CN101368961A - 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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- CN101368961A CN101368961A CN 200810103266 CN200810103266A CN101368961A CN 101368961 A CN101368961 A CN 101368961A CN 200810103266 CN200810103266 CN 200810103266 CN 200810103266 A CN200810103266 A CN 200810103266A CN 101368961 A CN101368961 A CN 101368961A
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- bladder cancer
- cancer antigen
- urine bladder
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- 102000036639 antigens Human genes 0.000 title claims abstract description 50
- 108091007433 antigens Proteins 0.000 title claims abstract description 50
- 206010005003 Bladder cancer Diseases 0.000 title claims description 47
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- 238000002360 preparation method Methods 0.000 title claims description 18
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- 238000004020 luminiscence type Methods 0.000 title claims description 12
- 238000004458 analytical method Methods 0.000 title claims description 11
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- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 claims description 5
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- QWXOJIDBSHLIFI-UHFFFAOYSA-N [3-(1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC2CC(Cl)(C4)C3)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 QWXOJIDBSHLIFI-UHFFFAOYSA-N 0.000 claims description 4
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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.Main to carcinoma of urinary bladder diagnosis and postoperative check at present by cystoscope and urinary cytology inspection.Though cystoscopy is accurate, is difficult to find carcinoma in situ or flat cancer; Though the urinary cytology inspection does not have wound, and specificity is better, and susceptibility is low, and for the tumour of lower, good differentiation by stages, its susceptibility only is 30% or lower, and is subjected to the influence of diagnostician's subjective factor bigger.The limitations restrict that cystoscope and urine exfoliative cytology are checked to the application of carcinoma of urinary bladder diagnosis, so it is very necessary to seek 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, easily causes false negative and false positive.Remarkable advantages such as the present invention adopts chemoluminescence method to detect UBC, and it is simple, easy to operate, highly sensitive to have an instrument and equipment, and linear response range is wide and easily be automated.Detect UBC with this method, than the enzyme-linked immunosorbent assay height, and to tumour early stage, good differentiation, its susceptibility is also higher to wellability tumor of bladder susceptibility, 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 height, technical requirement height, 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 purpose of the present 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 luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen of the present invention by urine bladder cancer antigen calibration object, urine bladder cancer antigen monoclonal antibody bag by solid phase carrier, chemical luminous substrate and the concentrated cleaning solution that urine bladder cancer antigen monoclonal antibody enzyme labeling thing, above-mentioned enzyme are acted on form.
According to kit of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle; Described enzyme is alkaline phosphatase or horseradish peroxidase; Described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol; Described 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; Described 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) with urine bladder cancer antigen monoclonal antibody bag by solid phase carrier; 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.
The method according to this invention, described bag be may further comprise the steps by solid phase carrier:
Be cushioned in the liquid at bag and add the urine bladder cancer antigen monoclonal antibody and make working fluid, it is carried on the solid phase carrier, 4 ℃ are spent the night, seal with confining liquid.
Wherein bag is cushioned the citrate buffer solution that liquid is selected 0.046M pH4.6 for use.
Confining liquid is selected the PBS damping fluid for use.
In said method, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
In said method, described enzyme is alkaline phosphatase or horseradish peroxidase.
In said method, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
In said method, described 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, described 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 combination, 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 substantially, is current at the widest class 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 by 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.
Urine bladder cancer antigen monoclonal antibody bag is finished by physisorption by solid phase.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 are less demanding, 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 the 1:4000 working concentration;
(1) horseradish peroxidase-labeled urine bladder cancer antigen MONOCLONAL ANTIBODIES SPECIFIC FOR
Get 10mg HRP and add 1ml0.1mol/L sodium acetate or water-soluble separating, fully mixing adds 0.08mol/L NaIO after about 5 minutes
4After aqueous solution 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) precipitation hydroformylation enzyme again, the centrifugal supernatant that goes, after the precipitation enzyme washs once with 6ml 80% ice-cold alcohol solution dipping again, the centrifugal ethanol that inclines, the precipitation 0.05mol/L sodium carbonate buffer 2ml dissolving of PH9.6, add the anti-human IgG of 2ml sheep or horse (about inner protein amount 20mg) then, stir, put in the refrigerator and spend the night, next day, taking-up added 10mg NaBH
4Mixing, add equivalent saturated ammonium sulphate enzyme conjugates after 3 hours, centrifugal, remove supernatant, precipitation is with the washing of 50% saturated ammonium sulfate once, centrifugal, remove supernatant again, precipitation changes in the bag filter with normal saline dialysis desalination (need change liquid 5 times) with the 0.01mol/LPBS3ml dissolving, need centrifugal removing if any precipitation, 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 product 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) bag quilt
Adopt the CT damping fluid of 0.046M pH4.6 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, described method for coating is:
| Trisodium citrate | 7.3g |
| Citric acid | 4.44g |
| Distilled water | 1000mL |
Behind the dissolving mixing, adjust pH value to 4.6, add 5.0mg urine bladder cancer antigen monoclonal antibody mixing, add then in each hole of microwell plate, 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
Using method: before using A liquid is mixed the back with B liquid in the 1:1 ratio and use.
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 |
Adjust 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, is assembled into finished product, 4 ℃ of preservations after qualified through inspecting by random samples.
To sum up, in research process of the present invention, the present inventor has at first carried out shaker test and Quality Identification to used starting material, then method for coating is studied, select optimal bag and be cushioned liquid and confining liquid, 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, dilute the enzyme labeling thing with the enzyme labelled antibody dilution with 1:2000, 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, and all the other all prepare quantitative determination reagent kit of the present invention with the method identical with embodiment 1.
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 all prepare quantitative determination reagent kit of the present invention with the method identical with embodiment 1.
, the urine bladder cancer antigen monoclonal antibody is connected in outside the magnetic-particle surface with classical glutaraldehyde method as carrier divided by magnetic-particle, all the other all prepare detection by quantitative kit of the present invention with the method identical with embodiment 1.
The using method of embodiment 6 kits of the present invention
The using method of embodiment 1 described kit is as follows:
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, the experiment with computing measurement result.
The using method of embodiment 7 kits of the present invention
The using method of embodiment 2 described 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 described using method 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 described using method, and enzyme linked immunological kit is outsourcing, and strictness illustrates the step use on schedule, and the performance evaluation index of two kinds of kits sees 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 better than 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 sees Table 2.
The clinical trial comparison of table 2 kit of the present invention and enzyme linked immunological kit
By table 2 data analysis as can be known, 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, and 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, a copy of it patient is diagnosed as urinary tract infection, another part is glomerulonephritis, and 1 part of false positive appears in enzyme linked immunological kit, illustrate 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 better than enzyme linked immunological kit, for the diagnosis of bladder cancer patients provides more reliable foundation.
Claims (10)
1. chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen, it is characterized in that, described kit by urine bladder cancer antigen calibration object, urine bladder cancer antigen monoclonal antibody bag by solid phase carrier, chemical luminous substrate, the concentrated cleaning solution that urine bladder cancer antigen monoclonal antibody enzyme labeling thing, above-mentioned enzyme are acted on form.
2. kit as claimed in claim 1 is characterised in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
3. kit as claimed in claim 1 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
4. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
5. kit as claimed in claim 1 is characterized in that, described concentrated cleaning solution is Tris-HCL cleansing solution or PBST cleansing solution.
6. kit as claimed in claim 4, it is characterized in that described 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.
7. a method for preparing the described kit of claim 1 is characterized in that may further comprise the steps: 1) preparation urine bladder cancer antigen calibration object; 2) with urine bladder cancer antigen monoclonal antibody bag by solid phase carrier; 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.
8. method as claimed in claim 7 is characterized in that, described bag is comprised quilt, sealing by the step of solid phase carrier.
9. method as claimed in claim 8 is characterized in that, bag is used the citrate buffer solution of 0.046M pH 4.6 and the urine bladder cancer antigen monoclonal antibody of debita spissitudo is mixed and made into coating buffer, and it is carried on the solid phase carrier; Confining liquid is selected the PBS-BSA damping fluid for use, carries out drying and shrouding then and handles.
10. method as claimed in claim 7 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
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