CN104142401A - Bladder tumor-associated antigen detection kit - Google Patents
Bladder tumor-associated antigen detection kit Download PDFInfo
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- CN104142401A CN104142401A CN201410359816.3A CN201410359816A CN104142401A CN 104142401 A CN104142401 A CN 104142401A CN 201410359816 A CN201410359816 A CN 201410359816A CN 104142401 A CN104142401 A CN 104142401A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3038—Kidney, bladder
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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 invention relates to a bladder tumor-associated antigen detection kit. The kit contains two specificity bladder tumor-associated antigen (BTAA) monoclonal antibodies. The coated monoclonal antibody on a perforated plate, the monoclonal antibody labeled by horseradish peroxidase (HRP), and a bladder tumor-associated antigen of a detected sample form a coated antibody-antigen-HRP labeled antibody sandwich compound structure. The HRP reacts with a substrate for generating signals used for analyzing the bladder tumor-associated antigen in a quantitative mode. By means of the kit, the content of the bladder tumor-associated antigen in the detected sample can be fast and sensitively detected in the quantitative mode.
Description
Technical field
The invention belongs to molecular immunology field, be specifically related to detect the kit of tumor of bladder related antigen content in sample.
Background technology
Carcinoma of urinary bladder is the modal urological cancer of China, and about 90% carcinoma of urinary bladder is Urothelial Carcinoma of Bladder.The recurrence rate of carcinoma of urinary bladder is very high, and the patient of 60%-70% may be recurred, and the patient of 11% recurrence can make progress as wellability tumour.For Diagnosis of Bladder and follow up a case by regular visits to and mainly depend on cystoscopy and urinary cytology inspection, patient compliance is poor at present, and price comparison is expensive, is difficult for as conventional microscopy; Although patient's specificity is high and without wound, the tumor sensitivity of minuent being shown to shallow property is low, easily produces false-negative result.Therefore the tumor of bladder mark that, searching susceptibility and specificity are high receives increasing concern as early diagnosis, detection and the prognosis evaluation of carcinoma of urinary bladder.
Tumor of bladder is the modal tumour of urinary system, wherein with TCCB (bladder transitional cell carcinoma, BTCC is the most common).BTCC biological behaviour is complicated and changeable, show as easy recurrence, multiple, infiltrate and shift, so these factors easily have influence on the diagnosis of carcinoma of urinary bladder and treatment.At present, the diagnosis of carcinoma of urinary bladder mainly relies on urine exfoliative cytology and cystoscopy, and cystoscopy belongs to invasive inspection, and patient is more painful, still has the possibility that causes urinary tract infections in some situation, thereby is not suitable for the group examination of carcinoma of urinary bladder.Other inspection methods, although as high in urine exfoliative cytology inspection specificity, sensitivity is poor, is easily subject to the interference of the factors such as urinary tract infections.In general, the feature that a kind of new clinical early diagnosis and detection method should have Noninvasive has good sensitivity and specificity simultaneously, and takes into account easy and simple to handlely, and use cost is low.Therefore, find highly sensitive, specificity is strong, and the non-invasive inspection method of being convenient to group examination is the focus of carcinoma of urinary bladder clinical research in recent years.
Set up a kind of easy fast, specificity has by force and not the early diagnosis that invasive inspection method is conducive to carcinoma of urinary bladder, to kind prognosis and to detect recurrence all significant.
Summary of the invention
According to some embodiments, a kind of kit that detects tumor of bladder related antigen in sample is provided, it comprises:
Be coated with the first tumor of bladder related antigen monoclonal antibody porous plate,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid.
In some embodiments, the hybridoma by CGMCC No.9317 or 9318 is produced by preserving number to be coated with in the porous plate of the first tumor of bladder related antigen monoclonal antibody the first tumor of bladder related antigen monoclonal antibody.
When the hybridoma that the monoclonal antibody on porous plate is CGMCC No.9317 by preserving number is produced, 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 CGMCC No.9318 by preserving number is produced, the hybridoma that monoclonal antibody in enzyme working fluid is CGMCC No.9317 by preserving number produces.
In some embodiments, sample diluting liquid comprises sodium chloride, lowlenthal serum, Tween-20 and antiseptic.In specific embodiments, sample diluting liquid comprises 30g/L sodium chloride, 25ml/L lowlenthal serum, 5ml/L Tween-20 and 0.5ml/L Proclin-300.Sample dilution can be mixed with concentrated type, can not be also.Technician is appreciated that other cycles of concentration is also included within the scope of the invention.
In some embodiments, the second tumor of bladder related antigen monoclonal antibody and antiseptic that enzyme working fluid comprises sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, calf serum, horseradish peroxidase-labeled.In some embodiments, by preserving number, the hybridoma by CGMCC No.9318 or 9317 is produced the second tumor of bladder related antigen monoclonal antibody.In specific embodiments, the second tumor of bladder related antigen monoclonal antibody that enzyme working fluid comprises 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/L Proclin-300 and 0.4 mg/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 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, can not be yet.In some embodiments, some reagent are mixed with concentrated type, and this can reduce transportation, reservoir volume.Technician is appreciated that other cycles of concentration except instantiation is also included within the scope of the invention.
In some embodiments, sample is from mammal, preferably from the mankind.Sample is selected from whole blood, blood plasma and serum.In specific embodiments, sample is human urine.
In some embodiments, kit of the present invention can also comprise calibration object as required.Calibration object is for the drafting of typical curve.In specific embodiments, the tumor of bladder related antigen sterling that calibration object comprises concentration known, the concentration of tumor of bladder related antigen is in 10ng/ml to 4000ng/ml scope.In specific embodiments, in calibration object, the concentration respectively of tumor of bladder related antigen is 400,200,50,10,5,0 ng/ml.Technician understands, and other concentration is applicable equally, for example, only need three concentration point to be just enough to set up a typical curve.
In some embodiments, kit of the present invention can also comprise quality-control product as required.In specific embodiments, quality-control product is that the high value urine specimen dilution of known tumor of bladder related antigen concentration forms.In specific embodiments, the concentration of high value Quality Control is in 160 ng/ml to 240ng/ml scopes; The concentration of low value Quality Control is in 16 ng/ml to 24ng/ml scopes.
According to some embodiments, a kind of tumor of bladder related antigen monoclonal antibody is provided, by preserving number, the hybridoma by CGMCC No.9317 is produced for it.
According to other embodiments, a kind of tumor of bladder related antigen monoclonal antibody is provided, by preserving number, the hybridoma by CGMCC No.9318 is produced for it.
According to other embodiments, provide to be selected from following one or both tumor of bladder related antigen monoclonal antibody and to detect the purposes in reagent in preparation: the tumor of bladder related antigen monoclonal antibody that the tumor of bladder related antigen monoclonal antibody that the hybridoma that is CGMCC No.9317 by preserving number produces and the hybridoma that is CGMCC No.9318 by preserving number produce.In a specific embodiments, the hybridoma that preserving number is CGMCC No.9317 and 9318 is produced two strain monoclonal antibodies jointly for the preparation of detecting reagent.Described detection reagent is selected from ELISA and detects 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 for detecting the detection reagent of tumor of bladder related antigen.
According to some embodiments, a kind of hybridoma is provided, its preserving number is CGMCCNo.9317.According to other embodiments, a kind of hybridoma is provided, its preserving number is CGMCC No.9318.
Accompanying drawing explanation
Fig. 1: the calibration curve that adopts kit of the present invention to draw.
Embodiment
In order to make easy to understand of the present invention, below in conjunction with specific embodiment, further set forth the present invention.Concrete material and the source thereof of in embodiment of the present invention, using are below provided.But, should be understood that, these are only exemplary, are not intended to limit the present invention, with the same or analogous material of type, model, quality, character or function of following reagent and instrument all can be for implementing the present invention.
Preparation and the evaluation of embodiment 1. monoclonal antibodies
1. immune Balb/c mouse
Choose the female Balb/c mouse of 6 week age, the about 20g of body weight, initial immunity, get Factor H 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, and the 14th and within 28 days, carry out for the second time respectively and immunizing dose is the same for the third time, add incomplete Freund's 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 Fusion of Cells
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, by sterile scissors, cut off skin, expose peritonaeum, with asepsis injector, inject the nutrient solution (forbidding to puncture intestinal tube) of 6ml precooling, repeatedly rinse, sucking-off washing fluid, washing fluid is put into 10ml centrifuge tube, the separated 6min of 1200 rpm/, with the nutrient solution suspendible of 20% (v/v) hyclone (FCS), adjust cell number to 1 * 10
5/ ml, adds 96 orifice plates, and 100 μ l/ holes, put into 37 ℃ of CO
2incubator is cultivated.
Prepare immune spleen cell: get the Balb/c mouse that immunity is good, draw neck to put to death, the aseptic spleen of getting, washes once with the incomplete nutrient solution of 10ml, and spleen grinds, cross 200 order cell sieves, splenocyte is transferred in 10ml centrifuge tube, centrifugal 10 min of 800rpm, cell is washed 2 times with 10ml nutrient solution, cell count, gets 1 * 10
8splenic lymphocyte suspension is standby.
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 standby.
Fusion of Cells: myeloma cell and splenocyte are mixed in the ratio of 1:10, in 50ml centrifuge tube, with the incomplete nutrient solution of serum-free, wash 1 time, centrifugal, 1200rpm, 8min; Abandon supernatant, with the suction pipe residual liquid that exhausts, in order to avoid affect polyglycol (PEG) concentration.At the bottom of attack centrifuge tube, make cell precipitation slightly loosening gently.
1ml 45% (g/100ml) PEG (molecular weight 4000) solution that adds 37 ℃ of pre-temperature, limit edged slightly shakes.37 ℃ of water-bath effect 90s.Add the incomplete nutrient solution of 37 ℃ of pre-temperature to stop PEG effect, every 2min, add respectively 1ml, 2ml, 3ml, 4ml, 5ml and 6ml.Centrifugal, 800rpm, 6min.Fill with clearly, with selecting nutrient solution resuspended 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 to 37 ℃, 5%CO
2in incubator, cultivate.
3. the screening of hybridoma
Splenocyte and myeloma cell are merged latter 5 days, form the mixture of various kinds of cell, add HAT nutrient culture media 100 μ l, within the 10th day, change HT medium culture.When hybridoma is covered with at the bottom of hole 1/5 area, can adopt indirect CLIA method to detect culture supernatant, screening positive clone.With Factor H sterling (Fitzgerald) coated elisa plate (5 μ g/ml) 100 μ l/ holes, 4 ℃ are spent the night, and by the liquid in ELISA Plate hole to the greatest extent, add PBST, repeated washing three times; Add confining liquid 200 μ l/ holes to seal, be placed in 37 ℃, 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 cleansing solution, in 37 ℃ of standing 2h.Add anti-(1:5000) the 100 μ l/ holes of HRP mark sheep anti mouse two, in 37 ℃ of standing 60min.Add PBST, repeated washing three times; Add substrate reactions liquid 100 μ l/ holes, put dark place reaction 10 minutes for 37 ℃.Add H
2sO
4(2mol/L) 50 μ l/ holes, cessation reaction.Microplate reader detects 450nm absorbance.The Factor H sterling (Fitzgerald) of take is antigen, immune mouse, successfully obtain the hybridoma cell strain (numbering 12D4 and 11E9) of 2 strains secretions BTAA monoclonal antibodies, this two strains monoclonal antibody is specific recognition in conjunction with Factor H sterling (Fitzgerald) respectively.
4. the cloning of hybridoma
The positive colony that obtains of screening, adopts limiting dilution assay to hybridoma cloning, clones first 1 day preparation feeder layer, and the hybridoma that will the clone full nutrient culture media that toos many or too much for use dries up gently in culture hole, counting.Adjusting 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 ℃, 5%CO
2in incubator.At the 7th day, change liquid, within later every 3 days, change liquid 1 time.Within 9 days, visible cell clone forms, and chemoluminescence method detects antibody titer.And by the cloning again of the strongest positive colony, until cell positive rate reaches 100%, can determine strain; The cell of determining strain expands to be 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 to 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 CGMCC No.9317, CGMCC No.9318.
A large amount of productions of embodiment 2. monoclonal antibodies
Possessed two strain monoclonal antibody hybridoma cell strains: get 1 * 10
7cell concentration be injected in Balb/c mouse peritoneal, after 10 days, collect ascites.By following steps monoclonal antibody purification:
1. the ascites of collecting gained is centrifugal with 2500 rpm, gets supernatant.The saturated ammonium sulfate that adds isopyknic PBS (pH7.4) and 1/2 volume, 4 ℃, standing 30min;
2. with 3000 rpm, 4 ℃, centrifugal 20min;
3. go precipitation, above reset and add isopyknic saturated ammonium sulfate, standing 30min;
4. with 3000 rpm, 4 ℃, centrifugal 20min;
5. get precipitation, add 5ml physiological saline and the standing 30min of 5ml saturated ammonium sulfate;
6. with 3000 rpm, 4 ℃, centrifugal 20min;
7. precipitation adds the PB damping fluid (PH7.4) of 5ml physiological saline and 5ml 0.02M;
8. the PB damping fluid balance Protein G gel column (purchased from GE company) that adds 8 times of column volumes;
9. the mixed liquor 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. use 0.2M pH2.8 glycine buffer wash-out antibody are also collected;
12. use regenerated liquid to clean pillar;
13. with equilibrium 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) coated: to adopt tumor of bladder related antigen monoclonal antibody (CGMCC No.9317) prepared by the embodiment 2 of 0.05M, carbonate buffer solution that pH value is 9.6 and debita spissitudo to be mixed and made into coated mixed liquor, and loaded on porous plate 4 ℃ of coated 12h;
Carbonate buffer solution standard recipe:
Natrium carbonicum calcinatum 1.600g
Sodium bicarbonate 2.940g
PH value 9.60～9.80 purified water are 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) sealing: confining liquid (sodium chloride 8.0g, lowlenthal serum 100ml, Proclin-3000.5ml, purified water is settled to 1000ml for sodium hydrogen phosphate 5.8g, sodium dihydrogen phosphate 0.593g) is loaded on porous plate, and room temperature 2 hours, dries dried overnight.
2. the preparation of enzyme working fluid:
2.1 markers step:
(1) taking 1 mg horseradish peroxidase (HRP, purchased from Sigma) is dissolved in 300 μ l distilled water.
(2) the 0.1M NaIO that adds 0.6 μ l newly to join in upper liquid
4solution, under room temperature, lucifuge stirs 20 minutes.
(3) above-mentioned solution is packed in bag filter, use the sodium-acetate buffer dialysis of 1mM pH 4.4,4 ℃ are spent the night.
(4) add 20 μ l 1M pH 9.5 carbonate buffer solutions, make the pH of HRP be elevated to 9.0 to 9.5, then add immediately 1 mg another strain antibody of the present invention (CGMCC No.9318) (in 1ml 0.01M carbonate buffer solution), room temperature lucifuge stirs 2 hours gently.
(5) add the 4mg/ml NaBH that 0.05ml newly joins
4liquid, mixes, then put 4 ℃ 2 hours.
(6) above-mentioned liquid is packed in bag filter, use 0.15M pH7.4 PBS dialysis, 4 ℃ are spent the night.
(7) add isopyknic glycerine ,-20 ℃ 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 dilution, be i.e. the ratio of 1:2500.
The preparation of 2.3 enzyme working fluids:
By the monoclonal antibody of the horseradish peroxidase-labeled of preparing in 2.1 steps, with the ratio of 1:2500, enzyme labelled antibody is dissolved in enzyme buffer liquid.Enzyme buffer formula of liquid is: 5.8g/L sodium hydrogen phosphate, 0.593 g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5 ml/LProclin-300).
3. the preparation of sample diluting liquid:
1000 ml sample diluting liquids comprise 30.0g sodium chloride, 25.0ml lowlenthal serum, 5.0ml Tween-20,0.5ml Proclin-300.
4. chemical luminescence for liquid: chemical luminescence for liquid A and chemical luminescence for liquid B be outsourcing reagent (purchased from: Roche), wherein chemical luminescence for liquid A contains luminol and luminescence enhancer, and chemical luminescence for liquid B contains superoxide and luminescence enhancer.Or according to chemiluminescence, preparation voluntarily.
5. the preparation of calibration object:
With sample diluting liquid, by the Factor H sterling dilution of Fitzgerald company, make that tumor of bladder related antigen concentration gradient in calibration object is respectively 400,200,50,10,5,0ng/ml.
As required, calibration object can be traced to the source to reference material or other reference material of Fitzgerald company.
6. the preparation of quality-control product:
The human urine sample (urine can be any commercially available urine specimen or collect from Clinical Institutions) of collecting the high value of tumor of bladder related antigen clinical detection, dilutes high value human urine to the concentration range of expectation with sample diluting liquid.High value Quality Control: 160ng/ml to 240ng/ml; Low value Quality Control: 16ng/ml to 24ng/ml.2-8 ℃ saves backup.
Above-mentioned each reagent set is dressed up to kit, can also in kit, be incorporated to the instruments such as operation instructions, shrouding film as required.
Test case
The using method of test case 1. tumor of bladder related antigen of the present invention chemical luminescence reagent kit
1.1. before experiment, prepare
1.1. in 4 ℃ of refrigerators, take out kit, all should equilibrate to room temperature (18-25 ℃).
1.2.20 purified water or 20 times of uses afterwards of distilled water diluting for times concentrated cleaning solution.
2. test method
2.1. application of sample incubation: get the coated plate of sufficient amount, be fixed on framework, calibration object hole, quality-control product hole and testing sample hole are set respectively, record each hole site; In testing sample hole, add 45 μ l sample diluting liquids, in calibration object hole, add in order calibration object 50 μ l, in quality-control product hole, add quality-control product 50 μ l, in sample well, add respectively the testing sample (be equal to sample and carried out 10 times of dilutions) of 5 μ l.Every hole adds enzyme working fluid 50 μ l (suggestion completes this step operation in 20min), and concussion mixes, and adds a cover shrouding film, 37 ℃ of incubation 30min.
Note: calibration object and quality-control product, without dilution, can directly be used.
2.2. wash plate:
Wash plate by hand: every hole adds 350 μ l washing lotions, standing 5-10 abandons after second to the greatest extent, repeats to rinse after 5 times, pats dry;
Wash plate machine washing plate: every hole adds 350 μ l washing lotions, each washroom, every 5-10 second, repeats to rinse after 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 mixes rear reading.
2.4. measure: put immediately and under Chemiluminescence Apparatus, measure RLU value.
2.5. calculate: according to the concentration of calibration object and corresponding RLU value, with calibration object S0 hole, return to zero, use double-log linear fit mode (log (X)-log (Y)) result of calculation, result is multiplied by extension rate (10 times), is sample ultimate density.
Linear equation is log (RLU)=4.513+1.065 log (concentration); R=1.00.
The methodology index of test case 2. kits of the present invention
1. accuracy: the recovery should be between 85% to 115%;
2. blank detection limit: should be not higher than 1 ng/ml;
3. the linearity of measuring system: in 10ng/ml to 4000ng/ml scope internal linear correlation coefficient r >=0.9900.
4. repeatability: variation within batch coefficient CV
in batchshould be no more than 10%;
5. difference between batch: interassay coefficient of variation CV
between batchshould be no more than 15%;
Table 1. accuracy, minimum detectability, system linear, repeated testing result
6. analyze specificity:
Detect respectively tumor of bladder related antigen negative sample N1, positive sample P1 urine specimen.
In N1, P1 sample, add respectively the cross antigen of three concentration gradients, detect the cross reaction situation of cross antigen to the antibody test of tumor of bladder related antigen:
People's factor I concentration (25 ng/ml, 12.5 ng/ml, 0 ng/ml),
People's factor D concentration (10ng/ml, 5 ng/ml, 0 ng/ml),
Ox factor H concentration (100 ng/ml, 50 ng/ml, 0 ng/ml)
Mouse factor H concentration (100 ng/ml, 50ng/ml, 0 ng/ml)
Interpretation of result: cross antigen conventionally and test substance there is higher homology, sequence similarity or there is the epi-position of structural similarity.Can imagine, if while there are these cross antigens in sample, antibody may be identified 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's factor I is not more than 25 ng/ml, people's factor D and is not more than 10ng/ml, Niu Yinzi H and mouse factor H and is not more than 100ng/ml, the testing result of tumor of bladder related antigen is not had to statistically significant impact.Show the good anti-cross reactivity of kit of the present invention.
7. stability: 37 ℃ of each components of kit can be placed 6 days.
Table 2.37 degree shelf stability experimental result
Table 3.4 ℃ preservation condition stability inferior experimental result
R-H, R-L represent respectively: high value accuracy reference material, low value accuracy reference material.
8. interfering material analysis:
8.1 urine Lactoferrins, experimental result is in Table 5.
Conclusion: according to result, when slight molten urine sample urine Lactoferrin≤200mg/ml, detect result substantially without affecting with this kit, testing result may cause false positive when serious molten urine, therefore should not be with serious molten urine sample.
8.2 albumin, experimental result is in Table 6.
Conclusion: when albumin concentration is in 1000 mg/dl, tumor of bladder related antigen antibody test result is not had a significant impact.
8.3 cholerythrin, experimental result is in Table 7.
Interpretation of result: when bilirubin concentration is not more than 20mg/dl, tumor of bladder related antigen antibody test result is affected without statistically significant.
8.4 uric acid, experimental result is in Table 8.
Interpretation of result: when uric acid concentration is not more than 100mg/dl, tumor of bladder related antigen antibody test result is affected without statistically significant.
8.5 ascorbic acid, experimental result is in Table 9.
Interpretation of result: when ascorbic acid concentrations is not more than 2g/dl, tumor of bladder related antigen antibody test result is affected without statistically significant.
8.6 human IgGs, experimental result is in Table 10.
Interpretation of result: when human IgG concentration is not more than 10 g/dl, tumor of bladder related antigen antibody test result is affected without statistically significant.
Brief summary:
1) utilize two strain specific antibodies of the present invention to prepare kit, adopt double antibody sandwich method to measure tumor of bladder related antigen level in urine, i.e. the sandwich complex structure of coated antibody and the tumor of bladder related antigen antigen formation " coated antibody-antigen-HRP labelled antibody " of HRP labelled antibody and tested sample on enzyme mark porous plate.The high sensitivity and the specificity that detect have been realized.
2) one aspect of the present invention can be measured the content of tumor of bladder related antigen in urine quickly and accurately, for early diagnosis, the early treatment of tumor of bladder, provides reliable clinical reference value.
3) on the other hand owing to using specific monoclonal antibody as coated solid phase, greatly improved detection sensitivity and the specificity of tumor of bladder related antigen, antijamming capability is strong.
4) the present invention have easy to use, detect the advantages such as quick, sensitive, stable, easy and simple to handle.Being applicable to vast middle and small hospital uses.
Claims (9)
1. a kit that detects tumor of bladder related antigen in sample, it comprises:
Be coated with the first tumor of bladder related antigen monoclonal antibody porous plate,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid;
Wherein:
Sample diluting liquid comprises: sodium chloride, lowlenthal serum, Tween-20 and antiseptic;
Enzyme working fluid comprises: the second tumor of bladder related antigen monoclonal antibody and antiseptic of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, 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 and luminescence enhancer, and chemical luminescence for liquid B contains superoxide and luminescence enhancer;
When described the first tumor of bladder related antigen monoclonal antibody hybridoma that is CGMCC No.9317 by preserving number is produced, by preserving number, the hybridoma by CGMCC No.9318 is produced the second tumor of bladder related antigen monoclonal antibody, or when described the first tumor of bladder related antigen monoclonal antibody hybridoma that is CGMCC No.9318 by preserving number is produced, by preserving number, the hybridoma by CGMCC No.9317 is produced the second tumor of bladder related antigen monoclonal antibody;
Sample is from mammal, preferably the mankind; Sample selection is human urine.
2. the kit of tumor of bladder related antigen in detection sample according to claim 1, also comprises calibration object,
The tumor of bladder related antigen sterling that calibration object comprises concentration known, the concentration of tumor of bladder related antigen is in 10ng/ml to 4000ng/ml scope.
3. the kit of tumor of bladder related antigen in detection sample according to claim 1, also comprises quality-control product,
Quality-control product is that the high value urine specimen dilution of known tumor of bladder related antigen concentration forms.
4. a CLIA kit that detects tumor of bladder related antigen in sample, it comprises:
Be coated with the first tumor of bladder related antigen monoclonal antibody porous plate,
Sample diluting liquid,
Enzyme working fluid,
Chemical luminescence for liquid;
Wherein:
Sample diluting liquid comprises: 30g/L sodium chloride, 25ml/L lowlenthal serum, 5ml/L Tween-20 and 0.5ml/L Proclin-300;
Enzyme working fluid comprises: the second tumor of bladder related antigen monoclonal antibody of 5.8g/L sodium hydrogen phosphate, 0.593g/L sodium dihydrogen phosphate, 8.0g/L sodium chloride, 200ml/L calf serum, 0.5ml/L Proclin-300 and 0.4 mg/L horseradish peroxidase-labeled;
Chemical luminescence for liquid is comprised of chemical luminescence for liquid A and chemical luminescence for liquid B; Described chemical luminescence for liquid A contains luminol and luminescence enhancer, and chemical luminescence for liquid B contains superoxide and luminescence enhancer;
When described the first tumor of bladder related antigen monoclonal antibody hybridoma that is CGMCC No.9317 by preserving number is produced, by preserving number, the hybridoma by CGMCC No.9318 is produced the second tumor of bladder related antigen monoclonal antibody, or when described the first tumor of bladder related antigen monoclonal antibody hybridoma that is CGMCC No.9318 by preserving number is produced, by preserving number, the hybridoma by CGMCC No.9317 is produced the second tumor of bladder related antigen monoclonal antibody.
5. a tumor of bladder related antigen monoclonal antibody, by preserving number, the hybridoma by CGMCC No.9317 is produced for it.
6. a tumor of bladder related antigen monoclonal antibody, by preserving number, the hybridoma by CGMCC No.9318 is produced for it.
7. be selected from following one or both tumor of bladder related antigen monoclonal antibody and detect the purposes in reagent in preparation:
The tumor of bladder related antigen monoclonal antibody that the hybridoma that is CGMCC No.9317 by preserving number produces and
The tumor of bladder related antigen monoclonal antibody that the hybridoma that is CGMCC No.9318 by preserving number produces;
Described detection reagent is selected from: ELISA detects reagent, immunoturbidimetry detects reagent, magnetic particle detection reagent, chemiluminescence detection reagent, immunofluorescence detection agent and radioimmunoassay test agent, preferably chemiluminescence detection reagent;
Described detection reagent is preferably the detection reagent that detects tumor of bladder related antigen.
8. a hybridoma, its preserving number is the hybridoma of CGMCC No.9317.
9. a hybridoma, its preserving number is the hybridoma of CGMCC No.9318.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410359816.3A CN104142401B (en) | 2014-07-25 | 2014-07-25 | Tumor of bladder related antigen detection kit |
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| CN104142401A true CN104142401A (en) | 2014-11-12 |
| CN104142401B CN104142401B (en) | 2016-04-20 |
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| Publication number | Publication date |
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| WO2016011852A1 (en) | 2016-01-28 |
| CN104142401B (en) | 2016-04-20 |
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