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.
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 |
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 |
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.
Embodiment 5 preparations chemical luminescence immune analysis quantitative measuring reagent kit for urine bladder cancer antigen of the present invention
, 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.