CN101368961B - Chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof - Google Patents
Chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chemiluminescence immunoassay quantitative analysis test kit and a preparing method thereof for urinary bladder cancer antigen. The test kit includes urinary bladder cancer antigen calibrating article, solid-phase carrier which is coated by urinary bladder cancer antigen monoclonal antibody, urinary bladder cancer antigen monoclonal antibody enzyme labeled compound, chemiluminescence zymolyte liquid on which the enzyme reacts, and condensed washing liquid. The preparing method of the test kit includes steps as following: 1) the calibrating article is prepared; 2) the solid-phase carrier is coated; 3) the antibody is labeled by the enzyme; 4) the chemiluminescence zymolyte liquid is prepared; 5) the condensed washing liquid is prepared; 6) all components above can be filled and packed through separate packing and then the components can be assembled into finished product finally. The test kit provides urinary bladder cancer diagnosing with an important way and evidence.
Description
Technical field
The invention discloses a kind of chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen and preparation method thereof, belong to the immunoassay medical domain.
Background technology
Tumor of bladder is the urinary system most common tumor, is transitional cell carcinoma more than 90%, and TCCB (transitional cell carcinoma of bladder) is the modal malignant tumour of urinary system, and pilosity is born in 50-70 between year.Be the shallow tumour of table more than 80% wherein, have 10%～20% to develop into invasive bladder cancer.At present carcinoma of urinary bladder diagnosis and main cystoscope and the urinary cytology of leaning on of postoperative check are checked.Though cystoscopy is accurate, is difficult to find carcinoma in situ or flat cancer; Though urinary cytology inspection does not have wound, and specificity is better, susceptibility is low, and for the tumour of lower, good differentiation by stages, its susceptibility is merely 30% or lower, and receives the influence of diagnostician's subjective factor bigger.The limitations restrict of cystoscope and urine exfoliative cytology inspection to the application of carcinoma of urinary bladder diagnosis, so seek very necessity of new diagnostic method.Therefore many scholars explore on macroscopic view, microcosmic and even gene level untiringly, have found this tumor of bladder knurl mark thing of urine bladder cancer antigen (UBC).
The essence of UBC is the CKs fragment 8 and 18 in tumor of bladder source, and at present, the immunologic detection method of UBC mainly is an enzyme-linked immunosorbent assay, but its sensitivity is low, and influence factor is more, is prone to cause false negative and false positive.The present invention adopts chemoluminescence method to detect UBC, and it is simple, easy to operate, highly sensitive to have an instrument and equipment, linear response range wide with remarkable advantage such as easily be automated.Detect UBC with this method, high to wellability tumor of bladder susceptibility than enzyme-linked immunosorbent assay, and also to tumour early stage, good differentiation, its susceptibility is also higher, and specificity fundamental sum enzyme-linked immunosorbent assay conforms to.
The invention solves shortcomings such as traditional detection method susceptibility is not high, complex steps, instrument requirement is high, technical requirement is high, patient suffering; Improve greatly than enzyme linked immunological experiment sensitivity again; Carry out this detection clinically and can assess the recurrence of classification and prediction tumor of bladder, can help treatment and improve prognosis.Though UBC can not be used for the diagnosis of tumor of bladder separately, can not substitute cystoscopy, can suitably reduce and prolong cystoscopic number of times and time according to its result, can be used as cystoscopic important supplementary means.
Summary of the invention
One of the object of the invention provides a kind of use chemiluminescence immunoassay quantitative measurement urine bladder cancer antigen kit, and the present invention also provides a kind of method for preparing the mentioned reagent box, and it is:
Chemical luminous substrate and concentrated cleaning solution that the solid phase carrier that chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen of the present invention is encapsulated by urine bladder cancer antigen calibration object, urine bladder cancer antigen monoclonal antibody, urine bladder cancer antigen monoclonal antibody enzyme labeling thing, above-mentioned enzyme are acted on are formed.
According to kit of the present invention, wherein, said solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle; Said enzyme is alkaline phosphatase or horseradish peroxidase; Said chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol; Said 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2--two oxidative ethanes (AMPPD), CSPD or CDP-Star; Said concentrated cleaning solution is Tris-HCL cleansing solution or PBST cleansing solution.
The invention provides a kind of method for preparing the mentioned reagent box, may further comprise the steps: 1) preparation urine bladder cancer antigen calibration object; 2) encapsulate solid phase carrier with the urine bladder cancer antigen monoclonal antibody; 3) with enzyme labeling urine bladder cancer antigen monoclonal antibody; 4) the preparation chemical luminous substrate liquid that above-mentioned enzyme acted on; 5) preparation concentrated cleaning solution; 6) the above-mentioned urine bladder cancer antigen calibration object of packing, the urine bladder cancer antigen monoclonal antibody of enzyme labeling, chemical luminous substrate and the concentrated cleaning solution that this enzyme acted on are assembled into finished product with each component at last.
According to the method for the invention, the said solid phase carrier that encapsulates may further comprise the steps:
In encapsulating damping fluid, add the urine bladder cancer antigen monoclonal antibody and process working fluid, it is carried on the solid phase carrier, 4 ℃ are spent the night, seal with confining liquid.
Wherein encapsulate damping fluid and select the citrate buffer solution of 0.046M pH 4.6 for use.
Confining liquid is selected the PBS damping fluid for use.
In said method, said solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
In said method, said enzyme is alkaline phosphatase or horseradish peroxidase.
In said method, said chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
In said method, said 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD), CSPD or CDP-Star.
In said method, said concentrated cleaning solution is Tris-HCL cleansing solution or PBST cleansing solution.
The inventor has adopted the research that experimentizes of horseradish peroxidase (HRP) and two systems of alkaline phosphatase (ALP); Carry out mark with HRP or ALP antagonist; Add determined antigen then and make it to combine, HRP or ALP catalytic luminescence substrate make it to decompose and the generation luminous signal.The substrate of HRP effect is luminol, different luminol derivant, and this type of material low price has been realized production domesticization basically, is current at one type of the widest luminous agent of clinical practice.The substrate of ALP effect is 1,2-two oxidative ethane analog derivatives (AMPPD), and it is a kind of novel chemiluminescence agent, belongs to the dioxetanes alkanes, and performance is very stable, and 5 ℃ of solid AMPPD that preserve down decompose hardly.The present invention has strengthened chemiluminescent intensity through using chemiluminescence intensifier, prolongs fluorescent lifetime.
When urine bladder cancer antigen monoclonal antibody enzyme labeling thing adopts horseradish peroxidase-labeled urine bladder cancer antigen monoclonal antibody, use improvement sodium periodate method to carry out mark; When urine bladder cancer antigen monoclonal antibody enzyme labeling thing adopts alkali phosphatase enzyme mark urine bladder cancer antigen monoclonal antibody, use glutaraldehyde method to carry out linkage flag.
The urine bladder cancer antigen monoclonal antibody encapsulates solid phase and accomplishes through physisorption.The inventor has investigated different buffer systems to the physisorption efficient of urine bladder cancer antigen monoclonal antibody on solid phase.
Remarkable advantages such as kit of the present invention is simple with its pinpoint accuracy, high sensitivity, operation steps, technology and instrument requirement are not high, with low cost; Solved purchase import reagent box problem of ultra-high price; Highly sensitive than enzyme linked immunological kit again, for clinical diagnosis and research work provide a kind of very valuable detection means.
Description of drawings
Fig. 1 is the calibration object linear graph in the prepared kit of embodiment 1.
Fig. 2 measures clinical blood sample comparison diagram for using kit of the present invention and enzyme linked immunological kit respectively.
Embodiment
The preparation of embodiment 1 chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen
One, enzyme labelled antibody preparation
Horseradish peroxidase-labeled urine bladder cancer antigen monoclonal antibody adopts the preparation of improvement sodium periodate method, dilutes enzyme labelled antibody with the enzyme labelled antibody dilution with 1: 4000 working concentration;
(1) horseradish peroxidase-labeled urine bladder cancer antigen MONOCLONAL ANTIBODIES SPECIFIC FOR
Get 10mg HRP and add 1ml 0.1mol/L sodium acetate or water-soluble separating, fully mixing adds 0.08mol/L NaIO after about 5 minutes
4, after WS 1.0ml mixes, put refrigerator interior 20 minutes; Add 0.4mol/L ethylene glycol solution 0.5ml and stop oxidation reaction, add 21%NaCL solution 0.3ml after 30 minutes, add the ice-cold absolute ethyl alcohol of 1.2ml (AR) deposition hydroformylation enzyme again; After the centrifugal supernatant that goes, deposition enzyme wash once with 6ml 80% ice-cold alcohol solution dipping again, the centrifugal ethanol that inclines; Deposition adds the anti-human IgG of 2ml sheep or horse (about inner protein amount 20mg) then with the 0.05mol/L sodium carbonate buffer 2ml dissolving of PH9.6, stirs; Put in the refrigerator and spend the night, next day, taking-up added 10mgNaBH
4Mixing adds equivalent saturated ammonium sulphate enzyme conjugates after 3 hours, centrifugal; Remove supernatant, deposition with the washing of 50% saturated ammonium sulfate once, and is centrifugal; Remove supernatant again, deposition changes in the bag filter with normal saline dialysis desalination (need change liquid 5 times) with 0.01mol/LPBS 3ml dissolving; Need centrifugal removing if any deposition, supernatant is light brown and is enzyme conjugates.Add 60% glycerine PBS, preserve below-20 ℃.
(2) enzyme labelled antibody dilution prescription:
| Sodium dihydrogen phosphate | 0.2g |
| Sodium hydrogen phosphate | 2.9g |
| Sodium chloride | 8.8g |
| Gelatin hydrolysate | 10g |
| Proclin300 | 1.0mL |
| Food Red | 1.0mL |
| Distilled water | 1000mL |
Two, the preparation of urine bladder cancer antigen calibration object
With animal blood serum or albumen damping fluid is matrix, and the pure article of adding urine bladder cancer antigen are formulated.The ultimate density of preparation is: 0ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL.
Three, the preparation of solid-phase coating plate
(1) encapsulates
Adopt the CT damping fluid of 0.046M pH 4.6 and the urine bladder cancer antigen monoclonal antibody of debita spissitudo to be mixed and made into coating buffer, and it is carried on the solid phase carrier;
Particularly, said method for coating is:
| Trisodium citrate | 7.3g |
| Citric acid | 4.44g |
| Distilled water | 1000mL |
Behind the dissolving mixing, adjustment pH value to 4.6 adds 5.0mg urine bladder cancer antigen monoclonal antibody mixing, add then in each hole of microwell plate, and every hole 110 μ L, 4 ℃ are spent the night.
(2) sealing
The confining liquid prescription is:
| NaH 2PO 4·2H 2O | 0.2g |
| Na 2HPO 4·12H 2O | 2.9g |
| Bovine serum albumin(BSA) | 10g |
| Proclin300 | 10g |
| Distilled water | 1000mL |
The mentioned reagent weighing is put into clean container well, adds the distilled water constant volume, the dissolving mixing, measuring pH value is 7.0, get rid of coating buffer after, every hole adds confining liquid 300 μ L respectively, room temperature placement 3 hours.Get rid of confining liquid, room temperature removal moisture drying 24 hours.Carry out envelope immediately, rearmounted 2～8 ℃ of preservations.
Four, chemical luminous substrate liquid
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention (HRP):
Chemical luminous substrate A liquid:
| Luminol | 10mM | 1.7716g |
| The 4-xenol | 0.3mM | 0.051g |
| 4-iodobenzene boric acid | 0.05mM | 0.012g |
| Boric acid | 11.4g | |
| Borax | 4.9g | |
| Distilled water | 1000mL |
The pH value is 8.0～10.0
Chemical luminous substrate B liquid:
| Urea peroxide | 3.5mM | 0.329g |
| Na 2HPO 4·12H 2O | 51.58g | |
| NaH 2PO 4·2H 2O | 8.74g | |
| Tween20 | 0.1％ | 1mL |
| Distilled water | 1000mL |
The pH value is 7.0～7.6
Method of application: before using A liquid and B liquid were used after the mixed by 1: 1.
Five, concentrated cleaning solution
The employed concentrated cleaning solution of horseradish peroxidase
| Na 2HPO 4·12H 2O | 58g |
| NaH 2PO 4·2H 2O | 2g |
| NaCl | 160g |
| Tween-20 | 1mL |
| Proclin?300 | 1mL |
| Distilled water | 1000mL |
Adjustment pH to 7.2～7.4
Dilute 20 times of uses with distilled water before using.
Six, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture, after sampling observation is qualified, is assembled into finished product, 4 ℃ of preservations.
To sum up; In research process of the present invention, inventor of the present invention has at first carried out shaker test and Quality Identification to used starting material, then method for coating is studied; Select optimal damping fluid and the confining liquid of encapsulating; Find best concentration conditions, and, made and can make the enzyme labeling thing keep active enzyme labelled antibody dilution for a long time.
The preparation of embodiment 2 chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen
Divided by glutaraldehyde method alkaline phosphatase is connected with the urine bladder cancer antigen monoclonal antibody; With the enzyme labelled antibody dilution with 1: 2000 dilution enzyme labeling thing; The employing composition is that Tris (24g), HCl (15mL), NaCl (160g), KCl (4g), distilled water (1000mL), pH value are 7.4 Tris-HCl concentrated cleaning solution and are outside the luminous substrate liquid with CSPD that all the other are all to prepare quantitative determination reagent kit of the present invention with embodiment 1 identical method.
Embodiment 3～4 preparations chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen of the present invention
Except that respectively with plastic bead, plastic tube as the carrier, all the other are all to prepare quantitative determination reagent kit of the present invention with embodiment 1 identical method.
The method of application of embodiment 6 kits of the present invention
The method of application of embodiment 1 said kit is following:
1) all detectable of balance and sample are to room temperature;
2) get the lath of expense;
3) every hole, pipe add 25 μ L calibration object/samples to be tested successively;
4) every hole, pipe add enzyme labeling thing 100 μ L successively, and vibration mixed it in 30 seconds on micro oscillator;
5) 37 ℃ of incubation 60min;
6) wash plate 5 times with the concentrated cleaning solution after the dilution, the coated antibody-antigen-hrp-antibody complex that is fixed on the solid phase carrier is separated with bond not;
7) every hole, pipe adding volume are the chemical luminous substrate liquid of 100 μ L, and room temperature lucifuge reaction 5min utilizes chemiluminescence detector to detect in 5～15min;
8) the double-log data fitting mode of use Log (x)-Log (y) is carried out the foundation of typical curve, and the result is measured in experiment with computing.
The method of application of embodiment 7 kits of the present invention
The method of application of embodiment 2 said kits is dezymotized label and is adopted alkaline phosphatase, and luminous substrate liquid consumption is the 50ul/ hole, and with outside the Chemiluminescence Apparatus measurement, all the other are all identical with embodiment 6 said method of application behind the room temperature lucifuge reaction 30min.
The methodology of embodiment 8 kits of the present invention and enzyme linked immunological kit is identified relatively
Kit of the present invention experimentizes by embodiment 5 said method of application, and enzyme linked immunological kit is outsourcing, and the step use is explained in strictness on schedule, and the performance evaluation index of two kinds of kits is seen table 1.
The performance index evaluation of two kinds of diagnostic kits of table 1
By table 1 data analysis, the present invention's's " chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen " accuracy, specificity and stability all reach the enzyme linked immunological kit level.And sensitivity significantly is superior to enzyme linked immunological kit.
Embodiment 9 chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen of the present invention and enzyme linked immunological kit are relatively
Collect hospital and make a definite diagnosis 50 parts of TCCB patients serums, urinary system benign disease patients serum 20 examples, 200 parts of normal human serums.Use the kit and the enzyme linked immunological kit of the embodiment of the invention 1 to carry out blood examination respectively, the statistical experiment result line correlation analysis of going forward side by side, coefficient R=0.9456.Contrast and experiment is seen table 2.
The clinical trial comparison of table 2 kit of the present invention and enzyme linked immunological kit
Can be known that by table 2 data analysis in 70 parts of TCCB patients, detect 68 parts of positives with kit of the present invention, enzyme linked immunological kit detects 66 parts of positives, the clinical coincidence rate of kit of the present invention will be higher than enzyme linked immunological kit; In urinary system benign disease patient; 2 parts of positives appear in kit of the present invention; 3 parts of positives appear in enzyme linked immunological kit, and a copy of it patient is diagnosed as urinary tract infection, and another part is glomerulonephritis; And 1 part of false positive appears in enzyme linked immunological kit, explain diagnose bladder move the shape cell cancer be will with the antidiastole of urinary system benign disease; In normal human serum, 1 part of positive appears in kit of the present invention, and 3 parts of positives appear in enzyme linked immunological kit, prove that once more the clinical coincidence rate of kit of the present invention is higher than enzyme linked immunological kit.In sum, kit of the present invention obviously is superior to enzyme linked immunological kit, for the diagnosis of bladder cancer patients provides more reliable foundation.
Claims (4)
1. chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen; It is characterized in that; Chemical luminous substrate, concentrated cleaning solution that the solid phase carrier that said kit is encapsulated by urine bladder cancer antigen calibration object, urine bladder cancer antigen monoclonal antibody, urine bladder cancer antigen monoclonal antibody enzyme labeling thing, above-mentioned enzyme are acted on are formed, and said enzyme is alkaline phosphatase or horseradish peroxidase, and said chemical luminous substrate is 1; 2-two oxidative ethane analog derivatives, luminol or different luminol
Wherein, the solid phase carrier that encapsulates of said urine bladder cancer antigen monoclonal antibody is through the following steps preparation:
1) encapsulates
Adopt the CT damping fluid of 0.046M pH 4.6 and the urine bladder cancer antigen monoclonal antibody of debita spissitudo to be mixed and made into coating buffer; The CT buffer formulation of described 0.046M pH 4.6 is the trisodium citrate of 7.30g and citric acid and the 1000mL distilled water of 4.44g; The pH value is 4.6; Damping fluid with preparation mixes with the monoclonal antibody of urine bladder cancer antigen then, and the gained coating buffer is carried on the solid phase carrier, and said solid phase carrier is microwell plate, plastic bead, plastic tube;
2) sealing
Confining liquid is selected the PBS-BSA damping fluid for use, and said confining liquid pH value is that 7.0 prescriptions do
0.2gNaH
2PO
42H
2O, 2.9gNa
2HPO
412H
2O, 10gBSA, Proclin300 10g and 1000mL distilled water.
2. kit as claimed in claim 1 is characterized in that, said concentrated cleaning solution is Tris-HCL cleansing solution or PBST cleansing solution.
3. kit as claimed in claim 1; It is characterized in that said 1,2-two oxidative ethane analog derivatives are (diamantane)-1; 2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
4. method for preparing the said kit of claim 1 is characterized in that may further comprise the steps:
1) preparation urine bladder cancer antigen calibration object;
2) encapsulate solid phase carrier with the urine bladder cancer antigen monoclonal antibody, wherein, the solid phase carrier that described urine bladder cancer antigen monoclonal antibody encapsulates prepares through following steps:
A) encapsulate
Adopt the CT damping fluid of 0.046M pH 4.6 and the urine bladder cancer antigen monoclonal antibody of debita spissitudo to be mixed and made into coating buffer; The CT buffer formulation of described 0.046M pH 4.6 is the trisodium citrate of 7.30g and citric acid and the 1000mL distilled water of 4.44g; The pH value is 4.6; Damping fluid with preparation mixes with the monoclonal antibody of urine bladder cancer antigen then, and the gained coating buffer is carried on the solid phase carrier, and said solid phase carrier is microwell plate, plastic bead, plastic tube;
B) sealing
Confining liquid is selected the PBS-BSA damping fluid for use, and said confining liquid pH value is that 7.0 prescriptions are 0.2gNaH
2PO
42H
2O, 2.9gNa
2HPO
412H
2O, 10gBSA, Proclin300 10g and 1000mL distilled water;
3) with enzyme labeling urine bladder cancer antigen monoclonal antibody, said enzyme is alkaline phosphatase or horseradish peroxidase;
4) the preparation chemical luminous substrate liquid that above-mentioned enzyme acted on, said chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol;
5) preparation concentrated cleaning solution;
6) the above-mentioned urine bladder cancer antigen calibration object of packing, the urine bladder cancer antigen monoclonal antibody of enzyme labeling, chemical luminous substrate and the concentrated cleaning solution that this enzyme acted on are assembled into finished product with each component at last.
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| 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 |
| CN103645310B (en) * | 2013-11-18 | 2016-06-01 | 洛阳莱普生信息科技有限公司 | A kind of Macrodantin chemiluminescence detection kit |
| CN103969447A (en) * | 2014-05-21 | 2014-08-06 | 张从从 | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof |
| 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 |
| CN104267197A (en) * | 2014-10-24 | 2015-01-07 | 浙江东方基因生物制品有限公司 | Nuclear matrix protein 22 chemiluminescent immunodetection reagent kit and preparing method thereof |
| CN105158483A (en) * | 2015-09-16 | 2015-12-16 | 北京九强生物技术股份有限公司 | Quantitative determination kit for hypersensitivity of Troponin I and detection method |
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Address after: 100094 Beijing Haidian District Yongfeng Base Fengxianzhong Road 7 Beike Modern Manufacturing Park Incubation Building Patentee after: Kemei Diagnostic Technology Co., Ltd Address before: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Patentee before: Beijing Kemei Biological Technology Co., Ltd. |