In immunofluorescent reagent item, inhale
Water cushion, chromatographic film, bonding pad, sample pad are sequentially arranged；Sample pad corresponds to sample application zone, and bonding pad corresponds to combined area；Chromatography
Film corresponds to detection zone；Chromatographic film is equipped with detection line T lines and nature controlling line C line；It is coated with anti-nuclear matrix albumen on detection line T lines
（NMP22）Monoclonal antibody N-C2；Sheep anti-mouse igg is coated in nature controlling line C line；Tiling has fluorescent material to mark anti-on bonding pad
NMP22 antibody complexes Cy3-N-C1.
2. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
T lines, C lines dilution formula of liquid are：0.01M PBS, 5% sucrose, 1%BSA；Bonding pad prescription for the treatment of liquid is：0.1M Tris-HCl,
0.1% Tween-20,0.5%PVA, 5% sucrose, 1%BSA；Cy3-N-C1 dilutes formula of liquid：0.01M PBS, 0.5%PVA, 5%
Sucrose, 1%BSA；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl, 0.1% Tween-20,1%BSA.
3. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
Sample pad, bonding pad, chromatographic film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is
2mm, sequentially close adhesion completes the examination that 3mm wide is cut into after bonding on bottom plate for water absorption pad, chromatographic film, bonding pad, sample pad
Agent item.
4. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
Water absorption pad is blotting paper；Chromatographic film is nitrocellulose（NC）Film；Bonding pad and sample pad are glass fibre element film.
5. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
The distance between detection line T lines and nature controlling line C line are 10mm.
6. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
Coated anti-NMP22 monoclonal antibodies N-C2 on detection line T lines, a concentration of 0.5 mg/mL, dosage are 3 μ l/cm.
7. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
Coated sheep anti-mouse igg in nature controlling line C line, a concentration of 0.5mg/mL, dosage are 3 μ l/cm.
8. a kind of immunofluorescent reagent item for measuring urine nuclear matrix protein according to claim 1, it is characterised in that：
Make anti-NMP22 antibody complexes Cy3-N-C1 spray films that the fluorescent material of a concentration of 1mg/mL marks on bonding pad, fluorescence
The antibody complex Cy3-N-C1 dosages of matter label are 6 μ l/cm；It is glimmering in the antibody complex Cy3-N-C1 of fluorescent material label
Stimulative substance is cyanine dyes Cy3.
9. a kind of preparation for measuring the immunofluorescent reagent item of urine nuclear matrix protein described in claim 1-8 any one
Method includes the following steps：
Sample pad processing：The sample pad of 20mm wide is soaked in sample pad treatment fluid, soaked overnight under the conditions of 4 DEG C, after taking-up
It is placed in drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Sample pad prescription for the treatment of liquid is：0.1M Tris-HCl,
0.1% Tween-20,1%BSA；
Bonding pad processing：The bonding pad of 10mm wide is soaked in bonding pad treatment fluid, soaked overnight under the conditions of 4 DEG C, after taking-up
It is placed in drying in 37 DEG C of thermostatic drying chambers, is preserved under the conditions of drying at room temperature；Bonding pad prescription for the treatment of liquid is：0.1M Tris-HCl,
0.1% Tween-20,0.5%PVA, 5% sucrose, 1%BSA；
It is prepared by the antibody complex Cy3-N-C1 of fluorescent material label：By the 0.15M NaCl solutions rooms anti-NMP22 antibody N-C1
Temperature dialysis 4 hours, then uses fresh 4 DEG C of dialysed overnights of 0.15M NaCl solutions, by N-C1 and 10mg/mL Cy3 according to 20:1
Molar ratio mixed room temperature be protected from light 45 minutes, be then protected from light dialysed overnight with 4 DEG C fresh of 0.15M NaCl solutions, then use
0.01M PBS solution room temperatures are protected from light dialysis 4 hours, then are protected from light dialysed overnight with 4 DEG C fresh of 0.01M PBS solutions, collect
Cy3-N-C1 is simultaneously preserved；
The antibody complex Cy3-N-C1 of fluorescent material label sprays film：Cy3-N-C1 is diluted to concentration with Cy3-N-C1 dilutions
For 1mg/mL Cy3-N-C1 solution and spray film on bonding pad, the dosage of the Cy3-N-C1 solution of 1mg/mL on bonding pad is
6μl/cm；Cy3-N-C1 dilutions：0.01M PBS, 0.5%PVA, 5% sucrose, 1%BSA；
T lines, the scribing line of C lines：It takes and draws T lines on the NC films of 30mm wide, the distance of C lines, T lines and C lines is 10mm；It is coated on T lines anti-
NMP22 monoclonal antibody N-C2 adjust a concentration of 0.5mg/mL of anti-NMP22 monoclonal antibodies N-C2 with T lines, C line dilutions, and dosage is 3 μ l/cm；
It is coated with sheep anti-mouse igg on C lines, adjusts a concentration of 0.5mg/mL of sheep anti-mouse igg with T lines, C line dilutions, dosage is 3 μ l/cm；
T lines, C line dilutions：0.01M PBS, 5% sucrose, 1%BSA；
Assembling：By water absorption pad, chromatographic film, bonding pad, sample pad, sequentially close adhesion is on bottom plate, sample pad, bonding pad, chromatography
Film, water absorption pad are a reagent strip unit, and adjacent reagent cells overlap zone length is 2mm, is cut into after completing bonding
The reagent strip of 3mm wide.
10. the immunofluorescent reagent item for measuring urine nuclear matrix protein described in claim 1-8 any one is as detection
Nuclear matrix protein in human urine（NMP22）Concentration reagent application.
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