CN1880954A - Macro creatine kinase agarose electrophoresis kit and preparation method thereof - Google Patents
Macro creatine kinase agarose electrophoresis kit and preparation method thereof Download PDFInfo
- Publication number
- CN1880954A CN1880954A CN 200510026832 CN200510026832A CN1880954A CN 1880954 A CN1880954 A CN 1880954A CN 200510026832 CN200510026832 CN 200510026832 CN 200510026832 A CN200510026832 A CN 200510026832A CN 1880954 A CN1880954 A CN 1880954A
- Authority
- CN
- China
- Prior art keywords
- agarose
- barbital
- preparation
- macro
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a huge creatine-kinase agarose electrophoresis agent box and making method in the medical biological domain, which comprises the following parts: agarose gel plate, Tris buffer, soluble bartitone-barbitone electrophoresis buffer, substrate, substrate buffer and color-developing agent. The invention can test serum sample to avoid false positive occurrence of CK-M B due to immune inhibiting method, which provides correct testing report for clinical.
Description
Technical field
The invention belongs to the medicine bioengineering field, relate to a kind of kit and preparation method thereof, more particularly, the present invention relates to a kind of a kind of macro creatine kinase agarose electrophoresis kit that can be used for the clinical disease detection and preparation method thereof.
Background technology
Creatine kinase (CK) has three kinds of isodynamic enzyme: CK-MM, CK-MB and CK-BB by the dimer that M and B subunit form in tenuigenin.In cell mitochondrial, also have another kind of isodynamic enzyme, be called CK-Mt (claiming huge CK again).Huge CK is divided into huge CK1 and huge CK2 again.Huge CK1 is the compound of a kind of CK isodynamic enzyme and autoantibody, because of its molecular weight is big, is difficult in vivo discharging, again because of it has the long half life period, so retention time is longer in blood.Document thinks that CK1 and disease do not have obviously and gets in touch, in addition have in healthy human blood, detect huge CK1.Huge CK2 is a kind of oligomeric mitochondria CK, can in healthy human blood, not occur, external numerous researcher is consistent to think that it and malignant tumour are closely related owing to its high recall rate in some tumour patient serum makes in recent years, can be used as a kind of new tumor markers.
CK-BB is present in healthy human brain tissue, in intestines and stomach and the uterine smooth muscle, does not have CK-BB in the normal human serum.Can occur serum CK-BB when necrosis or some malignant tumour and raise, expansion of prompting tumour or replying treatment usually raise in serum.
By wide clinical application, its measuring principle is immunosuppression assay for detecting CK-MB as the specific index of early diagnosis acute myocardial infarction AMI: with anti-CK-M antibody the M subunit is suppressed, measure the B subunit, the result takes advantage of 2 to be the CK-MB activity.And when occurring the normal person is not contained or content is very little huge CK or CK-BB in the serum, because they are not suppressed by anti-M subunit antibody, its activity is absolutely measured, be multiplied by coefficient 2 again, the phenomenon that " CK-MB " be equal to or higher than total CK activity (in fact, the CK-MB activity ratio that accounts for total CK activity seldom occurs more than 30%) will appear.Very easily healthy people or tumor patient mistaken diagnosis are hospitalized for treatment for the heart disease patient, cause the mental burden of serious patient, the early diagnosis of wasting unnecessary medical expense and incuring loss through delay tumor patient.
Along with the continuous improvement of inspection technology, full automatic biochemical apparatus is also more and more universal at small-middle hospital at present, and huge CK or CK-BB are more and clearer and more definite to the interference that the clinical examination result is caused.Had a strong impact on the correctness of assay, caused false judgment, even caused unnecessary medical tangle the enzymatic determination result.Therefore, need to find a kind of detection method of false-positive generation more accurate, that caused in the time of can avoiding because of immunosuppression assay for detecting CK-MB clinically.
At present, the development of the inferior company in U.S. Helen that and French Thebe also can be used for detecting macrocreatine kinase except supporting full-automatic (semi-automatic) electrophoresis apparatus agarose electrophoresis kit.Its primary structure is: full-automatic (semi-automatic) electrophoresis apparatus, agarose gel plate, sample cup (sample comb), reagent substrate, developer etc.Blood serum sample adds to (in the sample comb circular hole) in the sample cup, place on the specimen holder standby, other gets a slice agarose gel plate and is placed on the electrophoresis plate, a carbon-point (soaking into the film bar of damping fluid) is respectively put in the agar plate both sides, other disposes a bottle reagent substrate and puts into reagent trough (being injected by the sample panel aperture), reserve the mensuration program in advance, press switch and promptly begin electrophoresis.
But, above-mentioned full-automatic or semi-automatic electrophoresis apparatus costs an arm and a leg, general hospital can not buy, its special-purpose assorted agent box can not detect by the short run sample in addition, and 8 samples of kit minimum package amount one-time detection of promoting are less than 8 parts as sample at present, cost will improve greatly, and find often that clinically a routine suspicious sample need identify at once, therefore, be not suitable for identifying and use.
Summary of the invention
The object of the present invention is to provide a kind of macro creatine kinase agarose electrophoresis kit and preparation method thereof, use macro creatine kinase agarose electrophoresis kit of the present invention and identify serum specimen, the false-positive generation that is caused in the time of can avoiding because of immunosuppression assay for detecting CK-MB, provide correct examining report for clinical, for the early diagnosis of tumour provides new tumor markers.
The present invention solves the problems of the technologies described above the technical scheme that is adopted to provide a kind of macro creatine kinase agarose electrophoresis kit, wherein contain agarose gel plate, Tris damping fluid, barbital sodium-barbital electrophoretic buffer, substrate, substrate buffer solution and developer, in the described Tris damping fluid of every 1000ml, contain 20mmolTris, 14 gram sucrose, 500 milligrams of Sodium azides, 4-6gL-aspartic acid dry powder;
Contain 1% agarose in the described agarose gel plate;
In every liter of described barbital sodium-barbital electrophoretic buffer, contain 10.3 gram barbital sodiums, 1.84 gram barbitals;
In every liter of described substrate, contain 4.0mmolADP, 10.0mmol LAMP, 20 μ mol, two adenosines, five phosphoric acid, 4.0mmolNADP, 60mmol creatine acid sodium, 〉=5.0U hexokinase, 〉=3.0U glucose-6-phosphate dehydrogenase, 40mmolN-acetylcysteine;
The imidazoles that in every liter of described substrate buffer solution, contains 20mmol, the glucose of 40mmol, the magnesium acetate of 20mmol, 4.0mmolEDTA;
Contain chlorination nitro blue tetrazolium 2mg in the described developer of every 1ml, azophenlyene dimethyl ester sulfate 0.1mg.
Preferably, the pH value of described Tris damping fluid is 7.4.
Preferably, the PH8.6 of described barbital sodium-barbital electrophoretic buffer.
Preferably, the pH value of described substrate buffer solution is 6.7.
The present invention also aims to provide a kind of preparation method of macro creatine kinase agarose electrophoresis kit, may further comprise the steps:
(1) preparation PH7.4Tris damping fluid: take by weighing 20mmolTris, 14 gram sucrose, 500 milligrams of Sodium azides are positioned in the container then, add about 800ml distilled water again and stir, container is placed in 37 ℃ of water tanks, add L-aspartic acid dry powder again in container, limit edged stirring and dissolving is with PH test strips coarse adjustment PH about 7.4, adjust pH value to 7.4 with the PH analyzer at last, add water to 1000ml;
(2) preparation 1% Ago-Gel:
Take by weighing 1 gram agarose, heating for dissolving in PH is 7.4 Tris damping fluid 100ml, packing 8ml/ pipe;
(3) preparation agarose gel plate: take out 8ml/ and manage 1% Ago-Gel, the water proof heating for dissolving, putting the transparent polyester film of a slice 110mm * 80mm on iron plate supports, press a centre to dig the silica gel sheet of 100mm * 70mm * big aperture of 1mm above, be adjacent to the polyester film water all around, the 8ml agarose of heating for dissolving is poured in the hole, take advantage of a situation with the pipe end agarose being paved, after cooling, throw off silica gel sheet cleaned standby seam, on gel slab, cover a preservative film, take off gel slab, put into the plastic casing of a sealing;
(4) preparation PH8.6,0.05mol/L barbital sodium-barbital electrophoretic buffer: take by weighing barbital sodium 10.3 grams, barbital 1.84 grams are dissolved in the 1L distilled water;
(5) preparation substrate: with 4.0mmol LADP, 10.0mmol LAMP, 20 μ mol, two adenosines, five phosphoric acid, 4.0mmolNADP, 60mmol Creatine Phosphate Sodium, 〉=5.0U hexokinase, 〉=3.0U6-glucose phosphate dehydrogenase, the 40mmolN-acetylcysteine adds in the 1L distilled water and mixes;
(6) preparation substrate buffer solution: 20mmol imidazoles, 40mmol glucose, 20mmol magnesium acetate, 4.0mmolEDTA are dissolved in 1L distilled water, regulate PH to 6.7 with acetic acid;
(7) preparation developer: take by weighing chlorination nitro blue tetrazolium (NBT) 2mg, azophenlyene dimethyl ester sulfate (PMS) 0.1mg, adding distil water is to 1ml before using.
The present invention also aims to provide the application of macro creatine kinase agarose electrophoresis kit on lesion detection, may further comprise the steps:
1) gets the 1% agarose gel plate a slice that has prepared, cover the wide filter paper bar of 2cm from cathode side 1.5cm place, inhale and remove part moisture, remove behind the filter paper application of sample pore membrane bar is placed on it, flicking application of sample pore membrane is adjacent to itself and offset plate, avoid bubble, then 5-7 μ l sample is added in the hand-hole, left standstill 5-10 minute, treat that sample diffusion enters in the glue, be placed on the application of sample fenestra with a new thieving paper, inhale gently and remove unnecessary sample, remove the striping bar then;
2) offset plate is put on the electrophoresis tank that both sides have been added with damping fluid, application of sample is sidelong in cathode side, and two ends are put up a bridge with four layers of gauze, and electrophoresis 20 minutes, deposition condition are every plate 40mA, voltage 150V;
3) dissolve substrate with substrate buffer solution, treat that electrophoresis finishes, take out gel slab, substrate is uniformly applied on the offset plate, cover above one with offset plate plastic sheeting of the same size, put in 37 ℃ of constant temperature water baths incubation 40 minutes, or 45 ℃ of incubations 20 minutes;
4) incubation is finished, and removes the plastic sheeting of covering, puts drying box and dries in interior 20 minutes, and bake out temperature is lower than 75 ℃;
5) percent of each isodynamic enzyme district band of sweep measuring on the fluorescence opacimeter, or whether Direct observation the hot spot band of the huge CK of hyacinthine, CK-BB or CK-MB occurs under the 340nm uviol lamp.
More particularly, macro creatine kinase agarose electrophoresis kit of the present invention is scanning with visible light when quantitative, at complete operation 4) after, 1ml developer shop is added on the gel slab, covered with plastic film once more, the room temperature lucifuge was placed 10-15 minute, with 5% acetic acid rinsing background, scanned in 530-570nm.
The cardinal principle of utilization of the present invention is: the CK isodynamic enzyme is electrophoretic separation on the agarose medium, CK-BB migration anode albumin zone, CK-MB is positioned at α 2 to the beta Globulin zone, and CK-MM is positioned at cathode side gamma Globulin district, huge CK1 is between CK-MB and CK-MM, huge CK2 mobility is lower, is positioned at the CK-MM cathode side.Electrophoresis is finished, and covers substrate, and is dry behind the incubation, detects in the scanning of 340nm opacimeter, or cover PMS-NBT again behind the substrate incubation, and the enzyme band is hyacinthine, and available visible light scanning detects.
Before kit of the present invention uses, need be positioned in 4 ℃ of refrigerators standby, can not be freezing.
The present invention compares with prior art, and cost is low, and is easy and simple to handle, is fit to short run or individual other detection.Need not special instrument and equipment, most of laboratory all can be finished.Ago-Gel stilt polyester film water white transparency in addition, can be under the 340nm uviol lamp Direct observation, take pictures, and can connect computer and keep image.And import reagent box Ago-Gel stilt is a black, can not be under the 340nm uviol lamp Direct observation, take pictures, keep image.Simultaneously, macro creatine kinase agarose electrophoresis kit of the present invention is identified serum specimen, the false-positive generation that is caused in the time of can avoiding because of immunosuppression assay for detecting CK-MB provides correct examining report for clinical, for the early diagnosis of tumour provides new tumor markers.
Description of drawings
Fig. 1 is CK isodynamic enzyme and the electrophoresis synoptic diagram of huge CK on the agarose medium.
Fig. 2 is the electrophoretogram that adopts a sample of huge kreatinase kit detection of the present invention.
Fig. 3 is the electrophoretogram that adopts another sample of huge kreatinase kit detection of the present invention.
Embodiment
The preparation of embodiment 1 huge kreatinase kit of the present invention
The reagent that huge kreatinase kit of the present invention comprises is as follows:
1) PH7.4Tris damping fluid: 20mmolTris, 14 gram sucrose, 500 milligrams of Sodium azides, the about 800ml of adding distil water places beaker in 37 ℃ of water tanks, directly adds L-aspartic acid dry powder in beaker, limit edged stirring and dissolving, with about 7.4 (approximately with about L-aspartic acid dry powder 5 grams) of PH test strips coarse adjustment PH, adjust PH to 7.4 with the PH analyzer at last, add water to 1000ml and place refrigerator standby.
2) 1% Ago-Gel: weighing 1 gram agarose, heating for dissolving is in PH7.4Tris damping fluid 100ml, and packing 8ml/ pipe places in 4 ℃ of refrigerators standby.
3) agarose gel plate: take out 8ml/ and manage 1% Ago-Gel, the water proof heating for dissolving, putting the transparent polyester film of a slice 110mm * 80mm on iron plate supports, press a centre to dig the silica gel sheet of 100mm * 70mm * big aperture of 1mm above, be adjacent to the polyester film water all around, space (otherwise agarose is easily overflowed) is not arranged, the 8ml agarose of heating for dissolving is poured in the hole, take advantage of a situation with the pipe end agarose being paved, after cooling, throw off silica gel sheet cleaned standby seam, on gel slab, cover a preservative film, take off gel slab, put into the built-in 4 ℃ of refrigerators of plastic casing of a sealing and preserve.
4) PH8.6,0.05mol/L barbital sodium-barbital electrophoretic buffer: take by weighing barbital sodium 10.3 grams, barbital 1.84 grams are dissolved in the 1L distilled water.
5) preparation substrate: with 4.0mmol LADP, 10.0mmolLAMP, 20 μ mol, two adenosines, five phosphoric acid, 4.0mmolNADP, 60mmol Creatine Phosphate Sodium, 〉=5.0U hexokinase, 〉=3.0U6-glucose phosphate dehydrogenase, the 40mmolN-acetylcysteine adds in the 1L distilled water and mixes;
6) preparation substrate buffer solution: 20mmol imidazoles, 40mmol glucose, 20mmol magnesium acetate, 4.0mmolEDTA are dissolved in 1L distilled water, regulate PH to 6.7 with acetic acid;
7) developer: chlorination nitro blue tetrazolium (NBT) 2mg, azophenlyene dimethyl ester sulfate (PMS) 0.1mg, adding distil water 1ml before using.
The sample detection of embodiment 2 huge kreatinase kits of the present invention
Electrophoresis apparatus that the present invention uses and electrophoresis tank are as sale is arranged on the market.The application of sample pore membrane bar that the present invention adopts is prepared as follows: with a rigidulous plastic sheeting, tailor into 150mm * 20mm size dimension.After the iron pin heating with a 2mm * 5mm size dimension plastic sheeting is burnt a hole, a plastic sheeting can scald ten holes.
The concrete following steps that adopt:
1) gets agarose gel plate a slice,, inhale and remove part moisture cover the wide filter paper bar of 2cm from cathode side 1.5cm place, remove behind the filter paper application of sample pore membrane bar is placed on it, flicking application of sample pore membrane is adjacent to itself and offset plate, avoids bubble, then 5-7 μ l sample is added in the hand-hole, note making in the hole all being full of equably, left standstill 5-10 minute, and treated that sample diffusion entered in the glue, be placed on the application of sample fenestra with a new thieving paper, inhale gently and remove unnecessary sample, remove striping then.
2) offset plate is put on the electrophoresis tank that both sides have been added with damping fluid, noted application of sample is sidelong in cathode side, two ends are put up a bridge (higher as room temperature, overhead frame one ice bag is lowered the temperature) with four layers of gauze, and electrophoresis 20 minutes, deposition condition are every plate 40mA, voltage 150V.
3) dissolve substrate with the 1ml substrate buffer solution before electrophoresis finishes.
4) electrophoresis is finished, and takes out gel slab, and substrate is uniformly applied on the offset plate, cover above one with offset plate plastic sheeting of the same size, note avoiding bubble, put in 37 ℃ of constant temperature water baths incubation 40 minutes, or 45 ℃ of incubations 20 minutes.
5) incubation is finished, and removes the plastic sheeting of covering, puts in the drying box and to dry in 20 minutes (maximum temperature can not above 75 ℃).
6) percent of each isodynamic enzyme district band of sweep measuring on the fluorescence opacimeter, but also under the 340nm uviol lamp Direct observation whether the hot spot band of huge CK, CK-BB or CK-MB appears.
7) as quantitatively, then after complete operation 4,1ml developer shop is added on the gel slab with visible light scanning, covered with plastic film once more, the room temperature lucifuge was placed 10-15 minute, with 5% acetic acid rinsing background, scanned in 530-570nm.
Fig. 1 is CK isodynamic enzyme and the electrophoresis synoptic diagram of huge CK on the agarose medium, the CK isodynamic enzyme is electrophoretic separation on the agarose medium, CK-BB migration anode albumin zone, CK-MB is positioned at α 2 to the beta Globulin zone, and CK-MM is positioned at cathode side gamma Globulin district, huge CK1 is between CK-MB and CK-MM, and huge CK2 mobility is lower, is positioned at the CK-MM cathode side.Fig. 2, Fig. 3 are the electrophoretograms that adopts two samples of huge kreatinase kit detection of the present invention, wherein, a represents quality-control sample, b is patient's sample, c is a normal control, Fig. 2 illustrated in this patient's the serum and contained CK-MM and huge CK2, and Fig. 3 has illustrated in this patient's the serum and contained huge CK1 and CK-MM.
The test of embodiment 3 clinical detection
Use macro creatine kinase agarose electrophoresis kit of the present invention and immunodepression, the inventor has identified 133 routine clinical samples, and result's contrast wherein sees Table 1.Adopt kit of the present invention, wherein 20 routine samples contain huge CK1, and 107 routine samples contain huge CK2 (wherein turning out to be malignant tumour person's 71 examples through pathology, cirrhosis 16, other disease 20 examples) and 6 routine samples contain CK-BB.And the employing immunosuppression assay for detecting, these samples all have a common characteristic, and promptly " CK-MB " accounts for total CK activity more than 50% in the serum, and total CK activity normally or slightly increases.Therefore adopt kit of the present invention, avoided the false-positive phenomenon of CK-MB occurring, cause misjudgment and clinical misdiagnosis owing to the interference of huge CK1, CK2, CK-BB.Thereby detect tumor patient and heart disease patient more accurately, provide correct survey report for clinical.
Table 1
| Immunodepression | The present invention | |||||
| Type | The example number | The active average U/L of total CK | " CK-MB " average U/L | Huge CK1 | Huge CK2 | CK-BB |
| The focal myositis pneumonia of Healthy People coronary angiography Postoperative hypertension ventricular aneurysm IgG increases IgE hepatocellular carcinoma breast cancer meninx metastatic tumor Cervix Squamous Cell cancer carcinoma of the rectum colon cancer cancer of the stomach lung cancer carcinoma of gallbladder nodular panniculitis leukaemia cirrhosis Other diseases ED-SCLC lung cancer and does not make a definite diagnosis | 5 4 4 1 3 1 1 1 13 4 3 2 5 6 2 4 2 1 1 10 20 4 1 1 | 228 236 140 240 371 242 198 146 209 185 231 131 163 99 130 164 149 218 379 280 140 67 53 462 | 230 180 100 386 420 101 162 101 200 196 115 202 120 56 86 156 80 200 262 150 60 39 34 370 | + + + + + + + + | + + + + + + + + + + + + + + | + + + |
Claims (7)
1. a macro creatine kinase agarose electrophoresis kit wherein contains agarose gel plate, Tris damping fluid, barbital sodium-barbital electrophoretic buffer, substrate, substrate buffer solution and developer, it is characterized in that:
Contain 1% agarose in the described agarose gel plate;
In every liter of described Tris damping fluid, contain 20mmolTris, 14 gram sucrose, 500 milligrams of Sodium azides, 4-6g L-aspartic acid dry powder;
In every liter of described barbital sodium-barbital electrophoretic buffer, contain 10.3 gram barbital sodiums, 1.84 gram barbitals;
In every liter of described substrate, contain 4.0mmolADP, 10.0mmol LAMP, 20 μ mol, two adenosines, five phosphoric acid, 4.0mmolNADP, 60mmol creatine acid sodium, 〉=5.0U hexokinase, 〉=3.0U glucose-6-phosphate dehydrogenase, 40mmolN-acetylcysteine;
The imidazoles that in every liter of described substrate buffer solution, contains 20mmol, the glucose of 40mmol, the magnesium acetate of 20mmol, 4.0mmolEDTA;
Contain chlorination nitro blue tetrazolium (NBT) 2mg in the described developer of every 1ml, azophenlyene dimethyl ester sulfate (PMS) 0.1mg.
2. macro creatine kinase agarose electrophoresis kit as claimed in claim 1 is characterized in that: the pH value of described Tris damping fluid is 7.4.
3. macro creatine kinase agarose electrophoresis kit as claimed in claim 1 is characterized in that: the PH8.6 of described barbital sodium-barbital electrophoretic buffer.
4. macro creatine kinase agarose electrophoresis kit as claimed in claim 1 is characterized in that: the pH value of described substrate buffer solution is 6.7.
5. the preparation method of the described macro creatine kinase agarose electrophoresis kit of claim 1 is characterized in that, may further comprise the steps:
(1) preparation PH7.4Tris damping fluid: take by weighing 20mmolTris, 14 gram sucrose, 500 milligrams of Sodium azides are positioned in the container then, add about 800ml distilled water again and stir, container is placed in 37 ℃ of water tanks, add L-aspartic acid dry powder again in container, limit edged stirring and dissolving is with PH test strips coarse adjustment PH about 7.4, adjust PH to 7.4 with the PH analyzer at last, add water to 1000ml;
(2) preparation 1% Ago-Gel: take by weighing 1 gram agarose, heating for dissolving in PH is 7.4 Tris damping fluid 100ml, packing 8ml/ pipe;
(3) preparation agarose gel plate: take out 8ml/ and manage 1% Ago-Gel, the water proof heating for dissolving, putting the transparent polyester film of a slice 110mm * 80mm on iron plate supports, press a centre to dig the silica gel sheet of 100mm * 70mm * big aperture of 1mm above, be adjacent to the polyester film water all around, the 8ml agarose of heating for dissolving is poured in the hole, take advantage of a situation with the pipe end agarose being paved, after cooling, throw off silica gel sheet cleaned standby seam, on gel slab, cover a preservative film, take off gel slab, put into the plastic casing of a sealing;
(4) preparation PH8.6,0.05mol/L barbital sodium-barbital electrophoretic buffer: take by weighing barbital sodium 10.3 grams, barbital 1.84 grams are dissolved in the 1L distilled water;
(5) preparation substrate: with 4.0mmol LADP, 10.0mmol LAMP, 20 μ mol, two adenosines, five phosphoric acid, 4.0mmolNADP, 60mmol Creatine Phosphate Sodium, 〉=5.0U hexokinase, 〉=3.0U6-glucose phosphate dehydrogenase, the 40mmol N-acetylcystein adds in the 1L distilled water and mixes;
(6) preparation substrate buffer solution: 20mmol imidazoles, 40mmol glucose, 20mmol magnesium acetate, 4.0mmolEDTA are dissolved in 1L distilled water, regulate PH to 6.7 with acetic acid;
(7) preparation developer: take by weighing chlorination nitro blue tetrazolium (NBT) 2mg, azophenlyene dimethyl ester sulfate (PMS) 0.1mg, adding distil water is to 1ml before using.
6. the application of the described macro creatine kinase agarose electrophoresis kit of claim 1 on lesion detection is characterized in that, may further comprise the steps:
1) gets the 1% agarose gel plate a slice that has prepared, cover the wide filter paper bar of 2cm from cathode side 1.5cm place, inhale and remove part moisture, remove behind the filter paper application of sample pore membrane bar is placed on it, flicking application of sample pore membrane is adjacent to itself and offset plate, avoid bubble, then 5-7 μ l sample is added in the hand-hole, left standstill 5-10 minute, treat that sample diffusion enters in the glue, be placed on the application of sample fenestra with a new thieving paper, inhale gently and remove unnecessary sample, remove the striping bar then;
2) offset plate is put on the electrophoresis tank that both sides have been added with damping fluid, application of sample is sidelong in cathode side, and two ends are put up a bridge with four layers of gauze, and electrophoresis 20 minutes, deposition condition are every plate 40mA, voltage 150V;
3) dissolve substrate with substrate buffer solution, treat that electrophoresis finishes, take out gel slab, substrate is uniformly applied on the offset plate, cover above one with offset plate plastic sheeting of the same size, put in 37 ℃ of constant temperature water baths incubation 40 minutes, or 45 ℃ of incubations 20 minutes;
4) incubation is finished, and removes the plastic sheeting of covering, puts drying box and dries bake out temperature 60-75 ℃ in interior 20 minutes;
5) percent of each isodynamic enzyme district band of sweep measuring on the fluorescence opacimeter, or whether Direct observation the hot spot band of the huge CK of hyacinthine, CK-BB or CK-MB occurs under the 340nm uviol lamp.
7. the application of macro creatine kinase agarose electrophoresis kit according to claim 6 on lesion detection, it is characterized in that, scan when quantitative with visible light, at complete operation 4) after, 1ml developer shop is added on the gel slab, covered with plastic film once more, the room temperature lucifuge was placed 10-15 minute, with 5% acetic acid rinsing background, scan in 530-570nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026832 CN1880954A (en) | 2005-06-16 | 2005-06-16 | Macro creatine kinase agarose electrophoresis kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026832 CN1880954A (en) | 2005-06-16 | 2005-06-16 | Macro creatine kinase agarose electrophoresis kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1880954A true CN1880954A (en) | 2006-12-20 |
Family
ID=37519224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510026832 Pending CN1880954A (en) | 2005-06-16 | 2005-06-16 | Macro creatine kinase agarose electrophoresis kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1880954A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102759559A (en) * | 2012-07-26 | 2012-10-31 | 上海迪安医学检验所有限公司 | Diagnostic reagent kit for separating serum protein with ultra-thin agarose gel electrophoresis slab and preparation method of diagnostic reagent kit |
| CN103529412A (en) * | 2013-10-18 | 2014-01-22 | 吴仁华 | Experiment method based on pH value-sensitive magnetization transfer on 1.5T magnetic resonance imaging |
| CN104597098A (en) * | 2013-12-20 | 2015-05-06 | 上海迪安医学检验所有限公司 | Agarose gel plate storage method |
| RU2600165C1 (en) * | 2015-06-24 | 2016-10-20 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт акушерства, гинекологии и репродуктологии имени Д.О. Отта" | Method of determining cerebral isoform of creatine phosphokinase in human blood |
| CN107870190A (en) * | 2017-11-21 | 2018-04-03 | 中国人民解放军军事科学院军事医学研究院 | The hereditary biochemical site-tag of cerebral ischemia inbred strais meriones unguiculatus and its application |
| RU2679656C1 (en) * | 2018-04-24 | 2019-02-12 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр имени В.А. Алмазова" Министерства здравоохранения Российской Федерации | Method for determining creatin kinase macroforms and mb-macroform in human blood serum |
-
2005
- 2005-06-16 CN CN 200510026832 patent/CN1880954A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102759559A (en) * | 2012-07-26 | 2012-10-31 | 上海迪安医学检验所有限公司 | Diagnostic reagent kit for separating serum protein with ultra-thin agarose gel electrophoresis slab and preparation method of diagnostic reagent kit |
| CN103529412A (en) * | 2013-10-18 | 2014-01-22 | 吴仁华 | Experiment method based on pH value-sensitive magnetization transfer on 1.5T magnetic resonance imaging |
| CN104597098A (en) * | 2013-12-20 | 2015-05-06 | 上海迪安医学检验所有限公司 | Agarose gel plate storage method |
| RU2600165C1 (en) * | 2015-06-24 | 2016-10-20 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт акушерства, гинекологии и репродуктологии имени Д.О. Отта" | Method of determining cerebral isoform of creatine phosphokinase in human blood |
| CN107870190A (en) * | 2017-11-21 | 2018-04-03 | 中国人民解放军军事科学院军事医学研究院 | The hereditary biochemical site-tag of cerebral ischemia inbred strais meriones unguiculatus and its application |
| RU2679656C1 (en) * | 2018-04-24 | 2019-02-12 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр имени В.А. Алмазова" Министерства здравоохранения Российской Федерации | Method for determining creatin kinase macroforms and mb-macroform in human blood serum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1880954A (en) | Macro creatine kinase agarose electrophoresis kit and preparation method thereof | |
| CN101275954B (en) | Breast cancer early warning chip for easily rapid measuring human I type thymidine kinase gene protein | |
| CN105866418B (en) | A kind of breast carcinoma three joint inspection diagnostic kit | |
| Naber et al. | Strategies for the analysis of oncogene overexpression: studies of the neu oncogene in breast carcinoma | |
| CN1967246A (en) | Aminoacylase 1 and application for examining liver cancer by antibody of same | |
| Aleyassine et al. | Natural fluorescence in serum of patients with chronic renal failure not to be confused with creatine kinase-BB isoenzyme. | |
| CN102841201B (en) | Combined general check protein chip for early-stage cancers mainly comprising lung cancer | |
| CN110850096B (en) | Biomarker group and application thereof, protein chip kit and ELISA kit | |
| CN1188235A (en) | Anti-human IV type collagen enzyme linked immunological quantitative determining kit and preparing method | |
| Tan et al. | Sources of variability in quantifying circulating thymosin beta-4: literature review and recommendations | |
| Krawitt et al. | Limitations of the usefulness of the d-xylose absorption test | |
| CN107083428B (en) | Application of PAK5 in cancer diagnosis prognosis treatment and drug screening | |
| WO2013054984A1 (en) | Composition for diagnosis of lung cancer and diagnosis kit of lung cancer | |
| CN110687288B (en) | Multi-joint detection kit for rapidly detecting liver diseases and preparation method thereof | |
| EP2817636B1 (en) | New method for rapid detection of hepatocyte growth factor in biological fluids | |
| CN107643283B (en) | Application of interferon-induced T cell α chemokine in preparation of primary biliary cholangitis diagnostic reagent or kit | |
| CN112083165A (en) | Application of human serum REG I alpha as detection target or standard substance in preparation of reagent or kit for predicting tumor | |
| CN112198314B (en) | Marker for detecting malignant progression risk of esophageal precancerous lesion or early screening of esophageal cancer and application of marker | |
| CN108761067A (en) | The biomarker and application thereof of idiopathic inflammatory myopathies detection | |
| Baillie | CK Isoenzymes: Part 1. Clinical Aspects | |
| CN110244059B (en) | Application of group of serum proteins in preparation of early screening reagent for detecting human pneumoconiosis | |
| CN110018309B (en) | Kit for judging colorectal cancer prognosis through protein markers | |
| CN107312836A (en) | Applications of the microRNA miRNA 146a 5p in relevant disease diagnosis of risk | |
| CN1266478C (en) | Reagent box for testing acute coronary artery complex by insulin growth factor combing protein-4 hydrolase | |
| WO2015026893A1 (en) | Predictive biomarkers for metabolic syndrome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |