CN103033625B - Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof - Google Patents
Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of immune analysis medicine, and particularly provides a kit for detecting human bladder cancer by a chemiluminescent analysis method on the basis of immune magnetic particles and a preparation method for the kit. According to the invention, the kit mainly comprises: 1) a bladder cancer EJ cell standard product; 2) magnetic particles which are coated by monoclonal antibodies capable of specifically identifying bladder cancer cell surface antigen; 3) enzyme-labeled monoclonal antibodies capable of specifically identifying bladder cancer cell surface antigen; 4) a chemiluminescent substrate which the enzyme in the 3) acts on; 5) a reaction tube or a microporous plate; and 6) a magnetic separator matched with the reaction tube or the microporous plate in the 5).
Description
Technical field
The present invention relates to immunoassay and clinical diagnosis medical domain, particularly, the invention provides chemiluminescence detection kit of a kind of human bladder cancer cell and preparation method thereof.
Background technology
Carcinoma of urinary bladder is modal tumour in urinary system.Blood urine is one of clinical early symptom of bladder cancer patients, has gross hematuria and microscopy blood urine.But blood urine also has generation under urinary tract inflammation and calculus symptom, it is not the specific symptom of carcinoma of urinary bladder.Not obvious just because of carcinoma of urinary bladder early symptom, to such an extent as to find that there is blood urine, patient's tumor of bladder General development is larger tumor nodule.Find special Diagnosis of Bladder method and have important clinical value to early stage Diagnosis of Bladder.
The method of early diagnosis of carcinoma of urinary bladder has following several:
1) urinalysis: first after blood urine occurs is carry out urinalysis, so that whether get rid of is because urinary tract inflammation causes, can not determine whether patient suffers from carcinoma of urinary bladder;
2) Urine exfoliative cell analysis: carry out basis of microscopic observation to the Urine exfoliative cell in arena, can differentiate and distinguish the tumour cell and normal cell that worsen.But sensitivity is not high, early stage carcinoma of urinary bladder is easily missed;
3) ultrasonic, CT or MRI imaging: directly can carry out image viewing to the size of tumour and deterioration degree, accuracy comparatively previous methods improves greatly.Although the continuous progress of imaging technique in recent years, sensitivity improves constantly, and observes still inadequate, easily undetected to less tumour;
4) cystoscope is observed: cystoscope enters bladder by urinary tract, carries out visible observation in bladder, thus finds early stage tumour.Can 2 be detected), 3) in undetected most of patient.
What current early diagnosis transitional cell bladder carcinoma cell line specificity was the highest is by cystoscopic observation, and the application of Fluorescence cystoscope improves detection sensitivity.But patient generally needs clinical trail to observe, thus can find that tumour occurs or recurrence status early.But cystoscope observation can cause uncomfortable to patient.In addition, it is not high that current Fluorescence cystoscope detects required fluorescent dye specificity, and have certain negative effect to human body.Therefore, in the early diagnosis of carcinoma of urinary bladder, people are exploring more special, sensitive detection method always, utilize special tumor markers realization detection easily and fast as much as possible.
Realize the detection of carcinoma of urinary bladder mark in urine sample or cell, carry out detecting in real time that there is important using value in clinical diagnosis.The mark of current business-like available carcinoma of urinary bladder has: the disappearance (UroVysion kit) in the adhesion molecule (the Immunocyt kit of Scimedx Corp company of the U.S.) that the carcinomebryonic antigen of CFH associated protein (bladder tumor antigen, BTA kit), high molecular is relevant with two transitional cell bladder carcinoma cell lines, NMP-22 (NMP22), 3,7 and No. 17 chromosomal heteroploiies and P16 TIF 9p21 site.BTA refers to people's recruitment factor H associated protein, and BTA detects needs professional operator to carry out in standard laboratory, and sensitivity reaches 57-83%, specificity 60-92%, and the specificity of blood urine patient can only reach 46%.The situation such as benign prostatic hyperplasis, kidney stone and urinary tract infections all affects the specificity that BTA detects in addition; Immunocyt kit is by Immunofluorescence test Urine exfoliative cell, although sensitivity and specificity are very high, but need to carry out a large amount of Urine exfoliative cell detections to reach higher accuracy, therefore in the treatment and prognosis observation of bladder cancer, there is very high using value, but it is barely satisfactory to be applied in early diagnosis aspect; Wherein NMP22 is carcinoma of urinary bladder mark most widely used at present, the marketization of ELISA kit achieves the convenient, fast detection of carcinoma of urinary bladder early diagnosis, detection sensitivity and specificity are also very high, but having in the situation such as virus infections, kidney/vesical calculus, still there will be false positive; What UroVysion kit adopted is fluorescence in situ hybridization (FISH) technology of many targets, although sensitivity and specificity are greatly improved, U.S. FDA is ratified, similar with Immunocyt kit, needs the Urine exfoliative cell that detection is a large amount of.
Draw in conjunction with the application of current carcinoma of urinary bladder mark and the diagnosis present situation of carcinoma of urinary bladder: on the one hand, new special carcinoma of urinary bladder mark is still the emphasis of research, prepare early diagnosis kit, realize detecting fast, in the diagnosis of carcinoma of urinary bladder, there is important clinical value; On the other hand, carry out the cell detection in urine sample, avoid the impact of the non-specific symptom such as inflammation, blood urine, accuracy is the highest.
Summary of the invention
(1) technical matters solved
The present invention is conceived to the early diagnosis of carcinoma of urinary bladder and the tracing observation of bladder cancer, a difficult problem for the Diagnosis of Bladder aspect that quasi-solution is certainly existing, namely special identification transitional cell bladder carcinoma cell line two strain monoclonal antibody is adopted, realize the double antibody identification of transitional cell bladder carcinoma cell line and catch, in conjunction with high-sensitive chemiluminescence immune analysis method, quantitative detection urine comes off transitional cell bladder carcinoma cell line, do not need expensive detecting instrument, be convenient to clinical expansion, there is good market using value, the deficiency of market available reagent box will be made up.
The object of this invention is to provide that a species specific transitional cell bladder carcinoma cell line is caught, recognition methods, and high-sensitive detecting kit.
Another object of the present invention is to provide a kind of kit for carcinoma of urinary bladder early diagnosis, monitoring after operation.
Another object of the present invention is to provide a kind of method preparing mentioned reagent box.
(2) technical scheme
The present invention specifically provides following kit:
[1] for detecting a human bladder cancer cell's kit, described kit comprises the monoclonal antibody of two kinds of specific recognition transitional cell bladder carcinoma cell line surface antigens.
[2] kit Gen Ju [1], described kit comprises:
1) effect of EJ cell line standard items;
2) by the magnetic particle of the monoclonal antibody bag quilt of specific recognition transitional cell bladder carcinoma cell line surface antigen;
3) monoclonal antibody of the specific recognition transitional cell bladder carcinoma cell line surface antigen of enzyme labeling;
4) chemical luminous substrate that the enzyme 3) acts on;
5) reaction tube or microwell plate, described reaction tube or microwell plate are preferably through the Seal treatment without protein blocking liquid, and described is fish gelatin hydrolysate containing 1.0% without protein blocking liquid, the 0.02M phosphate buffer of pH 7.2;
6) with 5) in reaction tube or the supporting magnetic separator of microwell plate.
[3] kit Gen Ju [2], wherein said effect of EJ cell line standard items are the stepwise dilution liquid of effect of EJ cell line.
[4] kit Gen Ju [2], wherein, 2) monoclonal antibody of the specific recognition transitional cell bladder carcinoma cell line surface antigen described in is monoclonal antibody BCMab2 prepared by the hybridoma cell strain being CGMCC No.6906 by preserving number, and it is combined by covalent bond with magnetic particle.
[5] kit Gen Ju [2], wherein, 2) to be particle diameter the be monox hydroxyl magnetic particle of 100nm to 1 μm of the magnetic particle described in.
[6] kit Gen Ju [2], wherein, 3) monoclonal antibody of the specific recognition transitional cell bladder carcinoma cell line surface antigen of the enzyme labeling described in is the BCMab1 monoclonal antibody of enzyme labeling, and prepared by the hybridoma cell strain that described BCMab1 monoclonal antibody is CGMCC No.3845 by preserving number (preparation about BCMab1 monoclonal antibody specifically can see Chinese Patent Application No.: 201010251384.6).
[7] kit Gen Ju [2], wherein, 3) enzyme described in is horseradish peroxidase, alkaline phosphatase or luciferase.
[8] kit Gen Ju [2], wherein, 5) reaction tube described in has the polystyrene tube of optical clarity, polyethylene pipe, polypropylene tube or glass tube; Described microwell plate is the white microwell plate of the detection being applicable to chemiluminescence reaction.
The present invention also provides the method method preparing described kit, said method comprising the steps of:
1) effect of EJ cell line standard items are prepared: stepwise dilution is carried out to make the stepwise dilution liquid of effect of EJ cell line to effect of EJ cell line solution;
2) the magnetic particle that streptavidin is modified is prepared;
3) the two strain monoclonal hybridoma strains preparations that use preserving number to be CGMCC No.3845 and CGMCC No.6906 respectively can the monoclonal antibody BCMab1 of specific recognition transitional cell bladder carcinoma cell line surface antigen and BCMab2;
4) biotinylated monoclonal antibody BCMab2 is prepared: by the free lysine generation coupling reaction of biotin-N-hydroxy-succinamide and described antibody under alkalescence condition;
5) use 2) in preparation streptavidin modify magnetic particle and 4) in preparation biotinylated monoclonal antibody BCMab2, reacted by the specific binding of streptavidin and biotin, prepare by the magnetic particle of biotinylated monoclonal antibody BCMab2 bag quilt;
6) the monoclonal antibody BCMab1 of enzyme labeling is prepared: adopt carbodiimides (EDC) coupling method, realize the combination of the amino on described antibody and the carboxyl on described enzyme molecule;
7) prepare 6) in the Chemoluminescent substrate that acts on of enzyme;
8) finished product is assembled into.
Preferably, described method is further comprising the steps of:
A) after the described preparation by the magnetic particle of biotinylated monoclonal antibody BCMab2 bag quilt, the prepared magnetic particle by biotinylated monoclonal antibody BCMab2 bag quilt is closed to reduce non-specific adsorption with confining liquid, described confining liquid comprise 0.2% ~ 1.0% bovine serum albumin(BSA), 0.5% ~ 1.0% casein, 0.5% ~ 1.0% fish gelatin hydrolysate, the 0.02M phosphate buffer of pH 7.2; With
B) close reaction tube and microwell plate with without protein blocking fluid-tight, described is fish gelatin hydrolysate containing 1.0% without protein blocking liquid, the 0.02M phosphate buffer of pH 7.2.
The transitional cell bladder carcinoma cell line that kit of the present invention can come off in sensitive, Fast Measurement urine, can detect the deterioration tumour cell of early fallout early, highly sensitively detects in detection urine composition measurement and urinary cytology in the market.Patent detection sensitivity of the present invention reaches the detection that each analysis realizes 1 pernicious transitional cell bladder carcinoma cell line.
The tumour cell that kit of the present invention is also applicable to Bladder Cancer vitellophag detects, and is also applicable to the detection of the transitional cell bladder carcinoma cell line circulated in blood.
Kit of the present invention adopts immune magnetic particle specially to catch, the double-antibody sandwich method of enzyme labeling monoclonal antibody specific recognition, achieves the specific detection of transitional cell bladder carcinoma cell line.This kit can be got rid of normally, inflammation epithelial cell, and the interference of other haemocyte, a specific recognition transitional cell bladder carcinoma cell line.
Patent of the present invention adopts immune magnetic particle method to catch the transitional cell bladder carcinoma cell line come off in urine, easy and simple to handle, detection time is short, is beneficial to clinical large-scale application.
The monoclonal antibody of two kinds of specific recognition transitional cell bladder carcinoma cell line surface antigens is used in the present invention:
1) BCMab1: prepared by the hybridoma cell strain being CGMCC No.3845 by preserving number, this hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on May 21st, 2010, China, Beijing), this antibody and preparation thereof specifically describe in Chinese Patent Application No.: 201010251384.6; And
2) BCMab2: prepared by the hybridoma cell strain being CGMCC No.6906 by preserving number, this hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on November 27th, 2012, China, Beijing).
Accompanying drawing explanation
Fig. 1. prepared kit with EJ cell for model obtains linear standard curve.
Fig. 2 .EJ, Lovo, HCV29 clone and PBS detect the luminous value contrast of gained by this method.EJ: bladder cancer cell lines; Lovo: rectum cancer cell system; HCV29: normal epithelium cell system.
Fig. 3. kit detection sensitivity.Bladder cancer cell lines: EJ, T24,5637, BIU-87; BCU-Ta:Ta phase transitional cell bladder carcinoma sample; BCU-T1:T1 phase transitional cell bladder carcinoma sample.
Fig. 4. kit of the present invention is applied to carcinoma of urinary bladder and normal pattern detection.
Fig. 5. the ROC curve of kit clinical assays result of the present invention.
Embodiment
Embodiment 1 prepares a kind of new transitional cell bladder carcinoma cell line chemiluminescence detection kit of the present invention
One, the preparation of standardized transitional cell bladder carcinoma cell line
EJ cells (CRL-2888
tM) purchased from American Type culture preservation institute (ATCC), cultivate with the DMEM nutrient culture media (Gibco, 12100-046) containing 10% hyclone.Cell good for the adhered state of cultivation is added 1-2mL pancreatin (Invitrogen, R-001-100), 37 DEG C digest 2 minutes, add appropriate nutrient culture media, after the cell centrifugation digested is abandoned supernatant, being resuspended in hyclone adds in the formulated cryopreserving liquid of 10% dimethyl sulfoxide (DMSO) (DMSO), with 10
6individual/mL cell concentration-80 DEG C is frozen, often props up frozen 500 μ L.Kit transportation, this composition needs to adopt the frozen transport of dry ice separately.
Two, the preparation of monoclonal antibody BCMab2 hybridoma
Get frozen EJ cell, be incubated at DMEM nutrient culture media, cultivation in good condition, after 3 weeks, gets the EJ cell 1 × 10 being in growth logarithmic phase
7individual, the BALB/c mouse (being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) of immunity 6 weeks sizes, weekly immunity 4 times, immunity, after 1 month, gets Mouse spleen cells.And prepare murine myeloma cell Sp2/0 (CRL-1772, ATCC) and Mouse spleen cells and merge, according to list of references (Kirk, A.D., et al.Nat Med 1999,5,686-693) described in carry out the monoclonal strain cell of hybridoma screening EJ cell.
Then, the positive hybridoma cell colonized culture (limiting dilution assay take Turnover of Mouse Peritoneal Macrophages as feeder cells) will selected.Take turns colonized culture through 2-3, obtain the stable hybridoma cell clone strain that can produce high-titer monoclonal antibody.Hybridoma cell clone strain is expanded and cultivates, and frozen conservation.Screening gained hybridoma cell strain can secrete monoclonal antibody BCMab2, specific recognition EJ cell and other transitional cell bladder carcinoma cell line.
Three, the preparation of monoclonal antibody BCMab1 and BCMab2
Possess two strain monoclonal hybridoma strains: preserving number is that the hybridoma cell strain of CGMCC No.3845 can obtain BCMab1 antibody (specifically see, Chinese Patent Application No.: 201010251384.6); Preserving number is that the hybridoma cell strain of CGMCC No.6906 can obtain BCMab2 antibody.Get 5 × 10
6the individual hybridoma being in exponential phase, with 1 × 10
7cell concentration be injected in BALB/c mouse abdominal cavity, collect ascites after 10 days.Monoclonal antibody by following steps purifying ascites:
1. the ascites of collecting gained is centrifugal with 2500r/min, gets supernatant, with 0.01M pH7.4PBS two-fold dilution ascites supernatant.
2. in above-mentioned ascites, add isopyknic saturated ammonium sulfate, stirring at room temperature 1 hour;
3., with 11000r/min, 4 DEG C centrifugal 20 minutes, abandons supernatant.Be resuspended in appropriate 0.01M pH7.4PBS, and add the saturated ammonium sulfate of half ascites volume, 4 DEG C of stirrings are spent the night.
4. above-mentioned ascites is with 11000r/min, and 4 DEG C centrifugal 20 minutes, abandons supernatant.Precipitation is dissolved in 0.01M pH7.4PBS, then dialyses with 0.01M pH7.4PBS, obtain the thick ball of antibody.
5.Protein-G gel column is installed to AKTA protein purification instrument;
6., by thick for antibody ball 11000r/min, 4 DEG C centrifugal 20 minutes, gets supernatant.Supernatant with 0.5mL/min flow velocity by advance with 0.02M pH7.0 containing the equilibrated Protein-G gel column of the PBS of NaCl of 0.15M, hatch 1 hour after loading;
7.0.02M pH7.0 is containing the PBS wash-out foreign protein of the NaCl of 0.15M;
8.0.2M pH2.8 glycine buffer antibody elution peak;
9. the antibody-solutions of wash-out is through ultrafiltration concentration, surveys antibody concentration, namely completes the purifying of antibody.
Four, the preparation of immune magnetic particle
1. the biotinylation of monoclonal antibody BCMab2
Get the 10mg monoclonal antibody BCMab2 (2mg/mL) that purifying is good, dialyse with the carbonate buffer solution of 0.1M, pH9.5, the antibody-solutions of carbonate buffer system must be in.Biotin-N-hydroxy-succinamide (NHS-Biotin, article No. H1759), purchased from Sigma-Aldrich, is dissolved in dimethyl sulfoxide (DMSO) (DMSO) with 1mg/mL concentration.Then in antibody-solutions, add 200uL biotin solution, 4 DEG C of stirring reactions 6 hours, add the NH of 20uL 1M
4cl solution cessation reaction.Reactant is dialysed at the phosphate buffer of 0.05M, pH7.4, must be in the biotinylated antibody BCMab2. of phosphate buffer
2. the streptavidin of magnetic particle is modified
Get 20mg monox hydroxyl magnetic particle (Shanghai Ao Run micro-nano company limited, SM1-015A, SM1-035, SM1-050, SM1-100), remove store buffer liquid under outside magnetic field effect, add 2mL 1.25% glutaraldehyde activated, stirring at room temperature, after 2 hours, under externally-applied magnetic field, leaves standstill 5min, abandon supernatant, clean three times with 0.01M pH 7.4 phosphate buffer, and be resuspended in 0.05M, pH9.5 carbonate buffer solution.Afterwards, 1mg streptavidin (purchased from Sigma-Aldrich, S4762) is added, stirring at room temperature 2 hours, cleans three times with the 0.01M phosphate buffer of pH 7.4, and is resuspended in phosphate buffer solution, determining concentration is 4mg/mL, obtains the magnetic particle that streptavidin is modified.
3. the preparation of immune magnetic particle
The magnetic particle (4mg/mL) that 20mg streptavidin is modified is placed in magnetic field, after the complete sedimentation of magnetic particle, removes supernatant, adds the biotinylated antibody BCMab2 (2mg/mL) of 10mg, and 4 DEG C are stirred 4 hours.Four times are cleaned afterwards with 0.01M pH 7.4 phosphate buffer, finally be resuspended in (0.01M pH 7.4 phosphate buffer, 2% bovine serum albumin(BSA), 0.05%Tween-20 in antibody buffer solution, 0.05%proclin-300), immune magnetic particle is obtained.Preferably, the prepared magnetic particle by biotinylated monoclonal antibody BCMab2 bag quilt is closed to reduce non-specific adsorption with confining liquid, described confining liquid comprise 0.2% ~ 1.0% bovine serum albumin(BSA), 0.5% ~ 1.0% casein, 0.5% ~ 1.0% fish gelatin hydrolysate, the 0.02M phosphate buffer of pH 7.2.Prepared immune magnetic particle with 4mg/mL concentration in 4 DEG C of preservations.
Five, the selection of magnetic grain diameter
Magnetic grain diameter is less, disperses more even in the solution, but when being less than 100nm, under outside magnetic field effect, settling velocity slows down, and can increase the wash time in kit application process, and therefore this kit selectes the magnetic particle being greater than more than 100nm.Magnetic particle is scattered in reaction system in immunoreaction process, can fast reaction speed to a certain extent.Luminol-H
2o
2in luminescence system, there is certain catalytic action, when particle diameter is at 100nm to 1 μm, luminol-H
2o
2the luminescence kinetics curve luminous platform sustainable time of system is between 30min to 2h.Increase to more than 1 μm, this catalytic capability declines, luminol-H
2o
2the luminescence kinetics curve luminous platform duration of system reduces (lower than 15min).
Six, the preparation of enzyme labeling monoclonal antibody BCMab1
With horseradish peroxidase (HRP, purchased from Sigma, P6782) labelled antibody is example, adopt carbodiimides (EDC) coupling method, idiographic flow is as follows: 10mg HRP is dissolved in the 0.1M citrate buffer solution of pH6.0 and (adds 78.84mg citric acid in 1L deionized water, 476.44mg sodium citrate), add carbodiimides (EDC is dissolved in Ma Linji ethyl sulfonic acid) and the carboxyl reaction on enzyme molecule; Add N-hydroxy thiosuccinimide (NHS) after 10min, EDC molecule will be replaced, enzyme molecule and NHS is intermolecular forms the carboxylic group activated; The enzyme molecular reaction of 1mg monoclonal antibody BCMab1 and activation, realizes the amino on antibody and enzyme molecule activation carboxyl reaction, room temperature 2 hours, obtains HRP labeled monoclonal antibody BCMab1; Complete in the antibody-solutions of mark and dropwise add saturated ammonium sulfate solution, limit edged stirs, until saturated ammonium sulfate concentration is reduced to 1/3; 4 DEG C of centrifugal 10min of standing 1h, 8000rpm, move to new pipe by supernatant, precipitation equal-volume PBS Eddy diffusion; Repeat aforesaid operations 3 times, collect the Horseradish Peroxidase Conjugates BCMab1 that namely supernatant obtains purification, add equal-volume glycerine ,-20 DEG C save backup.
The coupling of alkaline phosphatase or luciferase and BCMab1, similar with above-mentioned steps, the concrete consumption of reagent suitably can carry out adjustment optimization.
Seven, the preparation of Sample dilution
Preparation sample diluting liquid, is specifically formulated as: in 1L deionized water, adds 3.628g Na
2hPO
412H
2o, 0.272g KH
2pO
4, 0.2g KCl, 8g NaCl, 18g urea, 0.05g uric acid, 1mL tire ox blood, 1mL proclin-300 vibration mixes, and regulates pH to 7.4, solution in 4 DEG C of preservations, for the dilution buffer of arena sample and freeze-stored cell.
Eight, Chemoluminescent substrate
The compound method of the Chemoluminescent substrate of horseradish peroxidase used in the present invention (HRP):
Based on chemical luminous substrate A liquid described in 1000mL, comprise 1.7716g luminol, 0.051g 4-xenol, 0.012g 4-iodobenzene boric acid, 11.4g boric acid, 4.9g borax, its pH value is 8.0 ~ 10.0;
Based on chemical luminous substrate B liquid described in 1000mL, comprise 0.329g urea peroxide, 1ml Tween-20,51.58g Na
2hPO412H
2o, 8.74g NaH
2pO
42H
2o, its pH value is 7.0 ~ 7.6.
Using method: A, B liquid bi-component reagent, mixes according to use amount equal-volume before use.
Nine, the Seal treatment of reaction tube and microwell plate
Preferably, close reaction tube and microwell plate with without protein blocking fluid-tight, described is fish gelatin hydrolysate containing 1.0% without protein blocking liquid, the 0.02M phosphate buffer of pH 7.2.
Embodiment 2 Exfoliated Cells In Urine Samples From Patients With Bladder Cancer chemical method of the present invention light immune analytic reagent kit using method
One, sample pre-treatments
Get people's urina sanguinis or morning secondary urine sample, as instant detection, sample without the need to process, direct-detection.As detected the same day, sample needed frozen: get urine 10mL, the centrifugal 5min of 3000rpm, be suspended from by arena in 500uL cells frozen storing liquid (hyclone containing 10%DMSO), be first placed in 4 DEG C 2 hours, then be placed in-80 DEG C of preservations, for subsequent use.
Two, detection method
Before using this kit to test, first constant temperature oven or water-bath are adjusted to 37 DEG C; Need first to take out magnetic particle solution, HRP labelled antibody and each buffer solution prepared in the present embodiment 1 afterwards, place to equilibrate to room temperature in room temperature; Again frozen sample and effect of EJ cell line standard items are taken out and be placed in 37 DEG C of water baths and melt again rapidly, centrifugally remove supernatant, be suspended from 1mL cell diluent again.EJ cell initial concentration 5 × 10
5individual/mL, then continues to carry out serial dilution with cell diluent, obtains a series of cell standard product, and namely 5 × 10
5individual/mL, 1 × 10
5individual/mL, 5 × 10
4individual/mL, 1 × 10
4individual/mL, 5 × 10
3individual/mL, 5 × 10
2individual/mL, 20/mL.Again, prepare 200-1000 μ L, 20-200 μ L, 1-10 μ L range micro sample adding appliance and corresponding suction nozzle and check whether Chemiluminescence Apparatus normally works.
The concrete operation step using kit of the present invention to carry out detecting is as follows:
(1) micro-pore plate type chemiluminescence detection kit
Extracting waste 96 microwell plate, every hole adds 50 μ L urine specimens or series of calibration cell solution (5 × 10
5individual/mL, 1 × 10
5individual/mL, 5 × 10
4individual/mL, 1 × 10
4individual/mL, 5 × 10
3individual/mL, 5 × 10
2individual/mL, 20/mL).Successively add Horseradish Peroxidase Conjugates solution and each 5 μ L of immune magnetic particle solution, 37 DEG C of oscillating reactions 60min again.Every hole adds cleansing solution 150 μ L, and on microwell plate oscillator, (MH-1, Kylin-Bell Instruments) shakes 10s mixing, is then placed on the supporting magnetic separator of 96 microwell plates (purchased from NEB company, S1511S), removes supernatant.Repeat washing step above, wash 4 times.Every hole adds Chemoluminescent substrate 100 μ L, fully mixes, and then on board-like Chemiluminescence Apparatus (Beijing Bin Song Photon B. V., BHP9504), sequentially measures luminous intensity (RLU).In theory every hole internal standard product cell number be respectively 25000/to analyze, 5000/analyze, 2500/analyze, 500/analyze, 250/analyze, 25/analyze, 1/analyze.See accompanying drawing 1 with the linear relationship of cell quantity and luminous value, wherein, ordinate RLU is relative luminous intensity, and horizontal ordinate is cell detection number (unit is cell/analysis).In 1 ~ 25000 cell/analyst coverage, coefficient R
2=0.9984, Y=12511+33.651X; In 1 ~ 500 cell/analyst coverage, coefficient R
2=0.9968, Y=1037+92.21X.
According to RLU size, select suitable standard curve range, the RLU of each sample to be tested is substituted into typical curve equation, obtains the number of tumour cell in urine sample to be measured.
(2) tube-type chemical luminescence detection kit
After reaction tube is numbered, add 200 μ L urine specimens or series of calibration cell solution wherein, mark each 20 μ L of BCMab1 antibody-solutions with magnetic particle solution and HRP and mix, 37 DEG C of oscillating reactions 60min.Be placed on multi-function magnetic separation vessel (Aorun Weina New Material Science and Technology Co., Ltd., Shanghai, MS-12) on, supernatant discarded after 3min, often pipe adds cleansing solution 300 μ L, abundant mixing, be placed on magnetic separator and leave standstill 3min, abandoning supernatant, repeat 4 times, finally discard cleansing solution, each pipe adds Chemoluminescent substrate 200 μ L, fully mixes, on the luminous measuring instrument (German Berthold Technologies GmbH & Co.KG) of tube-type chemical, then sequentially measure the luminous intensity (RLU) of each pipe.The number of tumour cell in urine sample to be measured is determined in the mode similar to above-mentioned ().
The methodology index of embodiment 3 kit of the present invention
According to manufacture conventional in this area and vertification regulation, the kit to preparation in embodiment 1 is examined and determine, and result is as follows:
1, kit precision measures
(1) EJ cell standard product Precision Experiment
The kit of preparation in embodiment 1 is extracted 10 kits, according to time-and-motion study 1 × 10 described in embodiment 2
3the EJ cell standard product of individual/mL 5 times.Calculate the measurement result coefficient of variation, measurement result is as shown in table 1, and the result display coefficient of variation is between 3.5% ~ 10%.
The experiment of table 1EJ cell standard product repeatability
(2) sample Precision Experiment
The kit of preparation in embodiment 1 is extracted 10 kits, according to two the bladder cancer patients Urine exfoliative cell of time-and-motion study described in embodiment 2.Calculate the coefficient of variation measuring concentration.The measurement result of three batches of kits in embodiment 1 is as shown in table 2, table 3, and result shows that the coefficient of variation is less than 9.0%.
The Urine exfoliative cell sample precision of table 2. patient 1 measures
The Urine exfoliative cell sample precision of table 3. patient 2 measures
2, kit accuracy determination
Get two routine bladder cancer patients Urine exfoliative cell samples, in embodiment 1, the tumour cell concentration of the routine sample of kit measurement two of preparation is respectively 4 × 10
3, 2 × 10
3individual/mL.Add isopyknic EJ cell standard product solution 4 × 10 wherein
3individual/mL and 2 × 10
3individual/mL, operates by according to described in embodiment 2, each concentration determination three times, calculates the recovery.Result is as shown in table 4, shows that the recovery is between 93.0% ~ 102.0%.
Table 4. accuracy determination
3, stabilization of kit experiment
To the kit of embodiment 1, after other reagent except frozen EJ cell standard product carries out 37 DEG C of 7 days Acceleration study, measure the result such as maximum, the minimum luminous intensity of kit, the recovery of standard addition of determinand and show that the kit index of embodiment 1 is all within normal range.Carry out 2 ~ 8 DEG C of 8 months tracking tests to the kit key component of embodiment 1, result shows that indices meets the requirements completely.Consider the generation of the freezing situation of kit, the kit of embodiment 1 is put into-20 DEG C freezing 7 days, measurement result also shows that the indices of kit is completely normal.-80 DEG C of frozen effect of EJ cell lines, after 8 months, recovery is cultivated, and is dyeed (the green skies, C0011), carry out living cells technology by Trypan Blue, and more than 95% cytoactive keeps good.Kit at least can preserve more than 6 months at 2 ~ 8 DEG C as can be seen from the above results.
4, kit specific test
Normal urinary tract epithelial cell line HCV29 or other tumor cell line is added as Hela (breast cancer, CCL-2 in Healthy People urine
tM), Jurkat (leukaemia, CRL-1990
tM), lovo (carcinoma of the rectum, CCL-219
tM), K562 (chronic myelogenous leukemia, CTL-243
tM), HepG2 (liver cancer, CRL-10741
tM), the cell concentration added reaches 10
5individual/mL, carries out specific test.Above clone is all purchased from American Type Culture and preserves institute (ATCC).By detecting luminous value, the impact added reaction system of more non-transitional cell bladder carcinoma cell line.Result as shown in Figure 2, adds normal Urothelial cell or other tumour cell, all produces lower background value in Healthy People urine.PBS damping fluid simulation plain buffer in addition, with effect of EJ cell line and T24 cell solution (10
3individual/mL) luminous value compare, also only produce low background, this method specificity can be obtained and sensitivity is all higher.
In the healthy Quality Control urine sample of 1mL, add human peripheral (taking from Healthy People venous blood) 10 μ L, obtain the visible blood urine sample of naked eyes; In addition, under high-power microscope visual field, add human peripheral mean constant of red blood cell reach 50, obtain microscopy blood urine sample, investigate blood urine to the impact of this kit.Measurement result shows, and magnetic particle is caught red blood cell is not non-specific, and BCMab1-HRP antibody does not have non-specific binding yet, and end product does not affect by erythrocytic.
5. kit sensitivity
Kit sensitivity is using the minimum tumor cell number that can detect as standard.Four kinds of bladder cancer cell lines EJ, T24 (HTB-4
tM, ATCC), 5637 (HTB-9
tM, ATCC), BIU-87 (Shanghai OEG cell research institute of the Chinese Academy of Sciences is so kind as to give), carry out accurate metering respectively by granular cell counter (U.S.'s Beckman, Z1), then carry out limiting dilution.Each sample is got 50 μ L and is reacted, obtain 1 through limiting dilution, 2,5 cells/analysis, then measured by this research method kit.Survey luminous value as shown in Figure 4.There is Tis, Ta, T1b, T2a, T2b Exfoliated Cells In Urine Samples From Patients With Bladder Cancer sample by stages in addition, first carry out Switzerland-Giemsa staining kit and carry out dyeing that (Science and Technology Ltd. is built up in Nanjing, D010), determine urine Exfoliated tumor cells concentration, then carry out limiting dilution.Each sample is got 50 μ L and is reacted, and obtains 1,2,5 cell/analyses (cell/assay), then measured by this research method kit through limiting dilution.Survey luminous value as shown in Figure 3.By Tu Ke get, the luminous value of individual cells is significantly higher than blank value (PBS does blank), namely single transitional cell bladder carcinoma cell line can be detected.
Prove through a large amount of experiments, kit method index of the present invention is as follows:
Sensing range: transitional cell bladder carcinoma cell line detectable concentration 20/mL ~ 5 × 10
6individual/mL, i.e. 1 cell/analysis ~ 25000 cell/analysis;
Sensitivity: minimum detecting is limited to 1 cell/analysis;
Precision: be less than 9% (n=5)
Utilize the inventive method to detect, highly sensitive, high specificity, sensing range is wide, simple to operate, no radioactivity pollute, and kit cost is low, and clinical applicability is strong, is more suitable for China's clinical detection Screening laboratory.Therefore the present invention is clinical detection carcinoma of urinary bladder Exfoliated tumor cells, and early diagnosis carcinoma of urinary bladder and dynamic monitoring, provide a kind of sensitive, accurate, quick, special method.
The application of embodiment 4 kit of the present invention in bladder cancer patients clinical sample mensuration
Collect transitional cell bladder carcinoma sample 205 example, and normal person's (comprising the optimum disease in the urological system patients such as healthy population, inflammation, calculus) urine specimen 389 example, apply kit of the present invention to detect, luminous value (RLU) more as shown in Figure 4.Visible, carcinoma of urinary bladder compares with normal person, survey luminous value there were significant differences.Therefore by the detection of Urine exfoliative cell, bladder cancer patients and normal population can be distinguished very well.According to ROC tracing analysis (as shown in Figure 5), get three different critical values respectively: (1) 3126 (RLU), sensitivity (sensitivity) is the highest, specificity (specificity) can accept, and obtains sensitivity, specificity is respectively 100% and 72%; (2) 20699 (RLU), sensitivity and specificity are all simultaneously optimum, obtain sensitivity, specificity is respectively 93% and 89%.56875 (RLU), specificity is the highest, and sensitivity can accept, and obtains sensitivity, specificity is respectively 72% and 99%.
The sensitivity in different critical value of table 5. kit of the present invention and specificity.
With EJ cell for standard items, quantitatively detect to obtain standard test curve (as shown in Figure 1).Adopt kit of the present invention to detect transitional cell bladder carcinoma sample, then according to pattern detection result and typical curve, tumor cell number in actual sample can be calculated.Prepare the cast-off cells smear of transitional cell bladder carcinoma sample simultaneously, carry out Urine exfoliative cell dyeing by quick auspicious Ji's Albert'stain Albert kit (Science and Technology Ltd. is built up in Nanjing, D010), statistics tumor cell number.Cell smear method and kit testing result of the present invention are compared, and result is as shown in table 6, and visible this method sensitivity is apparently higher than traditional cell smear method.
The urine Exfoliated tumor cells number testing result of table 6. cell smear and kit of the present invention
Claims (8)
1., for detecting a human bladder cancer cell's kit, described kit comprises:
1) effect of EJ cell line standard items;
2) by the magnetic particle of the monoclonal antibody BCMab2 bag quilt of specific recognition transitional cell bladder carcinoma cell line surface antigen, BCMab2 is prepared by the hybridoma cell strain being CGMCC No.6906 by preserving number, and it is combined by covalent bond with magnetic particle;
3) the monoclonal antibody BCMab1 of the specific recognition transitional cell bladder carcinoma cell line surface antigen of enzyme labeling, BCMab1 are prepared by the hybridoma cell strain being CGMCC No.3845 by preserving number;
4) chemical luminous substrate that the enzyme 3) acts on;
5) reaction tube or microwell plate;
6) with 5) in reaction tube or the supporting magnetic separator of microwell plate.
2. kit according to claim 1, wherein said effect of EJ cell line standard items are the stepwise dilution liquid of effect of EJ cell line.
3. to be particle diameter the be monox hydroxyl magnetic particle of 100nm to 1 μm of the magnetic particle kit according to claim 1, wherein, 2).
4. the enzyme kit according to claim 1, wherein, 3) is horseradish peroxidase, alkaline phosphatase or luciferase.
5. the reaction tube kit according to claim 1, wherein, 5) has the polystyrene tube of optical clarity, polyethylene pipe, polypropylene tube or glass tube; Described microwell plate is the white microwell plate of the detection being applicable to chemiluminescence reaction.
6. kit according to claim 1, wherein, described reaction tube or microwell plate through the Seal treatment without protein blocking liquid, described without protein blocking liquid be contain 1.0% fish gelatin hydrolysate, the 0.02M phosphate buffer of pH 7.2.
7. prepare the method for kit according to claim 1, said method comprising the steps of:
1) effect of EJ cell line standard items are prepared: stepwise dilution is carried out to make the stepwise dilution liquid of effect of EJ cell line to effect of EJ cell line solution;
2) the magnetic particle that streptavidin is modified is prepared;
3) the two strain monoclonal hybridoma strains preparations that use preserving number to be CGMCC No.3845 and CGMCC No.6906 respectively can the monoclonal antibody BCMab1 of specific recognition transitional cell bladder carcinoma cell line surface antigen and BCMab2;
4) biotinylated monoclonal antibody BCMab2 is prepared: by the free lysine generation coupling reaction of biotin-N-hydroxy-succinamide and described antibody under alkalescence condition;
5) use 2) in preparation streptavidin modify magnetic particle and 4) in preparation biotinylated monoclonal antibody BCMab2, reacted by the specific binding of streptavidin and biotin, prepare by the magnetic particle of biotinylated monoclonal antibody BCMab2 bag quilt;
6) the monoclonal antibody BCMab1 of enzyme labeling is prepared: adopt carbodiimides (EDC) coupling method, realize the combination of the amino on described antibody and the carboxyl on described enzyme molecule;
7) prepare 6) in the Chemoluminescent substrate that acts on of enzyme;
8) finished product is assembled into.
8. method according to claim 7, described method is further comprising the steps of:
A) after the described preparation by the magnetic particle of biotinylated monoclonal antibody BCMab2 bag quilt, the prepared magnetic particle by biotinylated monoclonal antibody BCMab2 bag quilt is closed to reduce non-specific adsorption with confining liquid, described confining liquid comprise 0.2% ~ 1.0% bovine serum albumin(BSA), 0.5% ~ 1.0% casein, 0.5% ~ 1.0% fish gelatin hydrolysate, the 0.02M phosphate buffer of pH 7.2; With
B) close reaction tube and microwell plate with without protein blocking fluid-tight, described is fish gelatin hydrolysate containing 1.0% without protein blocking liquid, the 0.02M phosphate buffer of pH 7.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210555378.9A CN103033625B (en) | 2012-12-19 | 2012-12-19 | Human bladder cancer cellular chemiluminescent detection kit and preparation method thereof |
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| CN103616507B (en) * | 2013-12-11 | 2015-08-19 | 山东博科生物产业有限公司 | Without albumen bag by plate confining liquid |
| 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 |
| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
| CN106198997B (en) * | 2016-06-28 | 2017-07-28 | 广州华弘生物科技有限公司 | A kind of kit of Quantitative detection troponin and creatine kinase isozyme |
| CN109406789A (en) * | 2018-11-23 | 2019-03-01 | 李翀 | A kind of bladder cancer circulating cells detection kit and application |
| CN115629059A (en) * | 2022-10-17 | 2023-01-20 | 重庆精准生物技术有限公司 | Application of cell, kit and method for improving chemiluminescence detection sensitivity |
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