CN102426243B - Preparation method of preeclampsia detection kit adopting Adipsin as detection index - Google Patents
Preparation method of preeclampsia detection kit adopting Adipsin as detection index Download PDFInfo
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Abstract
The invention relates to a preeclampsia (PE) detection kit adopting Adipsin as a detection index. The PE detection kit comprises an anti-human Adipsin monoclonal antibody coated porous plate, a biotin-labeled anti-human Adipsin monoclonal antibody detection solution, avidin-horseradish peroxidase, a coloration substrate 3',3',5,5'-tetramethyl benzidine and a Adipsin protein standard substance, wherein the avidin-horseradish peroxidase can be binded with the biotin-labeled anti-human Adipsin monoclonal antibody. The preparation method for the kit comprises the following process steps: (1) preparing the anti-human Adipsin monoclonal antibody coated porous plate; (2) preparing the biotin-labeled anti-human Adipsin monoclonal antibody detection solution; (3) preparing the avidin-horseradish peroxidase, the coloration substrate 3',3',5,5'-tetramethyl benzidine and the Adipsin protein standard substance.
Description
Technical field
The invention belongs to medical kit field, particularly a kind of kit for detection of pregnancy women preeclampsia (preeclampsia is called for short PE) and preparation method thereof.
Background technology
Be the obstetric complication betiding after pregnant 20 weeks preeclampsia (preeclampsia is called for short PE), is pregnant woman and the one of the main reasons of enclosing died.Preeclampsia, the incidence of disease in pregnant and lying-in women in worldwide was 2-8%, and the incidence of disease in China pregnant and lying-in women can reach 10%.Can there is the serious clinical manifestations such as hypertension, oedema and severe albuminuria in serious preeclampsia patient, the easy badness come-off such as Complicated with Pulmonary oedema, renal failure, HELLP syndrome also, the risk of suffering from angiocardiopathy, kidney trouble and metabolic disease long term also obviously increases.For fetus, pregnant Zhou Yue little, survival rate is lower, premature before pregnant 32 weeks, mortality ratio is up to 50%, and the probability of the severe complications such as neonate suffocates, intracranials hemorrhage, infection reaches 5-10%, and metabolic disease at a specified future date, the incidence of disease of angiocardiopathy also increase, have a strong impact on the health of filial generation, brought heavy psychology and health financial burden to family and society.
Prior art need meet following three foundations about the clinical diagnosis of preeclampsia: (1) pregnant and lying-in women's private prosecution has the clinical symptoms such as headache, dizziness; (2) through clinician, detect, pregnant and lying-in women's blood pressure meets Evaluation of Diagnostic Criteria of Hypertension, systolic pressure/diastolic pressure >=140/90mmHg; (3) the quantitative testing result of twenty-four-hour urine albumin is 24 hours albuminuria >=0.3g/day.Twenty-four-hour urine albumin quantitatively detects needs the around-the clock urine sample of collection patient to detect.The method of this method collecting sample is loaded down with trivial details, length consuming time.
Adipsin (have another name called factor D, or complement factor D, Chinese " Complement Factor D " by name) be a kind of serine protease that adipocyte produces, be present in blood circulation, closely related with the fat metabolism of body.Healthy gravid woman and suffer from the Adipsin stable content in gravid woman's blood circulation of preeclampsia, does not have significant difference between two groups.But, owing to suffering from the gravid woman of preeclampsia, there is blood vessel endothelium injury, the vascular endothelial cell damage of kidney causes kidney infiltration and reclaims function impaired, cause the Adipsin of high concentration to appear in the gravid woman's who suffers from preeclampsia urine sample, therefore, can be based on this Research foundation the diagnosis and prediction index using Adipsin as preeclampsia.At present not yet find to using that Adipsin content in urine sample is as the report of the detection index of preeclampsia, do not find take yet Adipsin content in urine sample be detect index preeclampsia detection kit and preparation method thereof report.
Summary of the invention
The object of the present invention is to provide that a kind of to take contained Adipsin in urine sample be novel preeclampsia of the detection kit and preparation method thereof that detects index, to simplify the trace routine of preeclampsia, reduce testing cost, alleviate patient's misery.
The inventor of present patent application found through experiments: Adipsin is stable content healthy gravid woman with in suffering from gravid woman's blood circulation of preeclampsia, the content of organizing Adipsin in blood preeclampsia is 2929.37 ± 814.08ng/mL, in healthy gestation group blood, the content of Adipsin is 2359.76 ± 667.63ng/mL, between two groups, there is no significant difference.But, owing to suffering from the gravid woman of preeclampsia, there is blood vessel endothelium injury, the vascular endothelial cell damage of kidney causes kidney infiltration and reclaims function impaired, cause the Adipsin of high concentration to appear in the gravid woman's who suffers from preeclampsia urine sample, protein chip screening experiment is found: puerpera's preeclampsia who makes a definite diagnosis compares with non-preeclampsia of the puerpera who makes a definite diagnosis that (two groups of puerperas' age mates with gestation all ages, foundation-free disease, without other pregnancy complications), Adipsin concentration in urine sample has notable difference, quantitative examination is found: the content of organizing Adipsin in urine sample preeclampsia is 349.04 ± 557.10ng/mL, in healthy gestation group urine sample, the content of Adipsin is 8.69 ± 6.34ng/mL, owing to there is significant difference in the content of Adipsin in preeclampsia group and healthy gestation group urine sample, therefore can be using the content of Adipsin in urine sample as judging whether gravid woman suffers from the detection index of preeclampsia.
Of the present inventionly take Adipsin as detecting detection kit preeclampsia of index, comprise the porous plate, biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) that are coated with anti-human Adipsin monoclonal antibody detect liquid, the Avidin-horseradish peroxidase with biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) combination, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Adipsin protein standard substance.
Experiment shows, the contained porous plate that is coated with anti-human Adipsin monoclonal antibody of mentioned reagent box, and preferred every hole is coated with anti-human Adipsin monoclonal antibody 0.9 μ g～1 μ g.
In mentioned reagent box, described biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) detects liquid and mainly biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb), bovine serum albumin(BSA), glycerine and phosphate buffer, consists of, the concentration of biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) is 24mg/mL, the concentration of bovine serum albumin(BSA) is 1.5g～3g/100mL, and the concentration of glycerine is 50mL/100mL.
Described biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) is coupled and is formed by biotin acyl-N maloyl imines ester and anti-human Adipsin monoclonal antibody, and the mass ratio of acyl-N maloyl imines ester and anti-human Adipsin monoclonal antibody is 0.5～1: 7.
In order to be user-friendly to, kit of the present invention also can be equipped with sample dilution, described sample dilution is at the phosphate buffer (PBS) of concentration 0.01mol/L～0.02mol/L or TRIS buffer (the Tris damping fluid of concentration 0.01mol/L～0.02mol/L, TBS) in, add bovine serum albumin(BSA) formulated, the concentration of bovine serum albumin(BSA) is 1g～2g/100mL.
Of the present invention take Adipsin be to detect index preeclampsia detection kit preparation method, processing step is as follows:
(1) preparation is coated with the porous plate of anti-human Adipsin monoclonal antibody
With the carbonate buffer solution of pH=8～10, by anti-human Adipsin monoclonal antibody dilution, be concentration 10 μ g/mL dilutions, in described anti-human Adipsin monoclonal antibody dilution, add absolute methanol, the addition of absolute methanol reaches 3mL/100mL with its concentration and is limited, then the described anti-human Adipsin monoclonal antibody dilution containing methyl alcohol is added in each hole of porous plate, at 4 ℃, be coated with at least 12 hours; After the coated time expires, the bovine serum albumin solution that is 1g/100mL by concentration adds each hole on porous plate and carries out capping at 37 ℃, and the reaction time is at least 1 hour; After capping finishes, the phosphate buffer washing porous plate with pH=7.2, obtains the porous plate that is coated with anti-human Adipsin monoclonal antibody after the unreacted reactant on porous plate is removed, and its storage temperature is 4 ℃;
(2) prepare biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) and detect liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that solute compound concentration is acyl-N maloyl imines ester solution of 50 μ g/ μ L, the carbonate buffer solution of pH=9.6 of take is solvent, anti-human Adipsin monoclonal antibody is that solute compound concentration is the anti-human Adipsin monoclonal antibody solution of 24mg/mL, by the mixed liquor of described acyl-N maloyl imines ester solution and described anti-human Adipsin monoclonal antibody solution under agitation in room temperature reaction at least 4 hours, obtain biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb), after reaction finishes, reactant liquor containing biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) is packed in bag filter, with the phosphate buffer of pH=9.2, at 4 ℃, dialyse, dialysis time is at least 12 hours, changes dislysate therebetween at least 3 times, continue after add bovine serum albumin(BSA) in described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 3g～6g/100mL with its concentration and is limited,
That 2. 1. step is prepared measures containing reactant liquor and glycerine after the dialysis of biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) and bovine serum albumin(BSA) for 1: 1 by volume, at room temperature mix and form biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) detection liquid, its storage temperature is-20 ℃;
(3) be equipped with Avidin-horseradish peroxidase, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Adipsin protein standard substance.Described Avidin-horseradish peroxidase, 3 ', 3 ', 5,5 '-tetramethyl benzidine and Adipsin protein standard substance are commercial goods, can directly from market, buy.
In the method for the invention, when preparation is coated with the porous plate of anti-human Adipsin monoclonal antibody, the addition of described anti-human Adipsin monoclonal antibody dilution is 100 μ L/ holes preferably, and the addition of the bovine serum albumin solution that described concentration is 1g/100mL is 120 μ L/ holes.
In the method for the invention, while preparing biotin labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb), preferably by mass ratio=0.5～1 of acyl-N maloyl imines ester and anti-human Adipsin monoclonal antibody: 7 measure described acyl-N maloyl imines ester solution and described anti-human Adipsin monoclonal antibody solution formation mixed liquor.
For the convenience of the user, the method of the invention has also been equipped with sample dilution, described sample dilution is at the phosphate buffer (PBS) of concentration 0.01mol/L～0.02mol/L or TRIS buffer (the Tris damping fluid of concentration 0.01mol/L～0.02mol/L, TBS) in, add bovine serum albumin(BSA) formulated, the addition of described bovine serum albumin(BSA) reaches 1g～2g/100mL with its concentration and is limited.
The present invention has following beneficial effect:
(1) detection that the present invention is preeclampsia provides a kind of kit that adopts new detection index and preparation method thereof.
(2) use kit of the present invention, can quantitatively detect the accurate content of Adipsin in gravid woman's urine sample.
(3) use kit of the present invention to make a definite diagnosis preeclampsia women and healthy gravid woman carry out sample detecting (seeing embodiment 2), testing result shows: the susceptibility of this kit is 90%, specificity is 80% left and right, thereby can improve the accuracy detecting preeclampsia.
(4) owing to detecting sample, be gravid woman's urine sample, because of but a kind of without wound detection.
Kit of the present invention is applicable to all pregnancy women.
Accompanying drawing explanation
Fig. 1 be the Adipsin of take of the present invention be to detect index preeclampsia porous plate in detection kit a kind of shape organigram.
Fig. 2 is A450 value-Adipsin concentration standard curve.
Embodiment
The detection kit and preparation method thereof preeclampsia that the Adipsin of take of the present invention be is detected to index below by embodiment is described further with using method.
The material source relating in following embodiment is as follows:
Mouse-anti people Adipsin monoclonal antibody, purchased from U.S. R & D company or RayBiotec company;
Adipsin protein standard substance, purchased from U.S. R & D company or RayBiotec company;
Carbonate buffer solution (CB), purchased from U.S. sigma company;
Phosphate buffer (PBS), purchased from U.S. sigma company;
TRIS buffer (Tris damping fluid, TBS), purchased from U.S. sigma company;
Bovine serum albumin(BSA), purchased from U.S. sigma company;
DMF, purchased from U.S. sigma company;
Acyl-N maloyl imines ester, purchased from U.S. sigma company;
The specification of glycerine is to analyze alcohol, purchased from U.S. sigma company;
Horseradish peroxidase, purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge;
3 ', 3 ', 5,5 '-tetramethyl benzidine, purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge;
Porous plate is the porous plate in 96 holes, and purchased from Denmark Nunc company, its shape and structure are as shown in Figure 1.
Embodiment 1
In the present embodiment, adopt following processing step preparation to take Adipsin as detecting detection kit preeclampsia of index:
(1) preparation is coated with the porous plate of mouse-anti people Adipsin monoclonal antibody
With the carbonate buffer solution of pH=9, by the dilution of mouse-anti people Adipsin monoclonal antibody, be the dilution of concentration 10 μ g/mL, in antibody diluent, add absolute methanol, the addition of absolute methanol reaches 3mL/100mL with its concentration and is limited, then the described mouse-anti people Adipsin monoclonal antibody dilution containing methyl alcohol is added by the amount in 100 μ L/ holes in each hole of 96 orifice plates, at 4 ℃, be coated with 12 hours; After the coated time expires, the bovine serum albumin solution of concentration 1g/100mL is added to each hole on porous plate and carries out capping at 37 ℃ by the amount in 120 μ L/ holes, the reaction time is 1 hour; After capping finishes, the phosphate buffer that is 0.05mol/L by pH=7.2, concentration washing porous plate, after unreacted reactant on porous plate is removed, obtain the porous plate that is coated with mouse-anti people Adipsin monoclonal antibody, be then placed in 4 ℃ of refrigerators and save backup;
(2) prepare biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb) and detect liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that solute compound concentration is acyl-N maloyl imines ester solution of 50 μ g/ μ L, with pH=9.6, concentration is that the carbonate buffer solution of 1mol/L is solvent, mouse-anti people Adipsin monoclonal antibody is that solute compound concentration is the mouse-anti people Adipsin monoclonal antibody solution of 24mg/mL, by acyl-N maloyl imines ester with mass ratio=1 of mouse-anti people Adipsin monoclonal antibody: 7 measure described acyl-N maloyl imines ester solution and described mouse-anti people Adipsin monoclonal antibody solution forms mixed liquor, by the mixed liquor of described acyl-N maloyl imines ester solution and described mouse-anti people Adipsin monoclonal antibody solution (magnetic stirring apparatus under agitation, 200 revs/min of stirring rates) in room temperature reaction 4 hours, obtain biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb), after reaction finishes, reactant liquor containing biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb) is packed in bag filter, with the phosphate buffer that pH=9.2, concentration are 0.05mol/L, at 4 ℃, dialyse, dialysis time is 12 hours, changes dislysate therebetween 3 times, continue after add bovine serum albumin(BSA) in described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 4g/100mL with its concentration and is limited,
That 2. 1. step is prepared measures containing reactant liquor and glycerine after the dialysis of biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb) and bovine serum albumin(BSA) for 1: 1 by volume, at room temperature mix and form biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb) detection liquid, its storage temperature is-20 ℃;
(3) each kit be equipped with Avidin-horseradish peroxidase 12mL, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine 12mL, Adipsin protein standard substance (concentration of Adipsin is 10ng/mL) 1mL and sample dilution 30mL, sample dilution is at the TRIS buffer of concentration concentration 0.02mol/L (Tris damping fluid, TBS) in, add bovine serum albumin(BSA) formulated, the addition of described bovine serum albumin(BSA) reaches 2g/100mL with its concentration and is limited; The volume ratio that described Avidin-horseradish peroxidase and biotin labeled mouse-anti people Adipsin monoclonal antibody detect liquid is 1: 1.
Embodiment 2
The present embodiment detects sample to be tested with detection kit preeclampsia of embodiment 1 preparation.
1, sample to be tested
(1) standard specimen is prepared
The Adipsin protein standard substance being equipped with in kit described in embodiment 1 is made as containing the highest standard specimen of Adipsin concentration, called after standard specimen 1, the concentration of its Adipsin is 6ng/mL, then with the sample dilution being equipped with in kit described in embodiment 1, described Adipsin protein standard substance is diluted to respectively to the standard specimen that Adipsin concentration is 2000pg/mL, 666.7pg/mL, 222.2pg/mL, 74.07pg/mL, 24.69pg/mL, 8.23pg/mL, called after standard specimen 2, standard specimen 3, standard specimen 4, standard specimen 5, standard specimen 6 and standard specimen 7, have 7 standard specimens altogether successively.
(2) sample
The pregnant woman of take West China No.2 Hospital, Sichuan University's outpatient service and being in hospital is experimental subjects, according to the testing result of existing detection method, experimental subjects is divided into eclampsia group and normal healthy controls group, collect two groups of pregnant woman's urine sample as sample, amount to 40 samples, wherein, sample 1-20 is taken from eclampsia group, and sample 21-40 is taken from normal healthy controls group.
(3) blank
The sample dilution being equipped with in kit described in embodiment 1---be blank.
2, pattern detection
(1) before detection, all reagent of kit, sample to be tested is slowly balanced to room temperature (18 ℃～25 ℃);
(2) standard specimen of step 1 being prepared, blank, sample add respectively in the corresponding well on 96 orifice plates that are coated with mouse-anti people Adipsin monoclonal antibody, and each standard specimen, blank, sample are all established the contrast of multiple hole; Application of sample amount in each well is 100 μ L.With glued membrane, hide described 96 orifice plates, and described 96 orifice plates are placed on jolting instrument under jolting state to incubated at room 2.5 hours, jolting speed is 100 revs/min.
(3) after incubation time expires, exhaust the liquid in each well, cleaning procedure is set on automatic washer, the 400 μ L/ hole washing lotions (the Tris damping fluid that the phosphate buffer of concentration 0.02mol/L or concentration are 0.02mol/L) of take are cleaned each well twice, during each cleaning, washing lotion need stop 10 seconds～15 seconds in hole; After cleaning, by 96 orifice plate upsets, make each well aperture downward, on absorbent filter, pat washing lotion residual in each well is removed completely.
(4) in each well on 96 orifice plates, add respectively 100 μ L biotin labeled mouse-anti people Adipsin monoclonal antibody (Biotin-AdipsinAb) to detect liquid, then with glued membrane, hide described 96 orifice plates, and described 96 orifice plates are placed on jolting instrument under jolting state to incubated at room 1.5 hours, jolting speed is 100 revs/min; Incubation time exhausts the liquid in each well after expiring, and by the described method of step (3), cleans described 96 orifice plates.
(5) in each well on described 96 orifice plates, add respectively 100 μ L Avidin-horseradish peroxidases, then with glued membrane, hide described 96 orifice plates, and described 96 orifice plates are placed on jolting instrument under jolting state to incubated at room 1.5 hours, jolting speed is 100 revs/min; Incubation time exhausts the liquid in each well after expiring, and by the described method of step (3), cleans described 96 orifice plates.
(6) in each well on described 96 orifice plates, add respectively 100 μ L 3 ', 3 ', 5,5 '-tetramethyl benzidine, then hatches 10 minutes in room temperature lucifuge, after incubation time expires, in each well, add respectively 100 μ L stop buffers, described stop buffer is the aqueous solution of sulfuric acid and sodium sulphite preparation, and the concentration of sulfuric acid is 2mol/L, and the concentration of sodium sulphite is 0.1mol/L.
(7) described 96 orifice plates are placed in microplate reader, by microplate reader, survey the A450 value (optical density value or absorbance) in each hole, measured result see the following form (the A450 value in table is the weighted mean value in two holes).
Table 1: the A450 value of standard specimen
| Standard specimen | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A450 value | 2.47895 | 1.34995 | 0.55605 | 0.2086 | 0.0639 | 0.0238 | 0.0092 |
Table 2: the A450 value of sample 1～20
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A450 value | 0.13265 | 0.06915 | 1.77485 | 0.70175 | 0.205 | 1.46035 | 2.2837 |
| Sample | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| A450 value | 0.3965 | 0.50175 | 0.13425 | 2.58855 | 0.088 | 0.2598 | 0.31965 |
| Sample | 15 | 16 | 17 | 18 | 19 | 20 | |
| A450 value | 0.0438 | 0.12975 | 0.24175 | 0.08385 | 0.1523 | 0.1299 |
Table 3: the A450 value of sample 21～40
| Sample | 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| A450 value | 0.2055 | 0.29965 | 0.4493 | 0.28165 | 0.8924 | 0.6017 | 0.1113 |
| Sample | 28 | 29 | 30 | 31 | 32 | 33 | 34 |
| A450 value | 0.83495 | 0.67255 | 0.5174 | 0.12425 | 0.1373 | 0.12635 | 0.21125 |
| Sample | 35 | 36 | 37 | 38 | 39 | 40 | |
| A450 value | 0.0694 | 0.32755 | 0.38015 | 0.0909 | 0.4242 | 0.1087 |
The A450 value of blank is 0.11045.
(8) according to the concentration of Adipsin in the A450 value of each standard specimen and each standard specimen, make A450 value-Adipsin concentration standard curve (seeing Fig. 2), and obtain computing formula y=0.0008x+0.0067, in formula, y is the A450 value of sample, and x is the content of Adipsin in sample.
(9) according to the A450 value of each sample of survey, with formula y=0.0008x+0.0067, calculate the concentration of Adipsin in each sample, result of calculation sees the following form:
Table 4: the result of calculation of sample 1～20 (Adipsin concentration unit: ng/mL)
Table 5: the result of calculation of sample 21～40 (Adipsin concentration unit: ng/mL)
The Adipsin mean concentration of sample 1～20 in reckoner 4, its result of calculation is 232.84 ± 489.25ng/mL.The Adipsin mean concentration of sample 21-40 in reckoner 5, its result of calculation is 4.21 ± 3.14ng/mL.From result of calculation, can find out, be taken from Adipsin mean concentration in the sample 1～20 of eclampsia group and there is statistical significance with the difference that is taken from the Adipsin mean concentration in the sample 21～40 of normal healthy controls group.
According to the result of calculation of the mean ± 2 * standard deviation of normal healthy controls group, be made as detectability, therefore, the Adipsin of kit setting of the present invention detects and is limited to 12ng/mL, be that in sample to be tested, Adipsin concentration is judged to be the positive (suffering from preeclampsia) higher than 12ng/mL, Adipsin concentration is judged to be feminine gender (not suffering from preeclampsia) lower than 12ng/mL.
Claims (2)
1. the contained Adipsin in urine sample of take be detect index preeclampsia detection kit a preparation method, it is characterized in that processing step is as follows:
(1) preparation is coated with the porous plate of anti-human Adipsin monoclonal antibody
With the carbonate buffer solution of pH=8～10, by anti-human Adipsin monoclonal antibody dilution, be the dilution of concentration 10 μ g/mL, in described anti-human Adipsin monoclonal antibody dilution, add absolute methanol, the addition of absolute methanol reaches 3mL/100mL with its concentration and is limited, then the described anti-human Adipsin monoclonal antibody dilution containing methyl alcohol is added in each hole of porous plate, its addition is 100 μ L/ holes, at 4 ℃, is coated with at least 12 hours; After the coated time expires, the bovine serum albumin solution that is 1g/100mL by concentration adds each hole on porous plate and carries out capping at 37 ℃, reaction time is at least 1 hour, and the addition of the bovine serum albumin solution that described concentration is 1g/100mL is 120 μ L/ holes; After capping finishes, the phosphate buffer washing porous plate with pH=7.2, obtains the porous plate that is coated with anti-human Adipsin monoclonal antibody after the unreacted reactant on porous plate is removed;
(2) prepare biotin labeled anti-human Adipsin monoclonal antibody and detect liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that solute compound concentration is acyl-N maloyl imines ester solution of 50 μ g/ μ L, the carbonate buffer solution of pH=9.6 of take is solvent, anti-human Adipsin monoclonal antibody is that solute compound concentration is the anti-human Adipsin monoclonal antibody solution of 24mg/mL, by mass ratio=0.5～1 of acyl-N maloyl imines ester and anti-human Adipsin monoclonal antibody: 7 measure described acyl-N maloyl imines ester solution and described anti-human Adipsin monoclonal antibody solution forms mixed liquor, by the mixed liquor of described acyl-N maloyl imines ester solution and described anti-human Adipsin monoclonal antibody solution under agitation in room temperature reaction at least 4 hours, obtain biotin labeled anti-human Adipsin monoclonal antibody,
After reaction finishes, the reactant liquor containing biotin labeled anti-human Adipsin monoclonal antibody is packed in bag filter, with the phosphate buffer of pH=9.2, at 4 ℃, dialyse, dialysis time is at least 12 hours, changes dislysate therebetween at least 3 times; Continue after add bovine serum albumin(BSA) in described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 3g～6g/100mL with its concentration and is limited;
That 2. 1. step is prepared measures containing reactant liquor and glycerine after the dialysis of biotin labeled anti-human Adipsin monoclonal antibody and bovine serum albumin(BSA) for 1: 1 by volume, at room temperature mixes and forms biotin labeled anti-human Adipsin monoclonal antibody detection liquid;
(3) be equipped with Avidin-horseradish peroxidase, chromogenic substrate 3', 3', 5,5'-tetramethyl benzidine and Adipsin protein standard substance.
According to claim 1 take contained Adipsin in urine sample be detect index preeclampsia detection kit preparation method, it is characterized in that being also equipped with sample dilution, described sample dilution is to add bovine serum albumin(BSA) formulated in the phosphate buffer of concentration 0.01mol/L～0.02mol/L or the TRIS buffer of concentration 0.01mol/L～0.02mol/L, and the addition of described bovine serum albumin(BSA) reaches 1g～2g/100mL with its concentration and is limited.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110365299.7A CN102426243B (en) | 2011-11-17 | 2011-11-17 | Preparation method of preeclampsia detection kit adopting Adipsin as detection index |
| PCT/CN2012/070010 WO2013071702A1 (en) | 2011-11-17 | 2012-01-04 | Preeclampsia detecting kit using adipsin as indicator and its preparing method |
Applications Claiming Priority (1)
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| CN1795387A (en) * | 2003-03-25 | 2006-06-28 | 普罗特奥格尼克斯公司 | Proteomic analysis of biological fluids |
| CN101351711A (en) * | 2005-12-29 | 2009-01-21 | 仁爱米拉索莱有限公司 | Diagnostic test for inflammatory endothelial dysfunctions in pregnancies |
| CN101889205A (en) * | 2007-07-27 | 2010-11-17 | 卡瓦迪斯有限责任公司 | The protein markers that is used for cardiovascular event |
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