CN102901824A - Method for detecting urine fibronectin, and kit thereof - Google Patents
Method for detecting urine fibronectin, and kit thereof Download PDFInfo
- Publication number
- CN102901824A CN102901824A CN 201110209641 CN201110209641A CN102901824A CN 102901824 A CN102901824 A CN 102901824A CN 201110209641 CN201110209641 CN 201110209641 CN 201110209641 A CN201110209641 A CN 201110209641A CN 102901824 A CN102901824 A CN 102901824A
- Authority
- CN
- China
- Prior art keywords
- urine
- hole
- incubation
- measured
- fibronectin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a method for detecting the urine fibronectin, and a kit thereof. The method comprises the steps of specimen acquisition, experiment preparation, sample addition, incubation, plate washing, enzyme addition, incubation, washing, color development and termination. A bladder cancer diagnosis technology having the advantages of simplicity, rapidness, non-invasion, low price, high sensitivity and strong specificity is established in the invention to determine the concentrations of the urine fibronectin of a bladder cancer patient in different pathological phases, and the index of the bladder cancer infiltration degree can be discriminated through the difference of the urine fibronectin.
Description
Technical field
The invention belongs to a kind of medical detection method field, more particularly, relate to a kind of for detection of the method for Urine fibronectin and the kit of preparation thereof.
Background technology
(Bladder transitional cell carcinoma, BTCC. is hereinafter to be referred as " carcinoma of urinary bladder " for TCCB.) be the modal malignant tumour of urinary system, also be one of whole body common cancer.But in recent years, China city carcinoma of urinary bladder incidence of disease has and increases year by year trend.The Main Means of Diagnosis of Bladder and Follow-up After is cystoscopy and the inspection of urine exfoliative cytology at present.Cystoscopy is a kind of inspection that wound property is arranged, and the person under inspection often has the psychology of fear; And cystoscopy has certain blind area, and it is relatively more difficult to flat low level carcinoma of urinary bladder and carcinoma in situ diagnosis.Even state-of-the-art soft lens also has up to 8.7% false negative rate and 7.5% infection rate.Simultaneously the bladder cancer recurrence rate is up to 50%-80%, after each recurrence frequently cystoscopy to check patient be a kind of undying torment, more caused heavy mental burden and economic pressures.
From the last century the nineties, biological marker becomes common practise as the research of Diagnosis of Bladder and monitoring recurrence in the urine to detect.The biological marker of having studied so far comprises: bladder tumor antigen (BTA), urine analyzes, NMP-22 (NMP22), Urine fibronectin (Fibronectin, FN), fluorescence in situ hybridization (FISH), ImmunoCyt, fibrin and fibrinogen degradation product (FDP) etc.
On the one hand, though other urine bladder cancer biological marker susceptibilitys commonly used increase than exfoliative cytology at present, but still do not reach the diagnosis requirement; And specificity is generally obviously on the low side, still can not replace at present the inspection of urine exfoliative cytology.Only have FN to reach higher sensitivity and specificity.Up to now, has U.S. food medicine surveillance authority (FDA) ratified BTA stat, BTA trak, UroVysion?, the non-test paper method such as ImmunoCyt urine biological marker object detecting method as the supplementary means of Diagnosis of Bladder and monitoring." NMP22BladderChek Test " test paper of listing in 2008 is the product that Ying Weilisi medical diagnosis group succeeds in developing, and is expensive for foreign patent protection product, is not suitable for the widespread use of population of China.
On the other hand, as a kind of desirable carcinoma of urinary bladder monitoring, diagnosing method, not only requirement can be made etiologic diagnosis, preferably can also judge simultaneously the risk that develops into invasive bladder cancer.As previously mentioned, the recurrence rate of NMIBC postoperative is up to 50-80%, but the ratio that final real progress is wellability and metastatic carcinoma is no more than 15%.There are some researches show that for the WD Ta phase carcinoma of urinary bladder of diameter less than 10mm, under the prerequisite of close observation, average retardation operation in 13.5 months is on its life cycle and not impact of tumour progression.Other there are some researches show that flesh layer invasive bladder cancer incured loss through delay 3 months capable cystectomies will obviously reduce survival rate.We think thus, and following up a case by regular visits to of carcinoma of urinary bladder not only is the early detection recurrence, the more important thing is the carcinoma of urinary bladder that early detection flesh layer infiltrates.Desirable carcinoma of urinary bladder urine biological marker should satisfy the needs of etiologic diagnosis, can reflect again the degree of risk of tumour progression, is beneficial to instruct clinical decision.
FN is the non-collagen sugar albumen of a kind of large molecule, and molecular weight is about 440KD, and it only is present in uroepithelial basilar memebrane and submucosa in urinary system.Basilar memebrane FN all has forfeiture in various degree in the carcinoma of urinary bladder, the tumor invading basilar memebrane and induce the proteinase of generation increased FN from basilar memebrane release and make FN content increase in the urine.Since (Sino-Japan medical congress) professor Shen Zhoujun in 1992 at first reported with urine FN diagnosing bladder cancer, many results of study all supported urine FN to can be used as the diagnosis index of BTCC, and FN becomes a kind of widely carcinoma of urinary bladder tumor markers of research; Research in 2005 finds, urine FN and tumor of bladder by stages, classification, tumor size, number, growth pattern etc. are closely related, show that urine FN has good using value to prediction bladder tumor recurrence and prognosis judgement.
Although urine FN has obtained consistent affirming as desirable Diagnosis of Bladder with the monitoring biological label, the detection method of each and result are not yet unified.At present, the clinical detection of FN remains in following problem: 1, not yet standardization of using method.2, step is more loaded down with trivial details, and the result is subject to the impact of operator's technical merit etc., and uncertain factor is more.3, get blood, the sampling both needed the professional, increased inspection charge; The patient is painful again, self-discipline check wish not high.
Summary of the invention
For defects, the object of the invention is to overcome or improves existing problem, is provided for detecting the method for Urine fibronectin.
Another purpose of the present invention has been to provide a kind of kit of the method for detection of Urine fibronectin.
For achieving the above object, technical solution of the present invention is:
A kind of method for detection of Urine fibronectin, its operation steps is as follows:
1) collection of specimens:
Get transitional cell bladder carcinoma, urine 2,000-3 after the centrifugal 15-30 of 000r/min minute, collects supernatant, gained urine specimen to be measured;
2) Preparatory work of experiment:
Then dilution did 10 times of dilutions with distilled water in 37 ℃ of water-bath 15-30 minutes;
Then cleansing solution did 20 times of dilutions with distilled water in 37 ℃ of water-bath 15-30 minutes;
Every standard items (512ng/ml) are accurately redissolved with the 1.0ml dilution.Get 250ul, make six doubling dilutions with dilution, and be diluted to one group of standard items;
3) application of sample: establish respectively blank hole, gauge orifice, testing sample hole, gauge orifice and testing sample hole add respectively variable concentrations standard items or urine specimen 100ul to be measured, add dilution 100ul in the blank hole;
4) incubation: at 37 ℃ of incubation 90-120 minutes;
5) wash plate: discard liquid in the reacting hole, fill with each hole with cleansing solution, leave standstill, dry, repeatedly pat dry after 3-5 time;
6) enzyme-added: described gauge orifice and testing sample hole add 100ul enzyme connection thing, gently mixing;
7) incubation: at 37 ℃ of incubation 60-90 minutes;
8) wash plate: discard liquid in the reacting hole, fill with each hole with described cleansing solution, leave standstill, drying pats dry after 3-5 time repeatedly;
9) colour developing: every substrate sheet is with the dissolving of 5ml substrate buffer solution before use, and every hole adds substrate solution 100ul, 37 ℃ incubation 15-20 minute;
10) stop: every hole adds the 50ul stop buffer, and mixing at microplate reader 492nm place, with the zeroing of blank hole, is measured each hole OD value gently.
Another aspect of the present invention, the concentration range of wherein said urine specimen to be measured is 1: 1000-1: 6000.Preferentially, the concentration range of wherein said urine specimen to be measured is 1: 2000-1: 4000.
Another aspect of the present invention, the sensitivity that described method is measured Urine fibronectin is 77%-100%.
Another embodiment of the present invention is: a kind of kit that detects Urine fibronectin.
Dilution of the present invention and cleansing solution are Enzyme-linked Immunosorbent Assay competition law (ELISA) common agents.
The specificity that the present invention urinates the FN diagnosing bladder cancer is 87%, and the positive prediction rate is 87%.
FN is the non-collagen sugar albumen of a kind of large molecule, and molecular weight is about 440KD, and it only is present in uroepithelial basilar memebrane and submucosa in urinary system.Basilar memebrane FN all has forfeiture in various degree in the carcinoma of urinary bladder, the tumor invading basilar memebrane and induce the proteinase of generation increased FN from basilar memebrane release and make FN content increase in the urine.Since (Sino-Japan medical congress) professor Shen Zhoujun in 1992 at first reported with urine FN diagnosing bladder cancer, many results of study all supported urine FN to can be used as the diagnosis index of BTCC, and FN becomes a kind of widely carcinoma of urinary bladder tumor markers of research.
Urine FN not only can be used as the biological marker of diagnosis and the monitoring of carcinoma of urinary bladder, with tumor of bladder by stages, classification, tumor size, number, growth pattern etc. are closely related, can also be as the objective indicator of estimating result for the treatment of.The present invention is intended to by utilizing the Elisa method to determine urine FN optimum detection method (double antibody sandwich method), best enzyme labeling concentration and optimum experimental condition.
TCCB is one of common cancer of China, and the carcinoma of urinary bladder method for quick especially urinates the detection method of usefulness and be at present the market vacancy phase, does not still have at present similar test paper, reagent class diagnostic method to be applied to clinical.
The present invention set up a kind of easy, quick, without wound, at a low price, the Diagnosis of Bladder technology of high sensitivity, high specificity, can measure different pathological stages transitional cell bladder carcinoma FN concentration, and by FN concentration difference, can differentiate the index of Basement membrane degree.The present invention can remedy the deficiency of existing diagnostic method, can greatly alleviate patient's misery, has very high clinical value, can also alleviate patient's financial burden simultaneously, has good economic benefit.If the present invention can will be very beneficial for the check of examination, diagnosis and the postoperative patient of carcinoma of urinary bladder by develop, and can provide useful reference function for quick, the easy diagnostic method of other uropoiesis epithelial tumours such as renal plevis, tumor of ureter and tumor of prostate.
The usefulness of technique scheme is:
(1) the present invention can analyze a plurality of samples simultaneously, respectively,
(2) the present invention detect FN in the urine have easy, without wound, inexpensive, and in the urine FN measure stronger than the FN specificity in the blood, susceptibility is higher,
(3) use the Elisa method and detect simultaneously not as a kind of that the method for synantigen has susceptibility and the high characteristics of specificity,
(4) number of times of cystoscopy be can effectively reduce in conjunction with the application of B ultrasonic, good society and economic benefit obtained.
(5) can differentiate the transitional cell carcinoma of bladder in different stadium stages.
Description of drawings
Fig. 1 is the operation steps for detection of the method for Urine fibronectin.
Embodiment
Below by specific instantiation explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by content disclosed in the present specification.The present invention also can be implemented or used by other different instantiations, and the every details in this instructions also can be based on different viewpoints and application, carries out various modifications and change under the purpose of the present invention not deviating from.
Embodiment 1
As shown in Figure 1:
A kind of method for detection of Urine fibronectin, its operation steps is as follows:
Detect principle:
Adopt the Enzyme-linked Immunosorbent Assay competition law, measure the content of fibronectin (Fibronectin, FN) in the human urine.The FN and the FN in the urine that are coated with in advance in the ELISA Plate are combined with the FN antibody competition, form antigen antibody complex behind the adding enzymic-labelled antibody, and in 492nm wavelength place colorimetric, FN content is directly proportional with OD value (492nm) in the urine to be determined.
Collection of specimens:
Leave and take for the first time urina sanguinis, advise patient to prohibit water after 10 o'clock evenings before that day when leaving and taking urine, urine sample 3 behind the centrifugal 10min of 000r/min, is collected supernatant, places clean container, and 2-8 ℃, can preserve 48 hours, can preserve 1 month for-20 ℃.
Preparatory work of experiment:
1. then dense dilution does 10 times of dilutions (the dense dilution of 1ml+9ml distilled water) with distilled water with being prepended to 37 ℃ of water-bath 15min (because the mother liquor salinity is high, the easy crystallization of Refrigerator store).
2. then dense cleansing solution does 20 times of dilutions (the dense dilution of 1ml+19ml distilled water) with distilled water with being prepended to 37 ℃ of water-bath 15min (because the mother liquor salinity is high, the easy crystallization of Refrigerator store).
3. every standard items (512ng/ml) are accurately redissolved with the 1.0ml dilution.Get 250ul, make six doubling dilutions with dilution, and be diluted to one group of standard items, as: 512,256,128,64,32,16,8ng/ml.
Operation steps:
Kit answers balance to room temperature (18-25 ℃) to test again.
Take out required reaction plate.
1. every hole adds variable concentrations standard items or urine specimen 100ul to be measured, adds dilution 100ul in the blank hole, 37 ℃ of incubations 90 minutes.
2. wash plate: discard liquid in the reacting hole, fill with each hole with cleansing solution, left standstill 3 seconds, dry, repeatedly pat dry after 3 times.
3. every hole adds 100ul enzyme connection thing, and mixing is 30 seconds gently, 37 ℃ of incubations 60 minutes.
4. wash plate: discard liquid in the reacting hole, fill with each hole with cleansing solution, left standstill 3 seconds, dry, repeatedly pat dry after 3 times.
5. colour developing: every substrate sheet is with the dissolving of 5ml substrate buffer solution before use, and every hole adds substrate solution 100ul, 37 ℃ incubation 15-20 minute.
6. termination colorimetric: every hole adds 50ul stop buffer (stop solution), and mixing is 30 seconds gently, and 15 minutes inherent microplate reader 492nm places with the zeroing of blank hole, measure each hole OD value.
As a result interpretation:
Take the OD value as horizontal ordinate, take standard items as ordinate, do typical curve at logarithmic paper, OD value per sample can be found its concentration from typical curve.Annotate: note the selection (semilog coordinate) of coordinate system! If Sample Dilution then the result will multiply by extension rate.
Embodiment 2
Measure different stadium transitional cell carcinoma of bladder patients, optimum urologic disease patient (not accompanying urinary tract infections is that A group, companion's urinary tract infections are the B group), clear-cell carcinoma, hamartoma and adrenal cortical tumor (c group) patient, non-urinary system malignant tumour (D group) patient and Normal group urinate cross, Serum Fibronectin FN concentration.
Before 5 routine BTCC operation in patients and postoperative 10-16 days equal collect specimens, collect specimen before other operation in patients.Patient and normal control leave and take and keep 2 littlely of above urine in urina sanguinis or the bladder, and the centrifugal 10min of 2,000r/min draws supernatant and puts in-20 ℃ of refrigerators.In the time of the preserving urine ripple, extract on an empty stomach PeV blood, separation of serum in 3 hours is put in-20 ℃ of refrigerators for subsequent use.
Experimental technique
According to the experimental technique of embodiment 1, purification goat-anti people FN specific antibody; The standby enzyme labelled antibody of improvement sodium periodate legal system, the ELISA method is measured urine FN concentration (g/L), and two-way agar diffusion method is measured serum FN concentration (mg/L).
Experimental result
One, the measurement result of urine FN:
1.BTCC patient's onset and recurrence urine FN concentration there was no significant difference (P＞0.05).
2.BTCC the patient is male, other urine of women FN concentration there was no significant difference (P＞0.05).
3.BTCC dung FN concentration there was no significant difference (P＞0.05) between each age group of patient.
4. I, II and III group BTCC Urinary FN concentration is respectively 57.54 ± 5.25ug/L, 284.45 ± 8.13ug/L and 1786.40 ± 7.41ug/L.III group concentration is significantly higher than I group (P＜0.01), and is significantly higher than II group (P＜0.05), and the II group is significantly higher than I group (P＜0.05).
5.A, each group of B, C, D and Normal group urine FN concentration is respectively: 56.23 ± 2.75,75.16 ± 2.82,40.83 ± 8.67,55.21 ± 5.13 and 70.30 ± 1.99ug/L there are no significant between them difference (P＞0.05).
6. III group BTCC Urinary FN concentration is significantly higher than A, B, C, D and Normal group (P＜0.01), II group BTCC Urinary FN is significantly higher than A, B, C, D and Normal group (P＜0.05), I group patient BTCC urinates FN A, B, C, D group and Normal group there was no significant difference (P＞0.05)
7. there were 5 routine BTCC Urinary FN concentration of contrast data to be respectively 825.68 ± 344.24 and 78.05 ± 15.80ug/L before the operation and after the operation in 10-16 days, on a declining curve.
Two, the measurement result of serum FN
1.BTCC patient's onset and recurrence serum FN there was no significant difference (P＞0.05)
Man, the other serum FN of women there was no significant difference (P＞0.05)
3. each age group valve serum FN there was no significant difference (P＞0.05).
4.BTCC serum FN concentration there was no significant difference (P＞0.05) between three groups of patients
5.BTCC patients serum Fn and A, B, C, the standby group of D serum FN there are no significant difference (P＞0.05).
6.BTCC patients serum FN is I150.63 soil 2.28mg/L, Normal group serum FN is 244.00 native 3.28mg/L, and BTCC patients serum FN significantly is lower than Normal group (P＜0.05).
Three, the straight line correlation of BTCC Urinary FN and serum FN
Recurrence and Correlation analysis show that its correlativity is very little, correlation coefficient r=0.0377, P＞0.05.
Embodiment 3
1. materials and methods
One, clinical data
1) carcinoma of urinary bladder group: 50 examples.Man's 34 examples, women 16 examples.Age 26-82 year, average 62 years old.Histological type is TCCB.According to the anticancer association of internation combination (UICC) by stages: T
129 examples, T
217 examples, T
34 examples.Pathological grading: G
118 examples, G
224 examples, G
38 examples.50 routine bladder cancer patients row partial cystectomy 42 examples, laser therapy 5 examples, full cystectomy 3 examples, all complete resection tumour.Reach month after operation before each operation in patients and all leave and take urine specimen.
2) non-carcinoma of urinary bladder group: other diseases of genito-urinary system patient is totally 30 examples.Man's 18 examples, women 12 examples.Age 34-76 year, average 60 years old.(1) optimum diseases of genito-urinary system group 20 examples wherein, male 12 examples, woman 8 example age 34-76 year, average 65 years old.Benign prostate hyperplasia 9 examples wherein, kidney stone 4 examples, uronephrosis 3 examples, renal cyst 2 examples, epididymitis 1 example, kidney abscess 1 example.(2) other genito-urinary system tumour 10 examples, male 6 examples, women 4 examples.Age 43-64 year, average 55 years old.Its carcinoma mesonephric 6 examples, prostate cancer 1 example, carcinoma of penis 1 example, carcinoma of renal pelvis 2 examples
3) Normal group: totally 20 examples.Man 12 falls women 8 examples.Age 27-80 year, average 60 years old.
Two, method
According to the experimental technique of embodiment 1, purification goat-anti people FN specific antibody; Urine specimen is taken from omnidistance urine for the first time in three groups of people early mornings, and-80 ℃ of low temperature save backup.
Measure urine Fn with the ELISA method, unit is ug/L, and the Fn standard items are Shanghai institute of Biological Products product.The anti-human Fn polyclonal antibody of rabbit, anti-Fn-HRP is Dakopatts company product, surveys simultaneously UCr, unit is umol/L.With the content that is compared to urine Fn of Fn and UCr, specific activity unit is ug/umol Cr, and the result processes through the t check.
Take the Fn normal concentration as horizontal ordinate, take the absorbance that records as ordinate, draw out the Fn typical curve, find tested sample concentration with the diluted urine specimen absorbance that is recorded and multiply by extension rate, be urine Fn content divided by UCr again.The urine composition varies widely because of the concentrated and dilution of urine, because of twenty-four-hour urine creatinine discharge rate substantially constant, urinates the Fn measurement result so mensuration is once urinated being compared to of sample content and UCr, and method is comparatively reliable.
2. experimental result
One, three groups of urine Fn content relatively
Transitional cell bladder carcinoma Fn content is up to 2.10ug/umol Cr, and minimum is 0.09ug/umol Cr, average 0.68 ± 0.49ug/umol Cr; Normal group is up to 0.15ug/umol Cr, and minimum is 0.07ug/umol Cr, average 0.10 ± 0.04ug/umol Cr: significant difference (P＜0.01) is arranged between two groups, show urine Fn content in bladder cancer patients apparently higher than the normal person.30 routine non-carcinoma of urinary bladder group Urine in Patients Fn content average out to: 0.20 ± 0.10ug/umol Cr.Wherein urine Fn is up to 0.34ug/umol Cr in the 20 routine optimum diseases of genito-urinary system groups, minimum 0.07ug/umol Cr, average 0.19 ± 0.06ug/umol Cr, with carcinoma of urinary bladder group difference conspicuousness (P＜0.01) is arranged, show urine Fn content in bladder cancer patients apparently higher than optimum diseases of genito-urinary system patient.The highest 0.53ug/umol Cr of urine Fn in 10 other genito-urinary system tumours of example, minimum 0.08ug/umol Cr, average 0.21 ± 0.14ug/umol Cr has conspicuousness (P＜0.01) with carcinoma of urinary bladder group difference.Wherein 2 routine carcinoma of renal pelvis urine Fn are respectively 0.53ug/umol Cr, 0.31ug/umol Cr.
Two, respectively organize comparison 50 routine bladder cancer patients urine Fn positive 40 examples (80%) of Fn positive rate, 50 routine non-bladder cancer patients and normal person's urine Fn negative patient 42 examples (84%), the susceptibility of urine Fn diagnosing bladder cancer is 80%, specificity is that 84% false negative rate is 20%, and false positive rate is 16%.
Three, carcinoma of urinary bladder urine Fn and tumor-infiltrated Degree of Accord Relation
Average 0.87 ± 0.50ug/umol the Cr of 21 routine invasive bladder carcinoma Urinary Fn, the average 0.55 ± 0.45ug/umol Cr of 29 routine shallow phenotype bladder cancer patients urine Fn.Show that urine Fn content is significantly higher than shallow phenotype carcinoma of urinary bladder (P＜0.01) in the patient of invasive bladder cancer, Fn is relevant with neoplasm staging.
Four, the pathological grading of carcinoma of urinary bladder urine Fn and tumour relation
This is organized 50 routine carcinomas of urinary bladder and is respectively by its mean urinary of pathological grading Fn content: G
118 examples, Fn is 0.40 ± 0.20ug/umolCr; G
224 examples, Fn is 0.78 ± 0.50ug/umol Cr; G
38 examples, Fn is 1.02 ± 0.64ug/umol Cr, P＜0.01.
Five, bladder cancer patients perioperatively urine Fn content relatively
Relatively 50 routine bladder cancer patients perioperatively urine Fn content are 0.68 ± 0.50ug/umol Cr before the art, and postoperative is 0.21 ± 0.11ug/umol Cr, and postoperative obviously reduces (P＜0.01) before than art.
So far invention has been described in conjunction with the embodiments.Those skilled in the art should be appreciated that in situation about not departing from the scope of the present invention with spirit, can easily make various other modifications to described embodiment.Therefore, the scope of appended claims is not limited to above-mentioned explanation, but will broadly explain claim.
Claims (5)
1. method for detection of Urine fibronectin, it is characterized in that: its operation steps is as follows:
1) collection of specimens:
Get transitional cell bladder carcinoma, urine 2,000-3 after the centrifugal 15-30 of 000r/min minute, collects supernatant, gained urine specimen to be measured;
2) Preparatory work of experiment:
Then dilution did 10 times of dilutions with distilled water in 37 ℃ of water-bath 15-30 minutes;
Then cleansing solution did 20 times of dilutions with distilled water in 37 ℃ of water-bath 15-30 minutes;
Every standard items 512ng/ml is accurately redissolved with the 1.0ml dilution.Get 250ul, make six doubling dilutions with dilution, and
Be diluted to one group of standard items;
3) application of sample: establish respectively blank hole, gauge orifice, testing sample hole, gauge orifice and testing sample hole add respectively variable concentrations standard items or urine specimen 100ul to be measured, add dilution 100ul in the blank hole;
4) incubation: at 37 ℃ of incubation 90-120 minutes;
5) wash plate: discard liquid in the reacting hole, fill with each hole with cleansing solution, leave standstill, dry, repeatedly pat dry after 3-5 time;
6) enzyme-added: described gauge orifice and testing sample hole add 100ul enzyme connection thing, gently mixing;
7) incubation: at 37 ℃ of incubation 60-90 minutes;
8) wash plate: discard liquid in the reacting hole, fill with each hole with described cleansing solution, leave standstill, drying pats dry after 3-5 time repeatedly;
9) colour developing: every substrate sheet is with the dissolving of 5ml substrate buffer solution before use, and every hole adds substrate solution 100ul, 37 ℃ incubation 15-20 minute;
10) stop: every hole adds the 50ul stop buffer, and mixing at microplate reader 492nm place, with the zeroing of blank hole, is measured each hole OD value gently.
2. method according to claim 1, it is characterized in that: the concentration range of wherein said urine specimen to be measured is 1: 1000-1: 6000.
3. method according to claim 2, it is characterized in that: the concentration range of wherein said urine specimen to be measured is 1: 2000-1: 4000.
4. method according to claim 1 is characterized in that: the sensitivity that described method is measured Urine fibronectin is 77%-100%.
One kind according to claim 1-4 arbitrary described method detect the kit of Urine fibronectin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110209641 CN102901824A (en) | 2011-07-26 | 2011-07-26 | Method for detecting urine fibronectin, and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110209641 CN102901824A (en) | 2011-07-26 | 2011-07-26 | Method for detecting urine fibronectin, and kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102901824A true CN102901824A (en) | 2013-01-30 |
Family
ID=47574175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110209641 Pending CN102901824A (en) | 2011-07-26 | 2011-07-26 | Method for detecting urine fibronectin, and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102901824A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675299A (en) * | 2013-12-24 | 2014-03-26 | 上海北加生化试剂有限公司 | Kit and method for detecting concentration of fibronectin in urine |
| CN111830023A (en) * | 2019-08-19 | 2020-10-27 | 杭州爱光医疗器械有限公司 | Sulfhydryl compound detection reagent, detection test paper, kit, test paper box and preparation method thereof |
-
2011
- 2011-07-26 CN CN 201110209641 patent/CN102901824A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675299A (en) * | 2013-12-24 | 2014-03-26 | 上海北加生化试剂有限公司 | Kit and method for detecting concentration of fibronectin in urine |
| CN111830023A (en) * | 2019-08-19 | 2020-10-27 | 杭州爱光医疗器械有限公司 | Sulfhydryl compound detection reagent, detection test paper, kit, test paper box and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11156613B2 (en) | Detection of cancer by elevated levels of bcl-2 | |
| Laurence et al. | Role of plasma carcinoembryonic antigen in diagnosis of gastrointestinal, mammary, and bronchial carcinoma | |
| JP6018923B2 (en) | How to predict the prognosis of sepsis | |
| Moonen et al. | Urinary NMP22® BladderChek® test in the diagnosis of superficial bladder cancer | |
| Sun et al. | CPA4 is a novel diagnostic and prognostic marker for human non-small-cell lung cancer | |
| CN102625914A (en) | Method for detection of, or the risk of, bladder cancer | |
| CN107406510A (en) | Prostate antigen standard items and application thereof | |
| CN102759623A (en) | Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof | |
| CN102072960A (en) | Method for detecting neutrophil gelatinase-associated lipocalin (NGAL) in sample | |
| CN105628932A (en) | SAA (serum amyloid A) immunochromatographic test strip and preparation method and test method thereof | |
| Wang et al. | Comparison of C3d immunohistochemical staining to enzyme-linked immunosorbent assay and immunofluorescence for diagnosis of bullous pemphigoid | |
| CN106680515B (en) | It is combined for the polymolecular marker of pulmonary cancer diagnosis | |
| Tsihlias et al. | The utility of fibrin/fibrinogen degradation products in superficial bladder cancer | |
| CN110726846A (en) | Application of HBP protein as diagnostic marker of Kawasaki disease | |
| JP6192122B2 (en) | Biomarkers for colorectal cancer diagnosis and prediction | |
| CN102901824A (en) | Method for detecting urine fibronectin, and kit thereof | |
| JP5801895B2 (en) | Method for detecting postoperative infections without systemic inflammatory response syndrome | |
| CN101493463B (en) | Stomach cancer diagnosis reagent and uses thereof | |
| Abdel-Razik et al. | A novel combination of C-reactive protein and vascular endothelial growth factor in differential diagnosis of ascites | |
| WO2014068018A1 (en) | Methods of prognosis and diagnosis of sepsis | |
| Svatek et al. | Role of urinary cathepsin B and L in the detection of bladder urothelial cell carcinoma | |
| WO2023065609A1 (en) | Molecular marker for determining very-early-stage onset risk of gastric cancer and evaluating progression risk of pre-cancerous lesion of gastric cancer, and use thereof in diagnostic kit | |
| CN107144688B (en) | CD19 positive excretion bodies are as application of the molecular labeling in preparing tumor diagnosis kit and kit | |
| CN113009135B (en) | Tubular magnetic particle chemiluminescence immune quantification kit for detecting CD47, and preparation method and application thereof | |
| Said et al. | Role of cancer antigen 125 in active pulmonary tuberculosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130130 |