CN107677832A - A kind of vWF ELISA detection reagent and its preparation method and application - Google Patents
A kind of vWF ELISA detection reagent and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of vWF ELISA detection reagent and its preparation method and application, and the detection reagent includes the latex beads system of two kinds of different-grain diameters.The vWF ELISA detection reagent prepared by preparation method of the present invention, repeatability is high, and precisely, quickly, limitations affect factor is few, and reliable results, cost is relatively low, beneficial to the application on clinical medicine for detection.
Description
Technical field
The invention belongs to field of clinical medical detection, and in particular to a kind of vWF ELISA detection reagent and its system
Preparation Method and application.
Background technology
Medical diagnosis on disease of the clotting assay to clinical departments has great importance, except the screening and diagnosis to hemorrhagic disease
Outside, the defects of being additionally operable to vWF ELISA identifies and haemophiliachemophiliac judgement.
VWF ELISA (von Willebrand factor, vWF) is closed by vascular endothelial cell and megacaryocyte
Into and secretion, be a kind of glycoprotein for being present in blood plasma, endothelial cell surface and Platelet alpha granule.Internal vWF is mainly with two
The connected dimeric forms of sulfide linkage are present, but also can be grouped to polymer, and molecular weight is up to million to more than ten million, and its defect will
Cause von Willebrand disease (von Willebrand disease, vWD).VWF can be with collagen and platelet membrane glycoprotein GPIb
Combined with a of II b- of GP III, in platelet adhesion reaction with being played an important role in assembling, and FF III (factors of F III) combination makes FF III live
Property become stable.In addition, vWF can also be combined with heparin, the endothelial cell of culture and smooth muscle cell matrix.
VWD is since nineteen twenty-four is reported first, because it is a kind of common hemorrhage of clinic thus is received significant attention,
China there is no vWD morbidity survey data at present, be calculated in the current ratio of vWD illness rates 1/1000, and China there are about 1,300,000 people trouble
This disease, but the vWD cases of the multiple hemorrhagic disease diagnosis and treatment center registrations in the whole nation are very few, and wherein reason has many aspects, such as to this
Disease lacks the clinical manifestation of characteristic, is easily covered by other diseases, and to the understanding deficiency of this disease and the limit of testing conditions
System make it that this disease is easily failed to pinpoint a disease in diagnosis.Thus, the detection to the vWF factors just seems most important.
CN104897910A discloses a kind of method and detection kit for determining von willebrand factor activity, the party
Method is to utilize sample and separation GPIb α albumen being mixed into a kind of detection sample, and measure vWF ELISA (FWF) is living
Property.But the method detects cumbersome, less stable, repeatability is relatively low, lacks reliability.
Therefore, need at present it is a kind of have the characteristics that it is repeated high, stably, sensitive, the quick and safe and reliable production of detection
Product.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of vWF ELISA detection reagent and
Its preparation method and application.The detection method reagent is simple, and detection reagent is stable, and reaction system is stable, influence factor
It is few, and detect precisely, it is simple to operate, safe and reliable, reproducible external diagnosis reagent is provided for clinical medicine.
To use following technical scheme up to this purpose, the present invention：
On the one hand, the invention provides a kind of vWF ELISA detection reagent, the detection reagent to include two kinds
The latex beads system of different-grain diameter.
In the present invention, compared to single particle size microballoon, the present invention uses the latex beads system of different-grain diameter, with difference
Aperture microballoon coupled antibody, is combined with antibody different loci, and antibody is fully coupled, and is improved coupling efficiency, is reduced cost, is increased
The strong repeatability of measured value, detection are highly stable.
Preferably, the latex beads is carboxyl polystyrene microsphere.
Preferably, the detection reagent includes 150nm latex beads system and 80nm latex beads system.
Preferably, the latex beads system of the 150nm：The volume ratio of 80nm latex beads system is 1:1.
Preferably, the mass concentration of the latex beads in the latex beads system of the 150nm is 1-10%, such as can be with
It is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 3-8%, more preferably 5%.
Preferably, the mass concentration of the latex beads in the latex beads system of the 80nm is 1-10%, such as can be with
It is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 3-8%, more preferably 5%.
Preferably, the detection reagent includes the buffer system that R1 and R2 is formed.
Wherein, the R1 is the MES buffer solutions that substance withdrawl syndrome is 5-200mM, also includes quality in the buffer solution
The Triton X-100. that the NaCl and quality volumn concentration that volumn concentration is 0.1-4% are 0.01-1%
The R2 is the MES buffer solutions that substance withdrawl syndrome is 5-200mM, also includes quality volume hundred in the buffer solution
The latex beads for the 80nm that the latex beads system for the 150nm that point content is 1-15%, quality volumn concentration are 1-15%
Anti-human FWF monoclonal antibodies of mouse that system, quality volumn concentration are 0.001-0.05%, quality volumn concentration are 1-
10% 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), quality volumn concentration are 0.1-3%
BSA and quality volumn concentration be 0.1-4% NaCl.
In the present invention, agents useful for same is simple, and suffered influence factor is few, and it is accurate to not only ensure assay, reduces again
Waste, reduce cost.
The quality volumn concentration refers to the quality (g) containing solid in 1 volume (L) liquid.
The substance withdrawl syndrome of the MES buffer solutions for example can be 5mM, 10mM, 20mM, 30mM, 40mM, 50mM,
60mM、70mM、80mM、90mM、100mM、110mM、150mM、130mM、140mM、150mM、160mM、170mM、180mM、
190mM or 200mM.
The quality volumn concentration of the NaCl can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,
0.7%th, 0.8%, 0.9%, 1%, 2%, 3% or 4%.
The quality volumn concentration of the Triton X-100 can be 0.01%, 0.02%, 0.03%, 0.05%,
0.08%th, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.
The quality volumn concentration of the latex beads system of the 150nm can be 1%, 2%, 3%, 4%, 5%,
6%th, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.
The quality volumn concentration of the latex beads system of the 80nm can be 1%, 2%, 3%, 4%, 5%,
6%th, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.
The quality volumn concentration of the mouse anti-human von willebrand disease factor monoclonal antibody can be 0.001%,
0.002%th, 0.003%, 0.005%, 0.006%, 0.008%, 0.01%, 0.02%, 0.03%, 0.04% or 0.05%.
The quality volumn concentration of the EDC is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
The quality volumn concentration of the BSA be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,
0.8%th, 0.9%, 1%, 2% or 3%.
Preferably, the pH of the R1 is 6-8, for example, can be 6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,
7th, 7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9 or 8, preferably 6.8-7.4.
Preferably, the pH of the R2 is 6-8, for example, can be 6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,
7th, 7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9 or 8, preferably 6.8-7.4.
Preferably, the R1 is the MES buffer solutions that substance withdrawl syndrome is 30-70mM, also includes matter in the buffer solution
Measure the NaCl that volumn concentration is 0.4-0.8% and the Triton X-100 that quality volumn concentration is 0.05-0.1%.
Further, the R1 is the MES buffer solutions that substance withdrawl syndrome is 50mM, also includes quality in the buffer solution
The Triton X-100 that the NaCl and quality volumn concentration that volumn concentration is 0.5% are 0.08%.
Preferably, the R2 is the MES buffer solutions that substance withdrawl syndrome is 30-70mM, also includes matter in the buffer solution
Measure the latex beads system for the 150nm that volumn concentration is 3-7%, the breast for the 80nm that quality volumn concentration is 3-7%
Glue microballoon system, quality volumn concentration be 0.003-0.007% mouse anti-human von willebrand disease factor monoclonal antibody,
The BSA and quality volume basis that EDC that quality volumn concentration is 2-4%, quality volumn concentration are 0.3-0.8% contain
Measure the NaCl for 0.3-0.8%.
Further, the R2 is the MES buffer solutions that substance withdrawl syndrome is 50mM, also includes quality in the buffer solution
The latex beads for the 80nm that the latex beads system for the 150nm that volumn concentration is 5%, quality volumn concentration are 5%
System, the mouse anti-human von willebrand disease factor monoclonal antibody that quality volumn concentration is 0.005%, quality volume basis
The NaCl that the BSA and quality volumn concentration that EDC that content is 2%, quality volumn concentration are 0.5% are 0.5%.
The present invention provides the preparation method of the 150nm latex beadses system, comprises the following steps：With EDC solution 100mL
With 37 DEG C of constant-temperature table concussion 20-30min of 150nm latex beadses 100mL, the microballoon 3000rpm centrifugation 10min after activation, abandon
Supernatant, with pH6.5-7.0MES buffer solution for cleaning 3-5 times；Add the mixing of mouse anti-human von willebrand disease factor monoclonal antibody
37 DEG C of incubation 5h, with pH6.5-7.0 MES buffer solution for cleaning 3-5 times after incubation；Adding pH6.5-7.0, to contain ox blood pure
Albumen MES buffer blinds 1-2h；Supernatant is abandoned in centrifugation, after pH7.0 MES buffer solution for cleaning 2-3 times, with 100mL pH7.0
MES buffer solutions ultrasound containing NaCl and BSA is resuspended, and it is stand-by to put 4 DEG C of preservations.
Preferably, the preparation of the 80nm latex beadses system comprises the following steps：By mouse anti-human von willebrand disease because
Sub- monoclonal antibody mixes with 80nm latex beadses, after being incubated 60-100min under normal temperature, adds EDC solution 100mL, is incubated 60-
150min, then the microballoon of coated antibody is subjected to 4-6 cleaning；BSA solution is added, closes 60-150min under normal temperature, centrifugation is gone
Supernatant, deionized water cleaning microballoon 4-6 times, it is resuspended with MES buffer solution ultrasounds of the 100mL pH7.0 containing NaCl and BSA, puts 4 DEG C
Preserve stand-by.
Preferably, the R2 is the 150nm latex beadses system and 80nm latex beadses system by volume 1:1 is mixed
Close.
On the other hand, the present invention provides a kind of detection reagent as described above in non-diagnostic and treatment detection vascular blood
The application method of friendly cause of disease, comprises the following steps：R1 and R2 reactions are added into detected sample, you can obtain being detected
VWF ELISA content.
In the present invention, the application method of the detection reagent is very simple, it is only necessary to which a step just can obtain accurately
As a result, it is workable, the time is saved, reduces cost.
Preferably, the testing sample:R1:R2 volume ratio is 5:2:15.
Preferably, the temperature that the detected sample and R1 are incubated is 35-40 DEG C, for example, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C,
39 DEG C or 40 DEG C, preferably 37 DEG C.
Preferably, the time that the detected sample and R1 are incubated is 40-100s, for example, 40s, 45s, 50s, 60s, 65s,
72s, 84s, 91s, 100s, preferably 60s.
On the other hand, the present invention provides detection reagent as described in relation to the first aspect, the reagent be used for it is haemophiliachemophiliac judge,
The preparation of the early warning detection kit of coronary heart disease or cancer.
Compared with prior art, the device have the advantages that：
(1) in the present invention, the latex beads system of different-grain diameter is used compared to single particle size microballoon, the present invention, used
Different pore size microballoon coupled antibody, is combined with antibody different loci, and antibody is fully coupled, and is improved coupling efficiency, is enhanced reality
The repeatability of measured value, detection are highly stable.
(2) reagent of the present invention is simple, and influence factor is few, under the premise of ensuring that assay is accurate, reduces and wastes, reduces
Cost.
(3) present invention detection is accurate, simple to operate, is provided for the detection on clinical medicine to vWF ELISA
Safe and reliable, reproducible external diagnosis reagent.
Embodiment
Technical scheme is further illustrated below by embodiment.Those skilled in the art should be bright
, the embodiment be only to aid in understand the present invention, be not construed as to the present invention concrete restriction.
Instrument in following examples：Automatic coagulometer：Crowd speeds great achievement XL3200.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
Embodiment 1
The preparation method of the detection reagent of the present embodiment vWF ELISA comprises the following steps：
(1) R1 is prepared：The MES buffer solutions that substance withdrawl syndrome is 50mM are first prepared, add quality volumn concentration
NaCl and quality volumn concentration 0.08%Triton X-100 for 0.5%, pH7.2 is adjusted after mixing.
(2) R2 is prepared：The preparation of 150nm latex beads systems comprises the following steps：It is 2% with quality volumn concentration
EDC solution 100mL and quality volumn concentration be 5% 37 DEG C of constant-temperature tables of 150nm latex beadses 100mL shake
25min, the microballoon 3000rpm centrifugation 10min after activation, abandons supernatant, with pH6.5-7.0MES buffer solution for cleaning 5 times；Add
The mouse anti-human von willebrand disease factor monoclonal antibody that quality volumn concentration is 0.005% mixes 37 DEG C of incubation 5h, incubates
With pH6.5-7.0 MES buffer solution for cleaning 5 times after educating；Add pH6.5-7.0 and contain quality volumn concentration for 0.5%
Bovine serum albumin(BSA) MES buffer blinds 2h；Supernatant is abandoned in centrifugation, after pH7.0 MES buffer solution for cleaning 3 times, uses 100mL
The MES buffer solutions for the BSA that the NaCl and quality volumn concentration that pH7.0 volumn concentrations containing quality are 0.5% are 0.5%
Ultrasound is resuspended, and it is stand-by to put 4 DEG C of preservations.
The preparation of 80nm latex beads systems comprises the following steps：The mouse that quality volumn concentration is 0.005% is resisted
Human von willebrand disease factor monoclonal antibody mixes with the 80nm latex beadses that quality volumn concentration is 5%, under normal temperature
After being incubated 80min, the EDC solution 100mL that quality volumn concentration is 5% is added, is incubated 100min, then by coated antibody
Microballoon carries out 6 cleanings；The BSA solution that quality volumn concentration is 0.5% is added, closes 100min under normal temperature, centrifugation is gone
Supernatant, deionized water cleaning microballoon 4-6 times, with the NaCl and quality that 100mL pH7.0 volumn concentrations containing quality are 0.5%
The MES buffer solutions ultrasound that volumn concentration is 0.5% BSA is resuspended, and it is stand-by to put 4 DEG C of preservations.
R2 is 150nm latex beadses system and 80nm latex beadses system by volume 1:1 mixing.
Embodiment 2
The preparation method of the detection reagent of the present embodiment vWF ELISA comprises the following steps：
(1) R1 is prepared：The MES buffer solutions that substance withdrawl syndrome is 10mM are first prepared, add quality volumn concentration
NaCl and quality volumn concentration 0.01%Triton X-100 for 0.1%, pH6.8 is adjusted after mixing.
(2) R2 is prepared：The preparation of 150nm latex beads systems comprises the following steps：It is 1% with quality volumn concentration
EDC solution 100mL and quality volumn concentration be 15% 37 DEG C of constant-temperature tables of 150nm latex beadses 100mL shake
30min, the microballoon 3000rpm centrifugation 10min after activation, abandons supernatant, with pH6.5-7.0MES buffer solution for cleaning 4 times；Add
The mouse anti-human von willebrand disease factor monoclonal antibody that quality volumn concentration is 0.001% mixes 37 DEG C of incubation 5h, incubates
With pH6.5-7.0 MES buffer solution for cleaning 1 time after educating；Add pH6.5-7.0 and contain quality volumn concentration for 0.1%
Bovine serum albumin(BSA) MES buffer blinds 1-2h；Supernatant is abandoned in centrifugation, after pH7.0 MES buffer solution for cleaning 2-3 time, use
The MES for the BSA that the NaCl and quality volumn concentration that 100mL pH7.0 volumn concentrations containing quality are 0.1% are 0.1%
Buffer solution ultrasound is resuspended, and it is stand-by to put 4 DEG C of preservations.
The preparation of 80nm latex beads systems comprises the following steps：The mouse that quality volumn concentration is 0.001% is resisted
Human von willebrand disease factor monoclonal antibody mixes with the 80nm latex beadses that quality volumn concentration is 15%, under normal temperature
After being incubated 100min, the EDC solution 100mL that quality volumn concentration is 1% is added, is incubated 150min, then by coated antibody
Microballoon carry out 4 times cleaning；The BSA solution that quality volumn concentration is 0.1% is added, 150min is closed under normal temperature, centrifuges
Remove supernatant, deionized water cleaning microballoon 5 times, with the NaCl and quality that 100mL pH7.0 volumn concentrations containing quality are 0.1%
The MES buffer solutions ultrasound that volumn concentration is 0.1% BSA is resuspended, and it is stand-by to put 4 DEG C of preservations.
R2 is 150nm latex beadses system and 80nm latex beadses system by volume 1:1 mixing.
Embodiment 3
The preparation method of the detection reagent of the present embodiment vWF ELISA comprises the following steps：
(1) R1 is prepared：The MES buffer solutions that substance withdrawl syndrome is 200mM are first prepared, add quality volumn concentration
NaCl and quality volumn concentration 1%Triton X-100 for 3%, pH7.4 is adjusted after mixing.
(2) R2 is prepared：The preparation of 150nm latex beads systems comprises the following steps：It is with quality volumn concentration
10% EDC solution 100mL shakes with 37 DEG C of constant-temperature tables of 150nm latex beadses 100mL that quality volumn concentration is 1%
20min is swung, microballoon 3000rpm after activation centrifugation 10min, abandons supernatant, with pH6.5-7.0MES buffer solution for cleaning 5 times；Again plus
Enter the mouse anti-human von willebrand disease factor monoclonal antibody that quality volumn concentration is 0.05% and mix 37 DEG C of incubation 5h, incubate
With pH6.5-7.0 MES buffer solution for cleaning 5 times after educating；It is 3% to add pH6.5-7.0 to contain quality volumn concentration
Bovine serum albumin(BSA) MES buffer blinds 1.5h；Supernatant is abandoned in centrifugation, after pH7.0 MES buffer solution for cleaning 2 times, uses 100mL
The MES buffer solutions ultrasound for the BSA that the NaCl and quality volumn concentration that pH7.0 volumn concentrations containing quality are 3% are 3%
It is resuspended, it is stand-by puts 4 DEG C of preservations.
The preparation of 80nm latex beads systems comprises the following steps：Quality volumn concentration is anti-human for 0.05% mouse
VWF ELISA monoclonal antibody mixes with the 80nm latex beadses that quality volumn concentration is 1%, is incubated under normal temperature
After educating 60min, the EDC solution 100mL that quality volumn concentration is 1% is added, is incubated 60min, then by the micro- of coated antibody
Ball carries out 4 cleanings；The BSA solution that quality volumn concentration is 3% is added, 60min is closed under normal temperature, supernatant is removed in centrifugation,
Deionized water cleaning microballoon 6 times, with the NaCl and quality volume basis that 100mL pH7.0 volumn concentrations containing quality are 3%
The MES buffer solutions ultrasound that content is 3% BSA is resuspended, and it is stand-by to put 4 DEG C of preservations.
R2 is 150nm latex beadses system and 80nm latex beadses system by volume 1:1 mixing.
Embodiment 4
The measure of repeatability：
VWF prepared by embodiment 1 reagent and commercially available vWF reagent while normal Quality Control vWF is determined, including it is following
Step：By 20 μ L R1,150 μ LR2 and 50 μ L sample to be tested after 37 DEG C, 60s mix incubation, surveyed on full automatic blood-coagulation
It is fixed.The repeated result of the comparison is as shown in table 1 below：
Table 1
Note：CF is the coefficient of variation.
As it can be seen from table 1 vWF of the present invention reagent is reproducible in 10 experiments, the coefficient of variation is only 1.01, and city
It is poor to sell reagent repeatability, the coefficient of variation 1.89.
The vWF reagents that embodiment 2 and embodiment 3 are prepared equally carry out replica test, this hair in 10 experiments
Bright vWF reagent is reproducible, and the coefficient of variation is respectively 1.08 and 1.05.
Embodiment 5
The measure of stability：
VWF detection reagents prepared by embodiment 1 and commercially available vWF detection reagents are positioned in 37 DEG C of insulating boxs, daily
Corresponding volume is taken to be detected according to detection method in the present invention, the stability result of the comparison is as shown in table 2：
Table 2
Embodiment 6
The measure of stability：
VWF detection reagents prepared by embodiment 2 and commercially available vWF detection reagents are positioned in 37 DEG C of insulating boxs, daily
Corresponding volume is taken to be detected according to detection method in the present invention, the stability result of the comparison is as shown in table 3：
Table 3
Embodiment 7
The measure of stability：
VWF detection reagents prepared by embodiment 3 and commercially available vWF detection reagents are positioned in 37 DEG C of insulating boxs, daily
Corresponding volume is taken to be detected according to detection method in the present invention, the stability result of the comparison is as shown in table 4：
Table 4
The vWF detection reagents that the present invention is can be seen that from table 2, table 3, the result of table 4 test knot under the conditions of 37 DEG C in 8 days
Fruit is stable, and repeatability is high, starts to change to the 8th talent, and commercial reagent began to produce change after the 3rd day, this
As a result it is extremely important especially for middle and small hospital for clinical laboratory, can under the premise of ensuring that assay is accurate
Wasted with reducing, so as to reduce cost.
Applicant states that the present invention illustrates that a kind of vWF ELISA of the present invention detects by above-described embodiment
Reagent and its preparation method and application, but the invention is not limited in above-mentioned method detailed, that is, do not mean that the present invention must be according to
Above-mentioned method detailed is relied to implement.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to this
The equivalence replacement of each raw material of invention product and the addition of auxiliary element, the selection of concrete mode etc., all fall within the protection of the present invention
Within the scope of scope and disclosure.
Claims (10)
1. a kind of vWF ELISA detection reagent, it is characterised in that the detection reagent includes two kinds of different-grain diameters
Latex beads system.
2. detection reagent according to claim 1, it is characterised in that the latex beads is carboxyl polystyrene microsphere；
Preferably, the detection reagent includes 150nm latex beads system and 80nm latex beads system；
Preferably, the volume ratio of the latex beads system of the 150nm and 80nm latex beads system is 1:1.
3. detection reagent according to claim 1 or 2, it is characterised in that the breast in the latex beads system of the 150nm
The mass concentration of glue microballoon is 1-10%, preferably 3-8%, more preferably 5%；
Preferably, the mass concentration of the latex beads in the latex beads system of the 80nm is 1-10%, preferably 3-8%,
More preferably 5%.
4. according to the detection reagent any one of claim 1-3, it is characterised in that the detection reagent includes R1 and R2
The buffer system of composition；
Wherein, the R1 is the MES buffer solutions that substance withdrawl syndrome is 5-200mM, also includes quality volume in the buffer solution
The Triton X-100 that the NaCl and quality volumn concentration that percentage composition is 0.1-4% are 0.01-1%；
The R2 is the MES buffer solutions that substance withdrawl syndrome is 5-200mM, is also contained in the buffer solution including quality volume basis
Measure the latex beads system of the 150nm for 1-15%, the latex beads system for the 80nm that quality volumn concentration is 1-15%,
Quality volumn concentration is 0.001-0.05% mouse anti-human von willebrand disease factor monoclonal antibody, quality volume basis
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides that content is 1-10%, quality volumn concentration are 0.1-
3% BSA and quality volumn concentration is 0.1-4% NaCl；
Preferably, the pH of the R1 is 6-8, preferably 6.8-7.4；
Preferably, the pH of the R2 is 6-8, preferably 6.8-7.4.
5. detection reagent according to claim 4, it is characterised in that the R1 is that substance withdrawl syndrome is 30-70mM's
MES buffer solutions, NaCl that quality volumn concentration is 0.4-0.8% is also included in the buffer solution and quality volume basis contains
Measure the Triton X-100 for 0.05-0.1%；
Preferably, the R1 is the MES buffer solutions that substance withdrawl syndrome is 50mM, also includes quality volume hundred in the buffer solution
The Triton X-100 that the NaCl and quality volumn concentration that point content is 0.5% are 0.08%.
6. detection reagent according to claim 4, it is characterised in that the R2 is that substance withdrawl syndrome is 30-70mM's
MES buffer solutions, the 150nm that quality volumn concentration is 3-7% latex beads system, quality are also included in the buffer solution
The mouse that the latex beads system for the 80nm that volumn concentration is 3-7%, quality volumn concentration are 0.003-0.007% resists
Human von willebrand disease factor monoclonal antibody, 1- (3- dimethylamino-propyls) -3- second that quality volumn concentration is 2-4%
The BSA and quality volumn concentration that base carbodiimide hydrochloride, quality volumn concentration are 0.3-0.8% are 0.3-
0.8% NaCl；
Preferably, the R2 is the MES buffer solutions that substance withdrawl syndrome is 50mM, also includes quality volume hundred in the buffer solution
Latex beads system, the matter for the 80nm that the latex beads system for the 150nm that point content is 5%, quality volumn concentration are 5%
Measuring mouse anti-human von willebrand disease factor monoclonal antibody, quality volumn concentration that volumn concentration is 0.005% is
2% 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, the BSA that quality volumn concentration is 0.5% and matter
Measure the NaCl that volumn concentration is 0.5%.
7. according to the detection reagent any one of claim 1-6, it is characterised in that the 150nm latex beadses system
Preparation comprise the following steps：With 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution 100mL and 150nm
37 DEG C of constant-temperature table concussion 20-30min of latex beads 100mL, the microballoon 3000rpm centrifugation 10min after activation, abandon supernatant, use
PH6.5-7.0MES buffer solution for cleaning 3-5 times；37 DEG C of mouse anti-human von willebrand disease factor monoclonal antibody mixing is added to incubate
5h is educated, with pH6.5-7.0 MES buffer solution for cleaning 3-5 times after incubation；Add pH6.5-7.0 and contain bovine serum albumin(BSA) MES
Buffer blind 1-2h；Supernatant is abandoned in centrifugation, after pH7.0 MES buffer solution for cleaning 2-3 time, with 100mL pH7.0 containing NaCl with
BSA MES buffer solutions ultrasound is resuspended, and it is stand-by to put 4 DEG C of preservations；
Preferably, the preparation of the 80nm latex beadses system comprises the following steps：By mouse anti-human von willebrand disease factor list
Clonal antibody mixes with 80nm latex beadses, after being incubated 60-100min under normal temperature, adds 1- (3- dimethylamino-propyls) -3- second
Base carbodiimide hydrochloride solution 100mL, 60-150min is incubated, then the microballoon of coated antibody is subjected to 4-6 cleaning；Add
BSA solution, 60-150min is closed under normal temperature, and centrifugation is removed supernatant, deionized water cleaning microballoon 4-6 times, contained with 100mL pH7.0
NaCl and BSA MES buffer solutions ultrasound is resuspended, and it is stand-by to put 4 DEG C of preservations；
Preferably, the R2 is the 150nm latex beadses system and 80nm latex beadses system by volume 1:1 mixing.
8. a kind of detection reagent as any one of claim 1-7 is in non-diagnostic and treatment detection von Willebrand disease
The application method of the factor, it is characterised in that comprise the following steps：R1 is added into detected sample and enters R2 reactions, you can
To the content for the vWF ELISA to be detected.
9. application method according to claim 8, it is characterised in that the testing sample:R1:R2 volume ratio is 5:2:
15；
Preferably, the detected sample and the temperature that R1 is incubated are 35-40 DEG C, preferably 37 DEG C；
Preferably, the detected sample and the R1 times being incubated are 40-100s, preferably 60s.
10. such as the detection reagent any one of claim 1-7, it is characterised in that the reagent is sentenced for haemophiliachemophiliac
The preparation of the early warning detection kit of fixed, coronary heart disease or cancer.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1773283A (en) * | 2005-11-11 | 2006-05-17 | 袁洪 | Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN104215770A (en) * | 2014-08-14 | 2014-12-17 | 上海睿康生物科技有限公司 | Two-particle-based retinol binding protein detection kit |
| CN104535770A (en) * | 2014-12-18 | 2015-04-22 | 江苏昊申医学科技有限公司 | Myoglobin determination kit of compound antibody |
| CN106932588A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Detection α1Kit of-microglobulin and preparation method thereof |
| CN107340395A (en) * | 2017-07-05 | 2017-11-10 | 深圳开立生物医疗科技股份有限公司 | A kind of Immunoturbidimetric kit for detecting Procalcitonin |
-
2017
- 2017-09-20 CN CN201710861688.6A patent/CN107677832A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1773283A (en) * | 2005-11-11 | 2006-05-17 | 袁洪 | Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN104215770A (en) * | 2014-08-14 | 2014-12-17 | 上海睿康生物科技有限公司 | Two-particle-based retinol binding protein detection kit |
| CN104535770A (en) * | 2014-12-18 | 2015-04-22 | 江苏昊申医学科技有限公司 | Myoglobin determination kit of compound antibody |
| CN106932588A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Detection α1Kit of-microglobulin and preparation method thereof |
| CN107340395A (en) * | 2017-07-05 | 2017-11-10 | 深圳开立生物医疗科技股份有限公司 | A kind of Immunoturbidimetric kit for detecting Procalcitonin |
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Application publication date: 20180209 |