CN101858917B - Kit for detecting premature rupture of membrane by taking Axl as detection index and preparation method thereof - Google Patents
Kit for detecting premature rupture of membrane by taking Axl as detection index and preparation method thereof Download PDFInfo
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Abstract
The invention discloses application of a regent for detecting Axl receptor tyrosine kinase to prepare a kit for detecting whether a pregnant woman has a premature rupture of membranes. The regent is a monoclonal antibody of the Axl receptor tyrosine kinase which is labeled by biotin. The kit includes a perforated plate coated with the monoclonal antibody of the Axl receptor tyrosine kinase, biotin-labeled monoclonal antibody detection liquid of the Axl receptor tyrosine kinase, avidin-horse radish peroxidase combined with biotin-labeled monoclonal antibody of the Axl receptor tyrosine kinase, a chromogenic substrate 3',3',5,5'-tetramethylbenzidine, and an Axl protein standard substance. A preparation method for the kit comprises the following steps of: (1) preparing the perforated plate coated with the monoclonal antibody of the Axl receptor tyrosine kinase; (2) preparing the biotin-labeled monoclonal antibody detection liquid of the Axl receptor tyrosine kinase; and (3) preparing avidin-horse radish peroxidase, the chromogenic substrate 3',3',5,5'-tetramethylbenzidine, and the protein standard substance of the Axl receptor tyrosine kinase.
Description
Technical field
The application that the present invention relates to the Axl monoclonal antibody reaches for detection of pregnancy women premature rupture of fetal membranes Sandwich ELISA kit and the preparation method of (Premature Rupture of Fetal Membrane is called for short PROM).
Background technology
Rupture of membranes (Rupture of Fetal Membrane is called for short ROM) may occur at any time in the pregnancy period, and the rupture of membranes that occurs before just before giving birth is called premature rupture of fetal membranes (PROM).(37 pregnant week) PROM incidence is about 10% after mature; The incidence of (before 37 pregnant weeks) PROM is 2-3.5% before mature.Premature rupture of fetal membranes is to cause that the obstetrical patient is antenatal, the principal element of postpartum complication, is to cause the fetus premature labor and main cause that the neonate must move in the intensive care unit.Because the medical worker has to ask balance between prolong pregnancy week and the risk that in utero reaches infection of pregnant women and Fetal Lung growth problem, thereby large to handling cost height, the difficulty of Patients with Preterm Premature Rupture of Membranes, it is most important accurately and timely to diagnose the pregnant woman whether premature rupture of fetal membranes to occur.
In the prior art, adopting goldstandard is a kind of accurate method of Diagnosis of Premature Rupture, namely in pregnant woman's amniotic cavity, inject dyestuff (such as methylene blue), if have in the vagina by the outflow of the liquid of dye coloring and can be diagnosed as premature rupture of fetal membranes, but adopt goldstandard to detect clinical very difficult operation, and cause patient's misery, so clinical seldom use.Usually adopt clinically and inquire that medical history, patient's private prosecution vagina have amniotic fluid to flow out, vaginal fluid pH value becomes meta-alkalescence (normal vaginal fluid pH is 4.5～5.5), microscopically is looked into the methods such as occurring amniotic fluid crystal in the vaginal fluid smear of seeing, although these methods are simple to operate, accuracy, susceptibility are all lower.
Axl (Axl receptor tyrosine kinase, the hereafter of instructions is " Axl ") belongs to Tyro3 receptor tyrosine kinase subfamily, and its part is Gas6 and Protein S.Their wide expression play an important role in neurodevelopment, immunological regulation, spermatogenesis, NK Cell Differentiation, platelet function and blood vessel occur in the tissues such as mammiferous nerve, immunity, reproduction, hematopoiesis and blood vessel.The genesis of the kinds of tumors such as the expression of research discovery Axl and Gas6 and gastroenteric tumor, prostate cancer is closely related.Axl has no report at present with the relation of gestation.
Summary of the invention
The object of the present invention is to provide the new purposes of Axl monoclonal antibody in the preparation kit for detecting premature rupture of membrane, a kind of kit for detecting premature rupture of membrane take Axl as the detection index and preparation method thereof is provided, the accuracy that detects to improve premature rupture of fetal membranes alleviates patient's misery.
The inventor of present patent application found through experiments: Axl appears at one of main protein in the amniotic fluid third trimester of pregnancy, and content is 15.28 ± 4.90ng/mL.To have amniotic fluid to leak out to vagina during premature rupture of fetal membranes from amniotic cavity, the Axl in the amniotic fluid then can appear in vaginal fluid/cervical mucus (Cervical-Vaginal Fluid, CVF).The protein chip screening experiment is found: the PROM puerpera who makes a definite diagnosis compares (two groups of puerperas' age and coupling in age in gestation week with the non-PROM puerpera who makes a definite diagnosis, the foundation-free disease, without pregnancy complication), the concentration of Axl has notable difference in vaginal fluid/uterine neck mucus sample.Therefore, can be with the detection index of Axl as premature rupture of fetal membranes.
Of the present invention take Axl as detecting the kit for detecting premature rupture of membrane of index, comprise the porous plate that is coated with the Axl monoclonal antibody, biotin labeled Axl monoclonal antibody (Biotin-AxlAb) detect liquid, the Avidin-horseradish peroxidase with biotin labeled Axl monoclonal antibody (Biotin-AxlAb) combination, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance.
Experiment shows, the contained porous plate that is coated with the Axl monoclonal antibody of mentioned reagent box, and preferred every hole is coated with Axl monoclonal antibody 0.9 μ g～1 μ g.
In the mentioned reagent box, described biotin labeled Axl monoclonal antibody (Biotin-AxlAb) detects liquid and mainly is comprised of biotin labeled Axl monoclonal antibody (Biotin-AxlAb), bovine serum albumin(BSA), glycerine and phosphate buffer, the concentration of biotin labeled Axl monoclonal antibody (Biotin-AxlAb) 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 Axl monoclonal antibody (Biotin-AxlAb) is coupled by biotin acyl-N maloyl imines ester and Axl monoclonal antibody and forms, and the mass ratio of acyl-N maloyl imines ester and Axl monoclonal antibody is 0.5～1: 7.
In order to be user-friendly to, kit of the present invention also can be equipped with the 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) it is formulated to add bovine serum albumin(BSA) in, and the concentration of bovine serum albumin(BSA) is 1g～2g/100ml.
Of the present invention take the preparation method of Axl as the kit for detecting premature rupture of membrane of detection index, processing step is as follows:
(1) preparation is coated with the porous plate of Axl monoclonal antibody
Carbonate buffer solution with pH=8～10 is the dilution of concentration 10 μ g/ml with the dilution of Axl monoclonal antibody, then described Axl monoclonal antibody dilution is added in each hole of porous plate, is coated with at least 12 hours at 4 ℃; After the coated time expires, be that the bovine serum albumin solution of 1g/100ml adds each hole on the porous plate and carries out capping at 37 ℃ with concentration, the reaction time is at least 1 hour; After capping finishes, with the phosphate buffer washing porous plate of pH=7.2, namely obtain to be coated with the porous plate of Axl monoclonal antibody after the unreacted reactant on the porous plate is removed, its storage temperature is 4 ℃;
(2) the biotin labeled Axl monoclonal antibody of preparation (Biotin-AxlAb) detects liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that the solute compound concentration is the acyl-N maloyl imines ester solution of 50 μ g/ μ l, take the carbonate buffer solution of pH=9.6 as solvent, the Axl monoclonal antibody is that the solute compound concentration is the Axl monoclonal antibody solution of 24mg/ml, the mixed liquor of described acyl-N maloyl imines ester solution and described Axl monoclonal antibody solution under agitation in room temperature reaction at least 4 hours, is namely obtained biotin labeled Axl monoclonal antibody (Biotin-AxlAb); After reaction finished, the reactant liquor that will contain biotin labeled Axl monoclonal antibody (Biotin-AxlAb) was packed in the bag filter, dialyses at 4 ℃ with the phosphate buffer of pH=9.2, and dialysis time is at least 12 hours, changes dislysate therebetween at least 3 times; Continue after add bovine serum albumin(BSA) in the described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 3g～6g/100ml with its concentration and is limited;
2. the metering in 1: 1 by volume of the dialysis afterreaction liquid that contains biotin labeled Axl monoclonal antibody (Biotin-AxlAb) and bovine serum albumin(BSA) that 1. step is prepared and glycerine, at room temperature mix and namely form biotin labeled Axl monoclonal antibody (Biotin-AxlAb) detection liquid, its storage temperature is-20 ℃;
(3) be equipped with Avidin-horseradish peroxidase, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance.Described Avidin-horseradish peroxidase, 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance be the commercial goods, can directly buy from market.
In the method for the invention, when preparation is coated with the porous plate of Axl monoclonal antibody, the preferred 100 μ l/ holes of the addition of described Axl monoclonal antibody dilution, described concentration is that the addition of the bovine serum albumin solution of 1g/100ml is 120 μ l/ holes.
In the method for the invention, when preparing biotin labeled Axl monoclonal antibody (Biotin-AxlAb), preferably measure described acyl-N maloyl imines ester solution by the mass ratio of acyl-N maloyl imines ester and Axl monoclonal antibody=0.5～1: 7 and described Axl monoclonal antibody solution forms mixed liquor.
For the convenience of the user, the method of the invention also has been equipped with the 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) it is formulated to add bovine serum albumin(BSA) in, and 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) the present invention provides a kind of kit that adopts new detection index and preparation method thereof for the detection of premature rupture of fetal membranes.
(2) use kit of the present invention, can quantitatively detect the accurate content of Axl in gravid woman's vaginal fluid/cervical mucus (Cervical-Vaginal Fluid, CVF).
(3) use kit of the present invention to making a definite diagnosis the rupture of membranes group and making a definite diagnosis the not broken group gravid woman of fetal membrane and carry out sample detecting (seeing embodiment 2), testing result shows: the susceptibility of this kit is 90%, specificity is about 95%, thereby can improve the accuracy that premature rupture of fetal membranes detects.
(4) because test sample is gravid woman's vaginal fluid, thereby be a kind of without the wound detection.
Kit of the present invention is applicable to all pregnancy women, the pregnancy women that especially exists premature delivery risk, belly to sustain damage.
Description of drawings
Fig. 1 be of the present invention take Axl as the kit for detecting premature rupture of membrane that detects index in a kind of shape organigram of porous plate.
Fig. 2 is A450 value-Axl concentration standard curve.
Embodiment
Below by embodiment kit for detecting premature rupture of membrane take Axl as the detection index of the present invention and preparation method thereof is described further with using method.
The material source that relates among the following embodiment is as follows:
The Axl monoclonal antibody is available from U.S. RayBiotec company;
The Axl protein standard substance is available from U.S. RayBiotec company;
Carbonate buffer solution (CB) is available from U.S. sigma company;
Phosphate buffer (PBS) is available from U.S. sigma company;
TRIS buffer (the Tris damping fluid, TBS), available from U.S. sigma company;
Bovine serum albumin(BSA) is available from U.S. sigma company;
DMF is available from U.S. sigma company;
Acyl-N maloyl imines ester is available from U.S. sigma company;
The specification of glycerine is to analyze alcohol, available from U.S. sigma company;
Horseradish peroxidase is available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing;
3 ', 3 ', 5,5 '-tetramethyl benzidine, available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing;
Porous plate is the porous plate in 96 holes, and its shape and structure are as shown in Figure 1.
Embodiment 1
In the present embodiment, adopt following processing step preparation take Axl as detecting the kit for detecting premature rupture of membrane of index:
(1) preparation is coated with the porous plate of Axl monoclonal antibody
Carbonate buffer solution with pH=9 is the dilution of concentration 10 μ g/ml with the dilution of Axl monoclonal antibody, then the amount of described Axl monoclonal antibody dilution by 100 μ l/ holes is added in each hole of 96 orifice plates, is coated with 12 hours at 4 ℃; After the coated time expires, the bovine serum albumin solution of concentration 1g/100ml is added each hole on the 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, be the phosphate buffer washing porous plate of 0.05mol/l with pH=7.2, concentration, i.e. acquisition is coated with the porous plate of Axl monoclonal antibody after the unreacted reactant on the porous plate is removed, and then is placed in 4 ℃ of refrigerators to save backup;
(2) the biotin labeled Axl monoclonal antibody of preparation (Biotin-AxlAb) detects liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that the solute compound concentration is the 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, the Axl monoclonal antibody is that the solute compound concentration is the Axl monoclonal antibody solution of 24mg/ml, by acyl-N maloyl imines ester with the mass ratio of Axl monoclonal antibody=measure described acyl-N maloyl imines ester solution at 1: 7 with described Axl monoclonal antibody solution forms mixed liquor, with the mixed liquor of described acyl-N maloyl imines ester solution and described Axl monoclonal antibody solution (magnetic stirring apparatus under agitation, 200 rev/mins of stirring rates) in room temperature reaction 4 hours, namely obtain biotin labeled Axl monoclonal antibody (Biotin-AxlAb); After reaction finishes, the reactant liquor that will contain biotin labeled Axl monoclonal antibody (Biotin-AxlAb) is packed in the bag filter, the phosphate buffer that with pH=9.2, concentration is 0.05mol/l is dialysed at 4 ℃, and dialysis time is 12 hours, changes dislysate therebetween 3 times; Continue after add bovine serum albumin(BSA) in the described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 4g/100ml with its concentration and is limited;
2. the metering in 1: 1 by volume of the dialysis afterreaction liquid that contains biotin labeled Axl monoclonal antibody (Biotin-AxlAb) and bovine serum albumin(BSA) that 1. step is prepared and glycerine, at room temperature mix and namely form biotin labeled Axl monoclonal antibody (Biotin-AxlAb) 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, Axl protein standard substance (concentration of Axl is 10ng/ml) 1ml and sample dilution 30ml, the sample dilution is at the TRIS buffer of concentration concentration 0.02mol/L (Tris damping fluid, TBS) it is formulated to add bovine serum albumin(BSA) in, and 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 Axl monoclonal antibody detect liquid is 1: 1.
Embodiment 2
Present embodiment is treated the side sample with the kit for detecting premature rupture of membrane of embodiment 1 preparation and is detected.
1, sample is prepared
(1) standard specimen
The Axl protein standard substance that is equipped with in the embodiment 1 described kit is made as contains the highest standard specimen of Axl concentration, called after standard specimen 1, the concentration of its Axl is 6000pg/ml, then with the sample diluting liquid that is equipped with in the embodiment 1 described kit described Axl protein standard substance is diluted to respectively the standard specimen that Axl 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
Reach puerpera in hospital as experimental subjects take West China No.2 Hospital, Sichuan University's outpatient service, according to the testing result of existing detection method experimental subjects is divided into rupture of membranes group and rupture of membranes group not, collect vaginal fluid/cervical mucus (the Cervical-Vaginal Fluid of two groups of puerperas when just before giving birth starting, CVF) as sample, amount to 40 samples, wherein, sample 1-20 is taken from the rupture of membranes group, and sample 21-40 is taken from not rupture of membranes group.
(3) blank
The sample dilution that is equipped with in the embodiment 1 described kit---be blank.
2, pattern detection
(1) detects front all reagent with kit, the slow equilibrium of sample to be tested 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 the Axl 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.Hide described 96 orifice plates with glued membrane, and described 96 orifice plates were placed on the jolting instrument under the jolting state incubated at room 2.5 hours, jolting speed is 100 rev/mins.
(3) after incubation time expires, exhaust the liquid in each well, at automatic washer cleaning procedure is set, take 400 μ l/ hole washing lotions (phosphate buffer of concentration 0.02mol/L or concentration are as the Tris damping fluid of 0.02mol/L) each well is cleaned twice, during each the cleaning, washing lotion needs to stop 10 seconds～15 seconds in the hole; Clean complete after, with 96 orifice plates upsets, make each well aperture downward, pat at absorbent filter washing lotion residual in each well removed fully.
(4) add respectively the biotin labeled Axl monoclonal antibodies of 100 μ l (Biotin-AxlAb) in each well on 96 orifice plates and detect liquid, then hide described 96 orifice plates with glued membrane, and described 96 orifice plates were placed on the jolting instrument under the jolting state incubated at room 1.5 hours, jolting speed is 100 rev/mins; Incubation time exhausts the liquid in each well after expiring, and cleans described 96 orifice plates by the described method of step (3).
(5) add respectively 100 μ l Avidin-horseradish peroxidases in each well on described 96 orifice plates, then hide described 96 orifice plates with glued membrane, and described 96 orifice plates were placed on the jolting instrument under the jolting state incubated at room 1.5 hours, jolting speed is 100 rev/mins; Incubation time exhausts the liquid in each well after expiring, and cleans described 96 orifice plates by the described method of step (3).
(6) add respectively 100 μ l 3 ' in each well on described 96 orifice plates, 3 ', 5,5 '-tetramethyl benzidine, then hatched 10 minutes in the 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 the microplate reader, survey the A450 value (optical density value or absorbance) in each hole with microplate reader, measured result see the following form (the A450 value in the 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 |
| The A450 value | 1.61275 | 1.0066 | 0.45615 | 0.194 | 0.122 | 0.09315 | 0.08065 |
[0072] Table 2: the A450 value of sample 1～20
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| The A450 value | 1.20565 | 0.89205 | 0.4422 | 0.2369 | 0.5826 | 0.1268 | 2.4718 |
| Sample | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| The A450 value | 0.5314 | 0.5908 | 0.169 | 0.5305 | 0.42755 | 1.1929 | 0.5233 |
| Sample | 15 | 16 | 17 | 18 | 19 | 20 | |
| The A450 value | 0.63705 | 1.35225 | 0.47385 | 0.39795 | 0.2968 | 0.9351 |
Table 3: the A450 value of sample 21～40
| Sample | 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| The A450 value | 0.07745 | 0.0696 | 0.1554 | 0.0787 | 0.38795 | 0.08865 | 0.0681 |
| Sample | 28 | 29 | 30 | 31 | 32 | 33 | 34 |
| The A450 value | 0.06345 | 0.17625 | 0.06325 | 0.13035 | 0.07865 | 0.0981 | 0.1015 |
| Sample | 35 | 36 | 37 | 38 | 39 | 40 | |
| The A450 value | 0.06495 | 0.21555 | 0.07375 | 0.09875 | 0.06075 | 0.1026 |
The A450 value of blank is 0.0811.
(8) according to the concentration of Axl in the A450 value of each standard specimen and each standard specimen, make A450 value-Axl concentration standard curve (see figure 2), and obtain computing formula y=0.0005x+0.0174, in the formula, y is the A450 value of sample, and x is the content of Axl in the sample.
(9) according to the A450 value of each sample of survey, calculate the concentration of Axl in each sample with formula y=0.0005x+0.0174, result of calculation sees the following form:
Table 4: the result of calculation of sample 1～20 (Axl concentration unit: pg/ml)
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Axl concentration | 4776.841 | 3425.601 | 1487.288 | 602.691 | 2092.243 | 128.2922 | 10232.43 |
| Sample | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| Axl concentration | 1871.633 | 2127.575 | 310.1235 | 1867.755 | 1424.164 | 4721.904 | 1836.731 |
| Sample | 15 | 16 | 17 | 18 | 19 | 20 | |
| Axl concentration | 2326.857 | 5408.511 | 1623.661 | 1296.623 | 860.7882 | 3611.095 |
[0081] Table 5: the result of calculation of sample 21～40 (Axl concentration unit: pg/ml)
| Sample | 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| |
0 | 0 | 194.1081 | 0 | 836.667 | 9.671195 | 0 |
| Sample | 28 | 29 | 30 | 31 | 32 | 33 | 34 |
| |
0 | 251.7188 | 0 | 124.8925 | 0 | 35.78249 | 45.17703 |
| Sample | 35 | 36 | 37 | 38 | 39 | 40 | |
| |
0 | 360.3086 | 0 | 37.57851 | 0 | 48.21644 |
The Axl mean concentration of sample 1～20 in the reckoner 4, its result of calculation are 2601.64 ± 2327.54pg/ml(2.60 ± 2.33ng/ml).The Axl mean concentration of sample 21～40 in the reckoner 5, its result of calculation are 97.21 ± 200.61pg/ml(0.10 ± 0.20ng/ml).Can find out that from result of calculation the difference that is taken from the Axl mean concentration in Axl mean concentration and the sample 21～40 that is taken from rupture of membranes group not in the sample 1～20 of rupture of membranes group has statistical significance.
Be made as detectability according to the result of calculation of the mean of rupture of membranes group ± 2* standard deviation not, the Axl of kit setting of the present invention detects and is limited to 600pg/ml(0.6ng/ml), be that Axl concentration is higher than 600pg/ml(0.6ng/ml in the sample to be tested) be judged to be the positive (premature rupture of fetal membranes namely occurs), Axl concentration is lower than 600pg/ml(0.6ng/ml) be judged to be feminine gender (premature rupture of fetal membranes does not namely occur).
Claims (10)
1. the reagent that detects Axl at content that preparation detects Axl in vaginal fluid or the cervical mucus to determine whether the pregnant woman application in the kit of premature rupture of fetal membranes occurs, it is characterized in that, the reagent of described detection Axl is the Axl monoclonal antibody, described Axl monoclonal antibody adopts biotin labeling, described kit comprises the porous plate that is coated with the Axl monoclonal antibody, biotin labeled Axl monoclonal antibody detects liquid, Avidin-the horseradish peroxidase of being combined with biotin labeled Axl monoclonal antibody, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance.
2. application according to claim 1 is characterized in that the every hole of described porous plate is coated with Axl monoclonal antibody 0.9 μ g～1 μ g.
3. application according to claim 1 and 2, it is characterized in that described biotin labeled Axl monoclonal antibody detects liquid and mainly is comprised of biotin labeled Axl monoclonal antibody, bovine serum albumin(BSA), glycerine and phosphate buffer, the concentration of biotin labeled Axl monoclonal antibody is 24mg/ml, the concentration of bovine serum albumin(BSA) is 1.5g～3g/100ml, and the concentration of glycerine is 50ml/100ml.
4. application according to claim 3, it is characterized in that described biotin labeled Axl monoclonal antibody is coupled by biotin acyl-N maloyl imines ester and Axl monoclonal antibody forms, and the mass ratio of biotin acyl-N maloyl imines ester and Axl monoclonal antibody is 0.5～1: 7.
5. one kind take Axl as detecting the kit for detecting premature rupture of membrane of index, it is characterized in that described kit comprise the porous plate that is coated with the Axl monoclonal antibody, biotin labeled Axl monoclonal antibody detect liquid, the Avidin-horseradish peroxidase of being combined with biotin labeled Axl monoclonal antibody, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance;
The every hole of described porous plate is coated with Axl monoclonal antibody 0.9 μ g～1 μ g, described biotin labeled Axl monoclonal antibody detects liquid and mainly is comprised of biotin labeled Axl monoclonal antibody, bovine serum albumin(BSA), glycerine and phosphate buffer, the concentration of biotin labeled Axl monoclonal antibody is 24mg/ml, the concentration of bovine serum albumin(BSA) is 1.5g～3g/100ml, and the concentration of glycerine is 50ml/100ml.
6. according to claim 5 take Axl as detecting the kit for detecting premature rupture of membrane of index, it is characterized in that described biotin labeled Axl monoclonal antibody is coupled by biotin acyl-N maloyl imines ester and Axl monoclonal antibody forms, and the mass ratio of biotin acyl-N maloyl imines ester and Axl monoclonal antibody is 0.5～1: 7.
7. according to claim 5 or 6 described take Axl as detecting the kit for detecting premature rupture of membrane of index, characterized by further comprising the sample dilution, described sample dilution is that the adding bovine serum albumin(BSA) is formulated in the TRIS buffer of the phosphate buffer of concentration 0.01mol/L～0.02mol/L or concentration 0.01mol/L～0.02mol/L, and the concentration of bovine serum albumin(BSA) is 1g～2g/100ml.
8. preparation method take Axl as the kit for detecting premature rupture of membrane that detects index is characterized in that processing step is as follows:
(1) preparation is coated with the porous plate of Axl monoclonal antibody
Carbonate buffer solution with pH=8～10 is the dilution of concentration 10 μ g/ml with the dilution of Axl monoclonal antibody, then described Axl monoclonal antibody dilution is added in each hole of porous plate, be coated with at least 12 hours at 4 ℃; After the coated time expires, be that the bovine serum albumin solution of 1g/100ml adds each hole on the porous plate and carries out capping at 37 ℃ with concentration, the reaction time is at least 1 hour; After capping finishes, with the phosphate buffer washing porous plate of pH=7.2, after being removed, the unreacted reactant on the porous plate namely obtains to be coated with the porous plate of Axl monoclonal antibody;
(2) the biotin labeled Axl monoclonal antibody of preparation detects liquid
1. with N, dinethylformamide is solvent, biotin acyl-N maloyl imines ester is that the solute compound concentration is the biotin acyl-N maloyl imines ester solution of 50 μ g/ μ l, take the carbonate buffer solution of pH=9.6 as solvent, the Axl monoclonal antibody is that the solute compound concentration is the Axl monoclonal antibody solution of 24mg/ml, the mixed liquor of described biotin acyl-N maloyl imines ester solution and described Axl monoclonal antibody solution under agitation in room temperature reaction at least 4 hours, is namely obtained biotin labeled Axl monoclonal antibody; After reaction finished, the reactant liquor that will contain biotin labeled Axl monoclonal antibody was packed in the bag filter, dialyses at 4 ℃ with the phosphate buffer of pH=9.2, and dialysis time is at least 12 hours, changes dislysate therebetween at least 3 times; Continue after add bovine serum albumin(BSA) in the described reactant liquor after dialysis, the addition of described bovine serum albumin(BSA) reaches 3g～6g/100ml with its concentration and is limited;
2. the metering in 1: 1 by volume of the dialysis afterreaction liquid that contains biotin labeled Axl monoclonal antibody and bovine serum albumin(BSA) that 1. step is prepared and glycerine at room temperature mixes and namely forms biotin labeled Axl monoclonal antibody detection liquid;
(3) be equipped with Avidin-horseradish peroxidase, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Axl protein standard substance.
9. according to claim 8 take the preparation method of Axl as the kit for detecting premature rupture of membrane of detection index, it is characterized in that also being equipped with the sample dilution, described sample dilution is that the adding bovine serum albumin(BSA) is formulated in the TRIS buffer of the phosphate buffer of concentration 0.01mol/L～0.02mol/L or 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.
10. it is characterized in that according to claim 8 or 9 described preparation methods take Axl as the kit for detecting premature rupture of membrane that detects index:
When (1) preparation is coated with the porous plate of Axl monoclonal antibody, the addition of described Axl monoclonal antibody dilution is 100 μ l/ holes, and described concentration is that the addition of the bovine serum albumin solution of 1g/100ml is 120 μ l/ holes;
(2) preparation is during biotin labeled Axl monoclonal antibody, measures described biotin acyl-N maloyl imines ester solution and described Axl monoclonal antibody solution formation mixed liquor by the mass ratio of biotin acyl-N maloyl imines ester and Axl monoclonal antibody=0.5～1: 7.
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| CN102297963A (en) * | 2011-05-30 | 2011-12-28 | 吉权 | Premature rupture of membrane colloidal gold test paper reaction buffer, and preparation method thereof |
| CN102426243B (en) * | 2011-11-17 | 2014-03-19 | 成都创宜生物科技有限公司 | Preparation method of preeclampsia detection kit adopting Adipsin as detection index |
| CN107759691B (en) * | 2017-12-04 | 2018-09-25 | 杭州尚健生物技术有限公司 | A kind of monoclonal antibody of specific binding AXL |
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| CN101210926A (en) * | 2006-12-27 | 2008-07-02 | 中国科学院大连化学物理研究所 | Reagent kit for detecting human serum tissue kallikrein and its preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2918605A1 (en) * | 2007-11-12 | 2015-09-16 | U3 Pharma GmbH | Axl antibodies |
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