CN104142401B - Tumor of bladder related antigen detection kit - Google Patents
Tumor of bladder related antigen detection kit Download PDFInfo
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- CN104142401B CN104142401B CN201410359816.3A CN201410359816A CN104142401B CN 104142401 B CN104142401 B CN 104142401B CN 201410359816 A CN201410359816 A CN 201410359816A CN 104142401 B CN104142401 B CN 104142401B
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to a kind of tumor of bladder related antigen detection kit.Test kit comprises two strain specificity tumor of bladder related antigen (BTAA) monoclonal antibodies.Tumor of bladder related antigen porous plate wrapping monoclonal antibody that the monoclonal antibody of quilt and horseradish peroxidase (HRP) mark and tested sample forms the sandwich complex structure of " coated antibody-antigen-HRP traget antibody ".HRP and then produce signal with substrate reactions, in order to carry out the quantitative analysis of tumor of bladder related antigen.By test kit of the present invention, to make it possible to rapidly, tumor of bladder related antigen content delicately in detection by quantitative sample.
Description
Technical field
The invention belongs to molecular immunology field, be specifically related to the test kit detecting tumor of bladder related antigen content in sample.
Background technology
Bladder cancer is the modal urological cancer of China, and the bladder cancer of about 90% is Urothelial Carcinoma of Bladder.The recurrence rate of bladder cancer is very high, and the patient of 60%-70% may be recurred, and the patient of 11% recurrence can be in progress as invasive tumor.At present for Diagnosis of Bladder and follow up a case by regular visits to and depend on cystoscopy and urinary cytology inspection, patient compliance is poor, and price comparison costliness, not easily as conventional microscopy; Although patient's specific degree is high and without wound, tumor sensitivity minuent being shown to shallow property is low, easily produce false-negative result.Therefore, find susceptibility and the high tumor of bladder mark of specific degree and receive increasing concern as the early diagnosis of bladder cancer, detection and prognosis evaluation.
Tumor of bladder is the modal tumour of urinary system, wherein with transitional cell carcinoma of bladder (bladdertransitionalcellcarcinoma, BTCC are the most common).BTCC biological behaviour is complicated and changeable, shows as easy recurrence, multiple, invasion and m etastasis, so these factors easily have influence on the Diagnosis and Treat to bladder cancer.At present, the diagnosis of bladder cancer mainly relies on urine sediment and cystoscopy, and cystoscopy belongs to invasive inspection, and patient is more painful, still has the possibility causing urinary tract infections in some cases, is not thus suitable for the group examination of bladder cancer.Other inspection methods, although as high in urine sediment inspection specific degree, sensitivity is poor, is easily subject to the interference of the factors such as urinary tract infections.In general, a kind of new clinical early diagnosis and detection method should have the feature of Noninvasive, and have good sensitivity and specific degree simultaneously, and take into account easy and simple to handle, use cost is low.Therefore, find highly sensitive, specific degree strong, and the woundless testing method being convenient to group examination is the focus of bladder cancer clinical study in recent years.
Set up a kind of easy fast, specific degree by force and not there is the early diagnosis that invasive inspection method is conducive to bladder cancer, to kind prognosis and to detect recurrence all significant.
Summary of the invention
Embodiment there is provided a kind of test kit detecting tumor of bladder related antigen in sample according to some, it comprises:
Be coated with the porous plate of the first tumor of bladder related antigen monoclonal antibody,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid.
In some embodiments, be coated with the first tumor of bladder related antigen monoclonal antibody in the porous plate of the first tumor of bladder related antigen monoclonal antibody to be produced by the hybridoma of preserving number by CGMCCNo.9317 or 9318.
When the hybridoma that the monoclonal antibody on porous plate is CGMCCNo.9317 by preserving number is produced, then the monoclonal antibody in enzyme working fluid is produced by another strain of hybridoma of the present invention; Vice versa, and when the hybridoma that the monoclonal antibody on porous plate is CGMCCNo.9318 by preserving number is produced, then the hybridoma that the monoclonal antibody in enzyme working fluid is CGMCCNo.9317 by preserving number produces.
In some embodiments, sample diluting liquid comprises sodium-chlor, lowlenthal serum, tween 20 and sanitas.In a particular embodiment, sample diluting liquid comprises 30g/L sodium-chlor, 25ml/L lowlenthal serum, 5ml/L tween 20 and 0.5ml/LProclin-300.Sample dilution can be mixed with concentrated type, may not be.Technician is appreciated that other cycles of concentration is also included within the scope of the invention.
In some embodiments, enzyme working fluid comprises the second tumor of bladder related antigen monoclonal antibody and sanitas of Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, sodium-chlor, calf serum, horseradish peroxidase-labeled.In some embodiments, the second tumor of bladder related antigen monoclonal antibody is produced by the hybridoma of preserving number by CGMCCNo.9318 or 9317.In a particular embodiment, enzyme working fluid comprises the second tumor of bladder related antigen monoclonal antibody of 5.8g/L Sodium phosphate dibasic, 0.593g/L SODIUM PHOSPHATE, MONOBASIC, 8.0g/L sodium-chlor, 200ml/L calf serum, 0.5ml/LProclin-300 and 0.4mg/L horseradish peroxidase-labeled.
In some embodiments, chemical luminescence for liquid comprises chemical luminescence for liquid A and chemical luminescence for liquid B.Described chemical luminescence for liquid A contains luminol,3-aminophthalic acid cyclic hydrazide and luminescence enhancer.Chemical luminescence for liquid B contains superoxide and luminescence enhancer.
Should be appreciated that each reagent of the present invention can be mixed with concentrated type, may not be.In some embodiments, some preparation of reagents become concentrated type, and this can reduce transport, reservoir volume.Technician is appreciated that other cycles of concentration except specific examples is also included within the scope of the invention.
In some embodiments, sample from Mammals, preferably from the mankind.Sample is selected from whole blood, blood plasma and serum.In a particular embodiment, sample is human urine.
In some embodiments, test kit of the present invention can also comprise calibration object as required.Calibration object is used for the drafting of typical curve.In a particular embodiment, calibration object comprises the tumor of bladder related antigen sterling of concentration known, and the concentration of tumor of bladder related antigen is in 10ng/ml to 4000ng/ml scope.In a particular embodiment, in calibration object the concentration respectively of tumor of bladder related antigen be 400,200,50,10,5,0ng/ml.Technician understands, and other concentration is applicable equally, such as, only need three concentration point to be just enough to set up a typical curve.
In some embodiments, test kit of the present invention can also comprise quality control product as required.In a particular embodiment, quality control product be known tumor of bladder related antigen concentration high level urine specimen dilution form.In a particular embodiment, the concentration of high level Quality Control is in 160ng/ml to 240ng/ml scope; The concentration of low value Quality Control is in 16ng/ml to 24ng/ml scope.
According to some embodiments, provide a kind of tumor of bladder related antigen monoclonal antibody, its hybridoma being CGMCCNo.9317 by preserving number is produced.
According to other embodiments, provide a kind of tumor of bladder related antigen monoclonal antibody, its hybridoma being CGMCCNo.9318 by preserving number is produced.
According to other embodiments, provide and be selected from one or both the purposes of tumor of bladder related antigen monoclonal antibody in preparation detection reagent following: the tumor of bladder related antigen monoclonal antibody that the hybridoma being CGMCCNo.9317 by preserving number produces and the tumor of bladder related antigen monoclonal antibody that the hybridoma being CGMCCNo.9318 by preserving number produces.In a specific embodiment, the hybridoma that preserving number is CGMCCNo.9317 and 9318 is produced two strain monoclonal antibodies jointly for the preparation of detection reagent.Described detection reagent is selected from ELISA detection reagent, immunoturbidimetry detection reagent, magnetic particle detection reagent, chemiluminescence detection reagent, immunofluorescence detection agent and radioimmunoassay test agent; Preferred chemiluminescence detection reagent; More preferably described detection reagent is detect the detection reagent of tumor of bladder related antigen.
According to some embodiments, provide a kind of hybridoma, its preserving number is CGMCCNo.9317.According to other embodiments, provide a kind of hybridoma, its preserving number is CGMCCNo.9318.
Accompanying drawing explanation
Fig. 1: adopt the working curve that test kit of the present invention is drawn.
Embodiment
In order to make easy to understand of the present invention, set forth the present invention further below in conjunction with specific embodiment.The following provide the concrete material and source thereof that use in embodiment of the present invention.But should be understood that, these are only exemplary, be not intended to limit the present invention, all may be used for implementing the present invention with the type of following reagent and instrument, model, quality, character or the same or analogous material of function.
The preparation of embodiment 1. monoclonal antibody and qualification
1. immune Balb/c mouse
Choose 6 week age, female Balb/c mouse that body weight is about 20g, initial immunity, get FactorH sterling (purchased from Fitzgerald, catalog number (Cat.No.) 30C-CP2038U) 20-50 μ g adds the subcutaneous multi-point injection of Freund's complete adjuvant, the 14th and within 28 days, carry out respectively second time and third time immunizing dose the same, add Freund's incomplete adjuvant intraperitoneal injection, merge first 3 days booster immunizations, dosage 20-50 μ g is advisable, and after 3 days, gets spleen and merges.
2. the step of cytogamy
Preparation feeder layer: get a non-immune Balb/c mouse, in 6 week age, draw neck to put to death, be immersed in 5min in 75% alcohol, cut off skin by sterile scissors, expose peritonaeum, inject the nutrient solution (forbidding to puncture intestinal tube) of 6ml precooling with asepsis injector, repeatedly rinse, sucking-off washing fluid, washing fluid puts into 10ml centrifuge tube, and 1200rpm/ is separated 6min, with the nutrient solution suspendible of 20% (v/v) foetal calf serum (FCS), adjustment cell count to 1 × 10
5/ ml, adds 96 orifice plates, and 100 μ l/ holes, put into 37 DEG C of CO
2incubator is cultivated.
Prepare immune spleen cell: get the Balb/c mouse that immunity is good, neck is drawn to put to death, asepticly get spleen, wash once with the incomplete nutrient solution of 10ml, spleen grinds, cross 200 order cell sieves, be transferred to by splenocyte in 10ml centrifuge tube, the centrifugal 10min of 800rpm, cell 10ml nutrient solution washes 2 times, cell counting, gets 1 × 10
8splenic lymphocyte suspension is for subsequent use.
Preparation myeloma cell SP2/0: the growth myeloma cell that takes the logarithm is centrifugal, washes 2 times with serum-free medium, and counting, obtains 5 × 10
7cell is for subsequent use.
Cytogamy: myeloma cell and splenocyte are mixed in the ratio of 1:10, washes 1 time with the incomplete nutrient solution of serum-free in 50ml centrifuge tube, centrifugal, 1200rpm, 8min; Abandon supernatant, to exhaust residual liquid with suction pipe, in order to avoid affect polyoxyethylene glycol (PEG) concentration.Gently at the bottom of attack centrifuge tube, cell precipitation is slightly loosened.
Add 1ml45% (g/100ml) PEG (molecular weight 4000) solution of 37 DEG C of pre-temperature, limit edged gentle agitation.37 DEG C of water-bath effect 90s.The incomplete nutrient solution adding 37 DEG C of pre-temperature, to stop PEG effect, adds 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2min.Centrifugal, 800rpm, 6min.Fill with clearly, select nutrient solution resuspended with containing 20% (v/v) calf serum HAT.By above-mentioned cell, be added in 96 orifice plates of existing feeder layer, every hole adds 100 μ l.Culture plate is put 37 DEG C, 5%CO
2cultivate in incubator.
3. the screening of hybridoma
After splenocyte and myeloma cell fusion 5 days, form the mixture of various kinds of cell, add HAT substratum 100 μ l, within the 10th day, change HT culture medium culturing.When hybridoma is covered with 1/5 area at the bottom of hole, indirect CLIA method can be adopted to detect culture supernatant, screening positive clone.With FactorH sterling (Fitzgerald) coated elisa plate (5 μ g/ml) 100 μ l/ hole, 4 DEG C are spent the night, and by the liquid in enzyme plate hole to the greatest extent, add PBST, repeated washing three times; Add confining liquid 200 μ l/ hole to close, be placed in 37 DEG C, 1 hour.Dry confining liquid, add 100 μ l cells and supernatant, positive control selects the immune serum of mouse, and negative control selects SP2/0 culture supernatant, and blank is washings, in 37 DEG C of standing 2h.Add HRP and mark sheep anti mouse two anti-(1:5000) 100 μ l/ hole, in 37 DEG C of standing 60min.Add PBST, repeated washing three times; Add substrate reactions liquid 100 μ l/ hole, put dark place for 37 DEG C and react 10 minutes.Add H
2sO
4(2mol/L) 50 μ l/ holes, termination reaction.Microplate reader detects 450nm absorbance.With FactorH sterling (Fitzgerald) for antigen, immune mouse, successfully obtain the hybridoma cell strain (numbering 12D4 and 11E9) of 2 strains secretion BTAA monoclonal antibodies, this two strains monoclonal antibody specific recognition in conjunction with FactorH sterling (Fitzgerald) respectively.
4. the cloning of hybridoma
Screen the positive colony that obtains, adopt limiting dilution assay to hybridoma cloning, clone first 1 day preparation feeder layer, the hybridoma that will the clone full substratum that cannots be used up dries up gently in culture hole, counting.Adjustment cell is 5 cell/ml.Get the Tissue Culture Plate of the feeder layer of preparation, every hole adds diluting cells 100 μ l.Hatch in 37 DEG C, 5%CO
2in incubator.Change liquid at the 7th day, within later every 3 days, change liquid 1 time.9 days visible cell Clone formation, chemoluminescence method detects antibody titer.And by the cloning again of the strongest positive colony, until cell positive rate reaches 100%, can strain be determined; The cell expansion determining strain is cultivated.And send the preservation of preservation center.
5. the preservation of hybridoma
Hybridoma cell strain (systematic name BTAA hybridoma cell strain) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on June 16th, 2014, and deposit number is respectively CGMCCNo.9317, CGMCCNo.9318.
A large amount of productions of embodiment 2. monoclonal antibody
Possess two strain monoclonal antibody hybridoma cell strains: get 1 × 10
7cell concn be injected in Balb/c mouse peritoneal, collect ascites after 10 days.By following steps monoclonal antibody purification:
1. the ascites of collecting gained is centrifugal with 2500rpm, gets supernatant.Add the saturated ammonium sulphate of isopyknic PBS (pH7.4) and 1/2 volume, 4 DEG C, standing 30min;
2. with 3000rpm, 4 DEG C, centrifugal 20min;
3. go precipitation, above reset and add isopyknic saturated ammonium sulphate, leave standstill 30min;
4. with 3000rpm, 4 DEG C, centrifugal 20min;
5. get precipitation, add 5ml physiological saline and 5ml saturated ammonium sulphate leaves standstill 30min;
6. with 3000rpm, 4 DEG C, centrifugal 20min;
7. precipitation adds the PB damping fluid (PH7.4) of 5ml physiological saline and 5ml0.02M;
8. add PB damping fluid balance ProteinG gel column (purchased from GE company) of 8 times of column volumes;
9. the mixed solution in the 7th step is added in gel column;
10. use the PB buffer solution elution foreign protein of 10 times of column volumes;
11. collect with 0.2MpH2.8 glycine buffer antibody elution;
12. use regenerated liquid cleaning pillar;
13. with balance liquid balance pillar.
The preparation of the chemiluminescence detection kit of embodiment 3. tumor of bladder related antigen of the present invention
1. preparation is coated with the porous plate of tumor of bladder related antigen monoclonal antibody:
A) bag quilt: adopt 0.05M, pH value to be that tumor of bladder related antigen monoclonal antibody (CGMCCNo.9317) prepared by the carbonate buffer solution of 9.6 and the embodiment 2 of proper concn is mixed and wraps mixed liquid, and being loaded on porous plate, 4 DEG C of bags are by 12h;
Carbonate buffer solution standard recipe:
Anhydrous sodium carbonate 1.600g
Sodium bicarbonate 2.940g
PH value 9.60 ~ 9.80 purified water is settled to 1000ml
B) wash plate: dilute 20 times of concentrated washing lotion to 1 times concentration, use 1 times of concentration washing lotion to wash plate 2 times; 20 times of concentrated washing lotion standard recipes:
C) close: by confining liquid (Sodium phosphate dibasic 5.8g, SODIUM PHOSPHATE, MONOBASIC 0.593g, sodium-chlor 8.0g, lowlenthal serum 100ml, Proclin-3000.5ml, purified water is settled to 1000ml) load on porous plate, room temperature 2 hours, dries, dried overnight.
2. the preparation of enzyme working fluid:
2.1 markers step:
(1) take 1mg horseradish peroxidase (HRP, purchased from Sigma) to be dissolved in 300 μ l distilled water.
(2) in upper liquid, add the 0.1MNaIO that 0.6 μ l newly joins
4solution, under room temperature, lucifuge stirs 20 minutes.
(3) load in dialysis tubing by above-mentioned solution, use the sodium-acetate buffer dialysis of 1mMpH4.4,4 DEG C are spent the night.
(4) 20 μ l1MpH9.5 carbonate buffer solutions are added, the pH of HRP is made to be elevated to 9.0 to 9.5, then add 1mg another strain antibody (CGMCCNo.9318) (in 1ml0.01M carbonate buffer solution) of the present invention immediately, room temperature lucifuge stirs 2 hours gently.
(5) 4mg/mlNaBH that 0.05ml newly joins is added
4liquid, mixing, then put 4 DEG C 2 hours.
(6) load in dialysis tubing by above-mentioned liquid, use 0.15MpH7.4PBS dialysis, 4 DEG C are spent the night.
(7) add isopyknic glycerine ,-20 DEG C of preservations, final horseradish peroxidase labeling antibody concentration is 1mg/ml.
2.2 enzymic-labelled antibody concentration are determined:
Adopt policy method to select the working concentration of enzyme labelled antibody, get 2 μ l enzyme labelled antibodies (1mg/ml) and join in 5ml enzyme diluent, be i.e. the ratio of 1:2500.
The preparation of 2.3 enzyme working fluids:
By the monoclonal antibody of horseradish peroxidase-labeled prepared in 2.1 steps, with the ratio of 1:2500, enzyme labelled antibody is dissolved in enzyme buffer liquid.Enzyme buffer liquid formula is: 5.8g/L Sodium phosphate dibasic, 0.593g/L SODIUM PHOSPHATE, MONOBASIC, 8.0g/L sodium-chlor, 200ml/L calf serum, 0.5ml/LProclin-300).
3. the preparation of sample diluting liquid:
1000ml sample diluting liquid comprises 30.0g sodium-chlor, 25.0ml lowlenthal serum, 5.0ml tween 20,0.5mlProclin-300.
4. chemical luminescence for liquid: chemical luminescence for liquid A and chemical luminescence for liquid B is outsourcing reagent (purchased from Roche), and wherein chemical luminescence for liquid A contains luminol,3-aminophthalic acid cyclic hydrazide and luminescence enhancer, chemical luminescence for liquid B contains superoxide and luminescence enhancer.Or according to chemiluminescence, prepare voluntarily.
5. the preparation of calibration object:
With sample diluting liquid, the FactorH sterling of Fitzgerald company is diluted, makes the tumor of bladder related antigen concentration gradient in calibration object be respectively 400,200,50,10,5,0ng/ml.
As required, calibration object can be traced to the source to the reference material of Fitzgerald company or other reference material.
6. the preparation of quality control product:
Collect the human urine sample (urine can be any commercially available urine specimen or collect from Clinical Institutions) of tumor of bladder related antigen clinical detection high level, with sample diluting liquid dilution high level human urine in the concentration range expected.High level Quality Control: 160ng/ml to 240ng/ml; Low value Quality Control: 16ng/ml to 24ng/ml.2-8 DEG C saves backup.
Above-mentioned each reagent set is dressed up test kit, the instrument such as working instructions, shrouding film can also be incorporated to as required in test kit.
Test case
The using method of test case 1. tumor of bladder related antigen of the present invention chemical luminescence reagent kit
1.1. prepare before experiment
1.1. in 4 DEG C of refrigerators, take out test kit, room temperature (18-25 DEG C) all should be equilibrated to.
1.2.20 doubly use after concentrated cleaning solution purified water or distilled water diluting 20 times.
2. test method
2.1. application of sample incubation: the bag getting sufficient amount, by plate, is fixed on framework, arranges calibration sample wells, Quality Control sample wells and testing sample hole respectively, records each hole site; 45 μ l sample diluting liquids are added in testing sample hole, calibration object 50 μ l is added in order in calibration sample wells, add quality control product 50 μ l in Quality Control sample wells, in sample well, add the testing sample (be equal to sample and carried out 10 times of dilutions) of 5 μ l respectively.Every hole adds enzyme working fluid 50 μ l (advising completing this step operation in 20min), and concussion mixing, adds a cover shrouding film, 37 DEG C of incubation 30min.
Note: calibration object and quality control product, without the need to dilution, can directly use.
2.2. plate is washed:
By hand wash plate: every hole adds 350 μ l washing lotions, abandon to the greatest extent after leaving standstill 5-10 second, after repeating flushing 5 times, pat dry;
Wash trigger and wash plate: every hole adds 350 μ l washing lotions, each washing room, every 5-10 second, after repeating flushing 5 times, pats dry.
2.3. luminous: every hole adds 50 μ l chemical luminescence for liquid A and 50 μ l chemical luminescence for liquid B, fully reading after mixing.
2.4. measure: measure RLU value under putting Chemiluminescence Apparatus immediately.
2.5. calculate: according to the concentration of calibration object and the RLU value of correspondence, return to zero with calibration object S0 hole, use double-log linear fit mode (log (X)-log (Y)) calculation result, result is multiplied by extension rate (10 times), is sample ultimate density.
Linear equation is log (RLU)=4.513+1.065log (concentration); R=1.00.
The methodology index of test case 2. test kit of the present invention
1. accuracy: the rate of recovery should between 85% to 115%;
2. blank detection limit: should not higher than 1ng/ml;
3. measuring system is linear: in 10ng/ml to 4000ng/ml scope internal linear correlation coefficient r >=0.9900.
4. repeatability: variation within batch coefficient CV
in batchshould 10% be no more than;
5. difference between batch: interassay coefficient of variation CV
between batchshould 15% be no more than;
Table 1. accuracy, minimum detectability, system linear, repeated detected result
6. analyze specificity:
Detect tumor of bladder related antigen negative sample N1, positive sample P1 urine specimen respectively.
In N1, P1 sample, add the cross antigen of three concentration gradients respectively, detect the cross reaction situation that cross antigen detects tumor of bladder associated antigen:
People factor I concentration (25ng/ml, 12.5ng/ml, 0ng/ml),
People's factor D concentration (10ng/ml, 5ng/ml, 0ng/ml),
Ox factor H concentration (100ng/ml, 50ng/ml, 0ng/ml)
Mouse factor H concentration (100ng/ml, 50ng/ml, 0ng/ml)
Interpretation of result: cross antigen usually and test substance there is higher homology, sequence similarity or there is the epi-position of structural similitude.Can imagine, if when there are these cross antigens in sample, antibody may identify and in conjunction with these cross antigens, cause false positive.Therefore, the cross reactivity of diagnostic kit is an important index.This reliability for clinical effectiveness is extremely important.
From following table 4, when people factor I be not more than 25ng/ml, people's factor D is not more than 10ng/ml, Niu Yinzi H and mouse factor H is not more than 100ng/ml time, do not have statistically significant to affect on the detected result of tumor of bladder related antigen.Show the anti-cross reactivity that test kit of the present invention is excellent.
7. stability: each component of test kit 37 DEG C can place 6 days.
Shelf stability experimental result spent by table 2.37
Table 3.4 DEG C preservation condition stability inferior experimental result
R-H, R-L represent respectively: high level accuracy reference material, low value accuracy reference material.
8. interfering substance analysis:
8.1 urine Lactoferrins, experimental result is in table 5.
Conclusion: according to result, as slight molten Urine specimens urine Lactoferrin≤200mg/ml, detect on result substantially without affecting with this test kit, when serious molten urine, detected result may cause false positive, therefore should not with serious molten Urine specimens.
8.2 albumin, experimental result is in table 6.
Conclusion: when albumin concentration is in 1000mg/dl, does not have a significant impact tumor of bladder associated antigen detected result.
8.3 bilirubin, experimental result is in table 7.
Interpretation of result: tumor of bladder associated antigen detected result is affected without statistically significant when bilirubin concentration is not more than 20mg/dl.
8.4 uric acid, experimental result is in table 8.
Interpretation of result: tumor of bladder associated antigen detected result is affected without statistically significant when uric acid concentration is not more than 100mg/dl.
8.5 xitix, experimental result is in table 9.
Interpretation of result: tumor of bladder associated antigen detected result is affected without statistically significant when ascorbic acid concentrations is not more than 2g/dl.
8.6 human IgGs, experimental result is in table 10.
Interpretation of result: when human IgG concentration is not more than 10g/dl, affects without statistically significant tumor of bladder associated antigen detected result.
Brief summary:
1) two strain specific antibodies of the present invention are utilized to prepare test kit, adopt double antibody sandwich method to measure tumor of bladder related antigen level in urine, the tumor of bladder related antigen antigen of the antibody and HRP traget antibody and tested sample that namely enzyme mark porous plate wrap quilt forms the sandwich complex structure of " coated antibody-antigen-HRP traget antibody ".Achieve highly sensitive and the specificity of detection.
2) one aspect of the present invention can measure the content of tumor of bladder related antigen in urine quickly and accurately, provides reliable clinical reference value for the early diagnosis of tumor of bladder, early treatment.
3) on the other hand owing to using specific monoclonal antibody as bag by solid phase, substantially increase detection sensitivity and the specificity of tumor of bladder related antigen, immunity from interference is strong.
4) the present invention have easy to use, detect the advantages such as quick, sensitive, stable, easy and simple to handle.Be applicable to vast middle and small hospital use.
Claims (7)
1. detect a test kit for tumor of bladder related antigen in sample, it comprises:
Be coated with the porous plate of the first tumor of bladder related antigen monoclonal antibody,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid;
Wherein:
Sample diluting liquid comprises: sodium-chlor, lowlenthal serum, tween 20 and sanitas;
Enzyme working fluid comprises: the second tumor of bladder related antigen monoclonal antibody and sanitas of Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, sodium-chlor, calf serum, horseradish peroxidase-labeled;
Chemical luminescence for liquid comprises: chemical luminescence for liquid A and chemical luminescence for liquid B; Described chemical luminescence for liquid A contains luminol,3-aminophthalic acid cyclic hydrazide and luminescence enhancer, and chemical luminescence for liquid B contains superoxide and luminescence enhancer;
The hybridoma that described first tumor of bladder related antigen monoclonal antibody is CGMCCNo.9317 by preserving number is produced, and the hybridoma that described second tumor of bladder related antigen monoclonal antibody is CGMCCNo.9318 by preserving number is produced, or
The hybridoma that described first tumor of bladder related antigen monoclonal antibody is CGMCCNo.9318 by preserving number is produced, and the hybridoma that described second tumor of bladder related antigen monoclonal antibody is CGMCCNo.9317 by preserving number is produced;
Described sample is from human urine.
2. the test kit of tumor of bladder related antigen in detection sample according to claim 1, also comprises calibration object,
Described calibration object comprises the tumor of bladder related antigen sterling of concentration known, and the concentration of tumor of bladder related antigen is in 10ng/ml to 4000ng/ml scope.
3. the test kit of tumor of bladder related antigen in detection sample according to claim 1, also comprises quality control product,
Described quality control product is that the high level urine specimen dilution of known tumor of bladder related antigen concentration forms.
4. detect a chemiluminescence immune detection reagent kit for tumor of bladder related antigen in sample, it comprises:
Be coated with the porous plate of the first tumor of bladder related antigen monoclonal antibody,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid;
Wherein:
Sample diluting liquid comprises: 30g/L sodium-chlor, 25ml/L lowlenthal serum, 5ml/L tween 20 and 0.5ml/LProclin-300;
Enzyme working fluid comprises: the second tumor of bladder related antigen monoclonal antibody of 5.8g/L Sodium phosphate dibasic, 0.593g/L SODIUM PHOSPHATE, MONOBASIC, 8.0g/L sodium-chlor, 200ml/L calf serum, 0.5ml/LProclin-300 and 0.4mg/L horseradish peroxidase-labeled;
Chemical luminescence for liquid is made up of chemical luminescence for liquid A and chemical luminescence for liquid B; Described chemical luminescence for liquid A contains luminol,3-aminophthalic acid cyclic hydrazide and luminescence enhancer, and chemical luminescence for liquid B contains superoxide and luminescence enhancer;
The hybridoma that described first tumor of bladder related antigen monoclonal antibody is CGMCCNo.9317 by preserving number is produced, and the hybridoma that described second tumor of bladder related antigen monoclonal antibody is CGMCCNo.9318 by preserving number is produced, or
The hybridoma that described first tumor of bladder related antigen monoclonal antibody is CGMCCNo.9318 by preserving number is produced, and the hybridoma that described second tumor of bladder related antigen monoclonal antibody is CGMCCNo.9317 by preserving number is produced.
5. the purposes be combined in preparation detection reagent of two kinds of tumor of bladder related antigen monoclonal antibodies below:
The tumor of bladder related antigen monoclonal antibody that the hybridoma being CGMCCNo.9317 by preserving number produces and
The tumor of bladder related antigen monoclonal antibody that the hybridoma being CGMCCNo.9318 by preserving number produces;
Described detection reagent is selected from: ELISA detection reagent, immunoturbidimetry detection reagent, magnetic particle detection reagent, chemiluminescence detection reagent, immunofluorescence detection agent and radioimmunoassay test agent.
6. purposes according to claim 5, wherein said detection reagent is chemiluminescence detection reagent.
7. purposes according to claim 5, wherein said detection reagent is detect the detection reagent of tumor of bladder related antigen.
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| CN201410359816.3A CN104142401B (en) | 2014-07-25 | 2014-07-25 | Tumor of bladder related antigen detection kit |
| PCT/CN2015/081159 WO2016011852A1 (en) | 2014-07-25 | 2015-06-10 | Bladder tumor-associated antigen detection kit |
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| CN201410359816.3A CN104142401B (en) | 2014-07-25 | 2014-07-25 | Tumor of bladder related antigen detection kit |
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| CN104142401B (en) * | 2014-07-25 | 2016-04-20 | 北京普恩光德生物科技开发有限公司 | Tumor of bladder related antigen detection kit |
| CN105017419B (en) * | 2015-07-06 | 2019-01-04 | 北京普恩光德生物科技开发有限公司 | The monoclonal antibody and its detection kit of anti-human NMP-22 |
| CN106370864A (en) * | 2016-08-31 | 2017-02-01 | 镇江华测金太医学检验所有限公司 | Chemiluminiscence box of neutrophile granulocyte gelatinase related lipid transport protein |
| CN106353508A (en) * | 2016-08-31 | 2017-01-25 | 镇江华测金太医学检验所有限公司 | Chemiluminescent enzyme-linked immunosorbent assay kit for bladder tumor-associated antigens |
| CN109100512A (en) * | 2018-08-02 | 2018-12-28 | 宁波奥丞生物科技有限公司 | A kind of chemical luminescence reagent kit detecting EGFR |
| CN115261401B (en) * | 2022-08-25 | 2023-10-20 | 杭州博岳生物技术有限公司 | Method for developing bladder tumor antigen monoclonal antibody by using yeast cell surface display technology |
| CN117089529B (en) * | 2023-10-16 | 2024-03-15 | 深圳普门科技股份有限公司 | Hybridoma cell, preparation method thereof, monoclonal antibody and kit |
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| CN1440462A (en) * | 2000-04-28 | 2003-09-03 | 麦康公司 | Method of identifying and producing antigen peptides and use thereof as vaccines |
| AU2007332473B2 (en) * | 2006-12-14 | 2012-09-27 | Forerunner Pharma Research Co., Ltd. | Anti-Claudin-3 monoclonal antibody, and treatment and diagnosis of cancer using the same |
| CN101368961B (en) * | 2008-04-02 | 2012-11-14 | 北京科美生物技术有限公司 | Chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof |
| CN102372773B (en) * | 2010-08-11 | 2013-06-05 | 中国科学院生物物理研究所 | Human bladder cancer tumor marker, antibody thereof and application thereof |
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| US20140072987A1 (en) * | 2012-09-12 | 2014-03-13 | Randox Laboratories Ltd. | Methods of detecing bladder cancer |
| CN103018461B (en) * | 2012-12-13 | 2015-03-18 | 中国科学院生物物理研究所 | Kit for early diagnosis of bladder cancer and preparation method of kit |
| CN103033625B (en) * | 2012-12-19 | 2014-12-24 | 中国科学院生物物理研究所 | Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof |
| CN103941017B (en) * | 2014-03-18 | 2016-01-20 | 北京普恩光德生物科技开发有限公司 | C reactive protein detection kit |
| CN104142401B (en) * | 2014-07-25 | 2016-04-20 | 北京普恩光德生物科技开发有限公司 | Tumor of bladder related antigen detection kit |
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