CN103439485A - Kit for efficiently detecting endogenous digitalis-like factor content and application method thereof - Google Patents
Kit for efficiently detecting endogenous digitalis-like factor content and application method thereof Download PDFInfo
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- CN103439485A CN103439485A CN2013103710065A CN201310371006A CN103439485A CN 103439485 A CN103439485 A CN 103439485A CN 2013103710065 A CN2013103710065 A CN 2013103710065A CN 201310371006 A CN201310371006 A CN 201310371006A CN 103439485 A CN103439485 A CN 103439485A
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Abstract
The invention relates to a kit for efficiently detecting endogenous digitalis-like factor content and an application method thereof. The kit comprises primary antibody goat anti-human digoxin polyclonal antibody, secondary antibody rat anti-goat IgG, 3H labeled uabain, polyethyleneglycol (PEG) 4000, phosphate buffer solution (PBS) with the pH value of 7.4, 3H scintillation solution, and tris(hydroxymethyl)metyl aminomethan buffer solution (Tris buffer solution) with the pH value of 7.4, and/or uabain. The kit provided by the invention is simple and convenient to operate, has the advantages of high sensitivity, high specificity and the like, and can perform stable and reliable early diagnosis on gestational period eclamptism, thereby providing favorable support for subsequent treatment.
Description
Technical field
The invention belongs to and belong to technical field of medical instruments, especially a kind of kit and using method of efficient detection EDF content.
Background technology
Pregnancy period pre-eclampsia (pregnancy period is epilepsy early) is the syndrome that a kind of disease is sent out the reason complexity, if treatment not in time can develop into eclampsia, threat to life.Cardinal symptom has hypertension, albuminuria, and oedema is fainted from fear, and renal dysfunction etc., become the highest a kind of disease of pregnant woman's fatal rate in the world, at present concrete etiology unknown.There are some researches prove a kind of material that causes susceptible women parent blood vessel endothelial dysfunction may be sick one of them reason of sending out of epilepsy early that placenta produces.It is that this disease the most significantly indicates that blood pressure raises, and causes the endothelium to the puerpera, the infringement of kidney and liver simultaneously.
In global pregnant woman, the incidence of disease is 6-8% to pre-eclampsia (early epilepsy), usually fallen ill in late period gestation second phase or pregnant three phases, and most of morbidity after 32 weeks pregnancy periods (before 32 weeks, morbidity is considered to early stage morbidity), only a few pregnant woman can fall ill 20 weeks pregnancy periods.First conceived pregnant woman's incidence of disease is the highest, and along with the increase of number of pregnancies, this disease incidence decrease.Early epilepsy is mainly in and has history of hypertension, diabetic history, and the obesity history, the autoimmune disease history is as lupus, and, in the pregnant woman of multifetation (twins or multiparity), its pathogenic process varies with each individual, and most of cases can be made a definite diagnosis before childbirth.The minority case was just made a definite diagnosis in minute six weeks puerperiums, was called " postpartum is epilepsy early ", after making a definite diagnosis, except caesarean birth or induced labor operation, there is no the other treatment mode.Early epilepsy is modal pregnancy syndrome, affects mother and fetus simultaneously, causes the premature even the mother and baby to be caused to life danger.
Early the sign of making a definite diagnosis of epilepsy is that protein content in pregnancy period hypertension (two measured values of independently at least being separated by six hours, systolic pressure is more than 140 and/or diastolic pressure is more than 90) and twenty-four-hour urine fluid samples is higher than 300 milligrams (albuminuria) traditionally.Than the systolic pressure of high 30 mm Hg of baseline blood pressure (BP) or the diastolic pressure of high 15 mm Hg, can not reach 140/90 absolute standard, still be considered to important morbidity signal, but can not make a definite diagnosis at this point.Swelling or oedema (especially at hand and face) once were considered to an early important symbol of epilepsy of diagnosis at first, but current medical practice proves to only have HP to can be used as diagnostic criteria.
Although early epilepsy is a kind of mortality disease, but its early stage morbidity is usually asymptomatic, and generally, early the signal of epilepsy is not specific, such as the pregnancy period faints from fear, in conceived process, other reasons more easily causes fainting from fear than eclampsia, and therefore early the early diagnosis of epilepsy is the difficult problem that medical circle can't be captured always.
In epilepsy patient's morning symptom, the rising of a kind of sodium potassium pump inhibiting factor-EDF (endogenousdigitalis-likefactor, EDLF) level, be the important symbol of epilepsy morning.This factor can cause blood pressure to raise, be present in human body and animal tissue and body fluid, its chemical constitution is similar to the steroids cardiotonic of extracting in the plant digitalis with biologically active, therefore named " digitalis like factor ", this factor can be to be attached on sodium potassium pump with similar mechanism of cardiotonic such as digoxin, and suppress its activity, but so far, the particular chemical of this type of factor is still unknown.Many hypertensions all find to follow the rising of EDF level, and therefore, this factor is likely and causes hypertensive reason.
Research shows, DigiTAb can reverse the sodium potassium pump inhibiting factor of plant origin and the effect of EDF, particularly a kind of once for digoxin excess intake treatment, the antibody fragment of Gaoxin specifically of Digibind by name, this antibody can be combined with EDF and suppress its effect, reduces blood pressure.In epilepsy women's morning clinical randomized double-blind experiment, women's blood pressure of accepting the DigiTAb treatment obviously reduces than placebo group, renal dysfunction also has significantly improvement, these evidences have further proved the effect of EDF in the morning epilepsy origin cause of formation, DigiTAb, by being combined with this factor, plays the effect of alleviating illness.
In order to realize the early diagnosis to the pregnancy early epilepsy, reach the purpose of early treatment, need to using DigiTAb as instrument, develop and a kind ofly can specificity accurately measure the method for EDF in sample, and make kit simple and efficient to handle.In decades after this factor is found, the greatest difficulty of research is, its content in vivo is very low, and, because structure also is not determined, does not also have at present method to separate its () in body.Therefore, the kit in the present invention provides a kind of the need clinically to be isolated, but still can this factor concentration of sensitive determination, and as the foundation of epilepsy early diagnosis early.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, kit and the using method of a kind of simple to operation, highly sensitive, high specificity, reliable and stable efficient detection EDF content are provided, the detection of this kit can reach nM be nanomole/liter, for successive treatment provides favourable support.
The present invention realizes that the technical scheme of purpose is as follows:
A kind of kit that detects EDF content, comprise following component
Primary antibodie goat-anti people digoxin polyclonal antibody, two anti-mouse-anti sheep IgG,
3the phosphate buffer of H mark unabain, Macrogol 4000, pH=7.4,
3the Tris damping fluid of H scintillation solution, pH=7.4.
And, also comprise unabain.
And, described primary antibodie goat-anti people digoxin Anti-TNF-α bulk concentration 12 ug/ml, described two anti-mouse-anti sheep IgG concentration 5 * 10
-7mol/L, described 3H mark unabain concentration is 10.0Ci/mmol, described Macrogol 4000 concentration is 30wt%.
And described kit is for detection of the content of the EDF in human serum, placenta tissue juice and freezing placenta tissue.
A kind of kit using method of detection EDF content as claimed in claim 1, step is as follows
(1) gather placenta, be cut into tissue block, liquid nitrogen frozen, be stored in-80 ℃;
(2) freezing placenta tissue piece is thinly sliced, be placed in homogenizer, put into liquid nitrogen container freezing 1 minute; With the speed of 1500rpm by tissue slice homogenate 5 minutes;
(3) collect the homogenate tissue, add the methyl alcohol of equivalent, mix, with the speed of 2000rpm centrifugal 5 minutes, remove albumen; Collect centrifuged supernatant, Vacuum Concentration, 4 ℃ of preservations;
(4) take out goat-anti people digoxin polyclonal antibody in kit, two anti-mouse-anti sheep IgG,
3the TRIS buffer of H mark unabain, pH=7.4, equivalent adds respectively, fully mixes incubated at room with the equivalent testing sample;
(5) the PEG4000 solution that adds again equivalent, centrifugal 10 minutes of 8000rpm, be precipitated thing;
(6) sediment is resuspended with the PBS of 500 microlitre pH7.4, adds
3the H scintillation solution;
(7) measure the sample exit dose, according to typical curve, calculate contained EDF concentration in sample.
Advantage of the present invention and good effect are:
1, the invention provides a kind of quick, simple to operation, high specificity, good stability and high kit of sensitivity of detecting, can effectively detect the content of EDF in sample, solve for a long time the significant difficulties of early diagnosis research to epilepsy morning.
2, this kit can be for measuring the concentration of placenta and blood serum sample EDF, for further the synthetic research of regulating in the mechanism of causing a disease of this factor, body being laid to good basis, and make the early pathological study work of epilepsy of pregnancy period stride forward major step.
The accompanying drawing explanation
Fig. 1 is that DigiTAb of the present invention reverses EDF inhibiting effect to sodium potassium pump in the morning epilepsy, and ordinate represents the repressed number percent of sodium potassium pump in erythrocyte, the % of unit; A representative contrast in figure, epilepsy clinical samples morning of DigiTAb is not used in the B representative, and epilepsy clinical samples morning after DigiTAb is used in the C representative.
Fig. 2 is typical curve of the present invention (obtaining according to the Specification Curve of Increasing method), and horizontal ordinate represents the log(mol/L of unabain solution concentration) value; Ordinate is radioactivity, and ordinate represents the contamination that standard unabain solution records through the method for the invention, y=0.0671e
-1.4038x, R
2=0.9979.
The concentration that Fig. 3 is the EDF secreted in the ten a independent placental samples measured of kit of the present invention, sample is from normal pregnancies, and wherein, horizontal ordinate 1-11 represents respectively ten a independent samples; Ordinate represents EDF concentration, unit receives and rubs/liter.
Fig. 4 is the concentration of kit of the present invention for definite placenta tissue EDF, and sample is from epilepsy pregnant woman morning, and wherein, horizontal ordinate 1-5 represents respectively 5 parts of independent samples; Ordinate represents EDF concentration, unit receives and rubs/liter.
Fig. 5 is kit of the present invention for measuring the early concentration of epilepsy pregnancy serum EDF, and wherein, horizontal ordinate 1-7 represents respectively 7 parts of independent samples; Ordinate represents EDF concentration, unit receives and rubs/liter.
Fig. 6 is that invention kit of the present invention is measured EDF concentration accumulation in time in people's placenta, and wherein, horizontal ordinate represents incubation time, unit hour; Ordinate represents EDF concentration, unit 10
-8rub/liter.
Fig. 7 is the comparison that GFAA method (Graphite Furnance Atomic Absorption Spectrometry, GFAAS (graphite furnace atomic absorption spectrometry)) and kit method therefor of the present invention detect EDF in identical placenta sample.Horizontal ordinate represents that the GFAA method records the active number percent of this factor in sample, the % of unit; Factor content in the sample that the ordinate representative is used kit of the present invention to record, unit: Na Mo/liter
Embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
Goat-anti people digoxin polyclonal antibody of the present invention (anti digoxin antibody) has another name called anti digoxin antibody purchased from GlaxoSmithKline(), two anti-mouse-anti sheep IgG are all purchased from Jackson ImmunoResearchLaboratories, Inc,
3h mark unabain purchased from PerkinElmer, Macrogol 4000 purchased from phosphate buffer (PBS damping fluid) autogamy of Calbiochem, pH=7.4,
3the H scintillation solution purchased from NationalDiagnostics, unabain purchased from the TRIS buffer (Tris damping fluid) of Sigma, pH=7.4 purchased from Sigma, the DMEM nutrient solution is purchased from Gibson.
This kit composed as follows:
Goat-anti people digoxin polyclonal antibody 12 ug/ml, two anti-mouse-anti sheep IgG5 * 10
-7mol/L,
3h mark unabain concentration be 10.0Ci/mmol, the Macrogol 4000 concentration phosphate buffer (PBS damping fluid) that is 30wt%, pH=7.4,
3the Tris damping fluid of H scintillation solution, unabain, pH=7.4.
Kit of the present invention needs to measure
3the radioactive instrument and equipment of H is measured.(equipment is called scintillationcounter.Any laboratory of doing the radioactivity experiment has, and operating process is for to put in sample, and operation software, directly go out result.)
Embodiment 1:
Kit is for measuring the EDF content of placenta tissue secretion
(1) gather the Freshman placenta that delivery time is less than 6 hours;
(2) push amnion aside, get the tissue block 4-5 piece of 5mmX5mmX5mm size, put into PBS and wash remained blood off;
(3) tissue block is cut into to particulate, removes the visible clot of naked eyes and blood vessel, be washed till without blood residually with PBS, blot PBS on aseptic filter paper, put into the DMEM nutrient solution, 37 ℃, 5%CO
2cultivate 48 hours;
(4) collect nutrient solution, centrifugal removal residual tissue piece, 4 ℃ of preservations;
(5) take out goat-anti people digoxin polyclonal antibody in kit, two anti-mouse-anti sheep IgG,
3h mark unabain, get respectively 100 microlitres, fully mixes incubated at room with 100 microlitre testing samples;
(6) add 400 microlitre PEG4000 solution, centrifugal 10 minutes of 8000rpm, be precipitated thing again;
(7) sediment is resuspended with the PBS of 500 microlitre pH7.4, adds 4 milliliters
3the H scintillation solution;
(8) measure the sample exit dose, according to typical curve, calculate contained EDF concentration in sample.
Result as shown in Figure 3, the EDF concentration of utilizing this kit to measure to secrete in independent placenta tissue, unit rubs for receiving/liter, this concentration can accurately be measured by kit provided by the invention.
Embodiment 2:
This kit is for measuring the content of cryonics placenta tissue EDF
(1) gather neonate's placenta, be cut into the tissue block of 2cm * 2cm * 2cm size, liquid nitrogen frozen, be stored in-80 ℃;
(2) freezing placenta tissue piece is thinly sliced, be placed in homogenizer, put into liquid nitrogen container freezing 1 minute; With the speed of 1500rpm by tissue slice homogenate 5 minutes;
(3) collect the homogenate tissue, add 5 ml methanol, mix, with the speed of 2000rpm centrifugal 5 minutes, remove albumen; Collect centrifuged supernatant, Vacuum Concentration, 4 ℃ of preservations;
Measure sample method with step in embodiment 1 (5)-(8).
As shown in Figure 4, this kit can be measured the concentration of EDF in freezing placenta tissue, in average out to embodiment 1 100 times, and sample source all is diagnosed as the early sick person of sending out of epilepsy.
Embodiment 3:
This kit is for measuring the content of human serum EDF
Measure sample method with step in embodiment 1 (5)-(8).
As shown in Figure 5, this kit can accurately measure the EDF of low content in human serum to result.
In pregnancy serum EDF concentration surpass 150 receive rub/liter, can tentatively be judged as early epilepsy patient.
Refer to EDF content in pregnancy serum herein, because hospital is before pregnant woman childbirth, chemical examination serum is most convenient.But, because this factor produces in placenta, placenta content will be far away higher than serum content, as Fig. 4 and Fig. 5 contrast; Fig. 3 and for example, show that the normal pregnancies placenta secretion is to the factor concentration in nutrient solution, with factor concentration tool comparability in epilepsy patient's serum early in Fig. 5, further confirmed in serum that content is far less than placenta, the low content of this factor in serum, and chemical examination serum is more convenient than placenta, is important innovations of the present invention.
Embodiment 4:
This kit is for measuring the accumulation of human placenta secretion EDF, and method is with embodiment 1.
As shown in Figure 6, along with the accumulation of organizing incubation time, the EDF content that this kit is measured people's placenta secretion progressively increases.
The Specification Curve of Increasing step is as follows:
(1) get concentration be respectively 50 receive rub/liter, 0.1 micro-rubbing/liter, 0.2 micro-rubbing/liter, 0.5 micro-rubbing/liter, 1 micro-rubbing/liter, 3 micro-rubbing/liter each 100 microlitres of unabain solution;
(2) take out the Tris damping fluid of goat-anti people digoxin polyclonal antibody in kit, two anti-mouse-anti sheep IgG, pH=7.4, add respectively 100 microlitres in the unabain solution of above-mentioned variable concentrations, fully mix incubated at room;
(3) add 400 microlitre PEG4000 solution, centrifugal 10 minutes of 8000rpm, be precipitated thing again;
(4) sediment is resuspended with the PBS of 500 microlitre pH7.4, adds the 3H scintillation solution;
(5) measure the sample exit dose, obtain contained EDF concentration in sample, curve plotting, obtain typical curve.
Because unabain can be with in conjunction with rate be attached to anti digoxin antibody on identical with EDF, can utilize the concentration of EDF in the concentration representative sample of known unabain standard solution.
The measurement factor content method of Fig. 7 is with embodiment 1, get the placenta tissue nutrient solution and measure exit dose, result shows that concentration and its activity of EDF in placenta tissue is linear, further the reliability of detection method described in the proved invention.
Claims (5)
1. a kit that detects EDF content, is characterized in that: comprise following component
Primary antibodie goat-anti people digoxin polyclonal antibody, two anti-mouse-anti sheep IgG,
3the phosphate buffer of H mark unabain, Macrogol 4000, pH=7.4,
3the Tris damping fluid of H scintillation solution, pH=7.4.
2. the kit of detection EDF content according to claim 1, is characterized in that: also comprise unabain.
3. the kit of detection EDF content according to claim 1, is characterized in that: described primary antibodie goat-anti people digoxin Anti-TNF-α bulk concentration 12 ug/ml, described two anti-mouse-anti sheep IgG concentration 5 * 10
-7mol/L, described 3H mark unabain concentration is 10.0Ci/mmol, described Macrogol 4000 concentration is 30wt%.
4. the kit of detection EDF content according to claim 1, it is characterized in that: described kit is for detection of the content of the EDF in human serum, placenta tissue juice and freezing placenta tissue.
5. the kit using method of a detection EDF content as claimed in claim 1, it is characterized in that: step is as follows
(1) gather placenta, chopping, liquid nitrogen frozen, be stored in-80 ℃;
(2) freezing placenta tissue piece is thinly sliced, be placed in homogenizer, put into liquid nitrogen container freezing 1 minute; With the speed of 1500rpm by tissue slice homogenate 5 minutes;
(3) collect the homogenate tissue, add the methyl alcohol of equivalent, mix, centrifugal removal albumen; Collect centrifuged supernatant, Vacuum Concentration, 4 ℃ of preservations;
(4) take out goat-anti people digoxin polyclonal antibody in kit, two anti-mouse-anti sheep IgG,
3the TRIS buffer of H mark unabain, pH=7.4, equivalent adds respectively, fully mixes incubated at room with the equivalent testing sample;
(5) add again the PEG4000 solution of equivalent, centrifugal, be precipitated thing;
(6) sediment is resuspended with the PBS of 500 microlitre pH7.4, adds
3the H scintillation solution;
(7) measure the sample exit dose, calculate the concentration of contained EDF in sample according to typical curve.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103439507A (en) * | 2013-08-22 | 2013-12-11 | 天津市康婷生物工程有限公司 | Kit for detecting endogenous digitalis-like factor content with digoxin monoclonal antibody and application method thereof |
CN105435617A (en) * | 2015-11-19 | 2016-03-30 | 史汉祥 | Device for flue gas desulfurization and denitrification |
CN113736744A (en) * | 2021-10-14 | 2021-12-03 | 江南大学 | Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof |
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2013
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US3975511A (en) * | 1974-03-01 | 1976-08-17 | Corning Glass Works | Solid phase radioimmunoassay |
EP0433074A1 (en) * | 1989-12-13 | 1991-06-19 | The General Hospital Corporation | Method for treating cardiac malfunction |
WO1994012210A1 (en) * | 1992-12-02 | 1994-06-09 | Bagrov Alexei Y | Anti-edlf antibody, composition thereof and method of diagnosing and treating cardiac arrhythmias, hypertension |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103439507A (en) * | 2013-08-22 | 2013-12-11 | 天津市康婷生物工程有限公司 | Kit for detecting endogenous digitalis-like factor content with digoxin monoclonal antibody and application method thereof |
CN103439507B (en) * | 2013-08-22 | 2015-03-25 | 天津市康婷生物工程有限公司 | Kit for detecting endogenous digitalis-like factor content with digoxin monoclonal antibody and application method thereof |
CN105435617A (en) * | 2015-11-19 | 2016-03-30 | 史汉祥 | Device for flue gas desulfurization and denitrification |
CN113736744A (en) * | 2021-10-14 | 2021-12-03 | 江南大学 | Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof |
CN113736744B (en) * | 2021-10-14 | 2023-07-18 | 江南大学 | Digitoxin monoclonal antibody hybridoma cell strain and application thereof |
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Address after: No.9 Saida South Road, Xiqing Economic and Technological Development Zone, Tianjin 300200 Patentee after: Tianjin Kangting Bioengineering Group Co., Ltd. Address before: No. 126 Youyi South Road, Saida Pharmaceutical Industrial Park, Xiqing Economic and Technological Development Zone, Tianjin 300200 Patentee before: Tianjin Kangting Biotechnology Co., Ltd. |