CN109142716A - The detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application - Google Patents
The detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application Download PDFInfo
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- CN109142716A CN109142716A CN201811000652.XA CN201811000652A CN109142716A CN 109142716 A CN109142716 A CN 109142716A CN 201811000652 A CN201811000652 A CN 201811000652A CN 109142716 A CN109142716 A CN 109142716A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The present invention provides the detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application, the reagent strip (1) includes the liquid storage cavity layer set gradually from top to bottom, flow channel layer and reaction cavity layer；The liquid storage cavity layer is provided at least one liquid storage chamber (4)；The reaction cavity layer is provided at least one reaction chamber (2)；The flow channel layer is provided with microchannel (8), for being connected to liquid storage chamber (4) and reaction chamber (2).Reagent strip of the invention is based on microflow control technique, it is integrated to will test reagent, avoid reagent contamination, reduce transportation cost, the reagent strip is loaded on detecting instrument, automation Molecular Detection is realized, experimental error caused by open system pollution that may be present and manual operation is eliminated, there is important application in the early detection of A β -40 and A β -42.
Description
Technical field
The invention belongs to Protein Detection field, it is related to the detection reagent item of joint-detection A β -40 and A β -42 a kind of and its answers
With more particularly to a kind of joint-detection A β -40 and A β -42 based on magnetic particle and chemiluminescence detection reagent item and its
Using.
Background technique
According to statistics, there is nearly 30,000,000 alzheimer's disease (Alzheimer disease, AD) patient in the whole world.The sea A Erzi
Silent disease makes a definite diagnosis rear mean survival time (MST) only 10 years, still falls within what medical field can not cure at present to the harm of the elderly and its serious
Pernicious old age mental disease.For 60 years old or more crowd, with advancing age, the disease incidence of alzheimer's disease was sharply risen
It is high.In China, the alzheimer's disease disease incidence of over-65s crowd is 7%, and the disease incidence of 85 years old or more crowd is 25%, and
The disease incidence of 95 years old or more crowds is then up to 60%.Early stage alzheimer's disease non-evident sympton, it is difficult to diagnose, easily be missed
It examines.
Alzheimer's disease research association thinks that the lesion growth of alzheimer's disease is slow, early diagnosis, early intervention
The generation that lesion can significantly be delayed, it is most important for reducing disease incidence, be conducive to the normal life for maintaining patient.Meanwhile
Early diagnosis helps to distinguish Alzheimer's and the doubtful patient of alzheimer's disease, improves diagnostic accuracy.
Amyloid beta (A β) is substrate of the amyloid protein precursor (APP) after followed by action of proteolytic enzymes, by No. 21
Chromosome coding is one of main pathological protein of A Ercimohai disease.APP is processed by least three kinds of enzymes, and cutting approach include
Secrete enzymatic pathway and secretion enzymatic pathway II.In secretion enzymatic pathway, APP is cracked by beta-secretase first, then in gamma-secretase
Under effect, the polypeptide containing 39-43 amino acid is formed by cutting.Wherein, A β -42 and A β -40 is the most common hypotype, A β -
42 protein fragments being made of 42-43 amino acid, are predominantly located at the intracerebral of A Ercimohai disease patient；In cerebrospinal fluid and
In blood, the content of A β -40 is much higher than β -42 A, but A β -42 is more toxic, is easier to assemble.
CN 1928564A discloses a kind of detection A β1-42The method and its kit of antibody, this method is including the use of A β1-42
Protein fragments solution is coated with solid support；Solid support is added in biological sample to be detected and is incubated for；Cleaning removal
The sundries such as unbonded albumen；Add A β1-42Monoclonal antibody solution is simultaneously incubated for；The unbonded A β of cleaning removal1-42Monoclonal
Labeled secondary antibody and incubation is added in antibody；Cleaning removes any unbonded secondary antibody；It detects in mixtures incubated
The immune amount in conjunction with compound of antigen-antibody.The detection method accuracy with higher, method is easy, practical,
Using competitive enzyme-linked immune detection method, quantitative detection can be made to antibody, method is easy, and suitable clinic generallys use.However still
There is not detection of the prior art for A β -40, and enzyme linked immunosorbent detection not can avoid the interference of extraneous excitation light source background signal,
Signal-to-noise ratio is low, poor sensitivity, is not suitable for the early diagnosis of alzheimer's disease.
Therefore it provides a kind of high sensitivity, high specificity, the A β -40 strong with automatic detection system compatibility are detected and are tried
Agent item is of great significance in alzheimer's disease research field.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provide a kind of joint-detection A β -40 and A β -42 detection reagent item and its
Using, the reagent strip is based on microflow control technique and magnetic microparticle chemiluminescence technology, and high specificity, high sensitivity, accuracy are good,
A β -40 and A β -42 detection reagent are loaded on reagent strip, realize the integrated of reagent strip, with detection and analysis equipment
It matches, realizes automation, highly sensitive, the fast joint detection of A β -40 and A β -42, be the early stage of alzheimer's disease
Diagnostic field is of great significance.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the reagent strip 1 includes the liquid storage set gradually from top to bottom the present invention provides a kind of reagent strip
Cavity layer, flow channel layer and reaction cavity layer；
The liquid storage cavity layer is provided at least one liquid storage chamber 4；
The reaction cavity layer is provided at least one reaction chamber 2；
The flow channel layer is provided with microchannel 8, for being connected to liquid storage chamber 4 and reaction chamber 2；
Detection reagent is housed in the liquid storage chamber 4；
The detection reagent includes A β -40 capture antibody coupling magnetic particle, A β -42 capture antibody coupling magnetic particle, cleaning
Liquid, A β -40 detect the combination of any one or at least two in antibody, A β -42 detection antibody or reaction solution.
Reagent strip of the invention not only increases detection efficiency using microchannel connection liquid storage chamber and reaction chamber, and
The consumption for reducing sample and reagent is conducive to be loaded into and realizes that the automation of molecule is controllably examined on automatic detection device
It surveys.
Preferably, the reagent strip 1 further includes at least one sealing plug 3, is loaded into liquid storage chamber 4.
In the present invention, sealing plug is placed in liquid storage chamber, the leakproofness of liquid storage chamber is not only ensure that, avoids detection reagent
It is contaminated in storage and transportational process, and is cooperated by sealing plug and power pressurizing unit, by the inspection in liquid storage chamber
Test agent indentation microchannel enters reaction chamber, realizes the detection function of reagent strip.
Preferably, at least one buckle 6 is provided on the outer surface of the sealing plug 3, on the inner surface of the liquid storage chamber 4
At least two card slots 7 are provided with, the position of sealing plug 3 is limited by connecting with buckle 6.
In the present invention, by buckling the connection with card slot, position of the sealing plug in liquid storage chamber is defined, when on sealing plug
Buckle be connected with the first card slot in liquid storage chamber, sealing plug is in liquid storage position, and detection reagent does not flow out, when on sealing plug
Buckle is connected with the second card slot in liquid storage chamber, and sealing plug is in liquid release position, and sealing plug, which squeezes liquid storing bag, makes liquid storing bag
Rupture, detection reagent outflow.
Preferably, the length of the reaction cavity layer is not less than the length of the liquid storage cavity layer.
In the present invention, sample to be tested is added as well relative to the part that liquid storage cavity layer is protruded in reaction cavity layer.
Preferably, the reaction cavity layer is provided with partition 9, and the height of the partition 9 is less than the height of the reaction cavity layer.
Reaction chamber interlayer is divided into different reaction chambers, it is therefore prevented that phase by the way that partition is arranged in reaction cavity layer by the present invention
Cross contamination occurs for adjacent reagent, increases the accuracy of reagent strip；In addition, the height of partition is less than the height of reaction cavity layer, make
On partition top, there are appropriate spaces, moved between differential responses chamber for magnetic particle.
Preferably, the upper surface of the partition 9 includes any one in plane, inclined-plane or curved surface, preferably inclined-plane.
Preferably, the bottom surface of the reagent strip 1 is provided with locating slot 5, for the reagent strip 1 to be fixed on detecting instrument
On.
Preferably, the volume of the liquid storage chamber 4 be 10-500 μ L, such as can be 10 μ L, 50 μ L, 100 μ L, 150 μ L,
200 μ L, 250 μ L, 300 μ L, 350 μ L, 400 μ L, 450 μ L or 500 μ L.
Preferably, the volume of the reaction chamber 2 be 10-500 μ L, such as can be 10 μ L, 50 μ L, 100 μ L, 150 μ L,
200 μ L, 250 μ L, 300 μ L, 350 μ L, 400 μ L, 450 μ L or 500 μ L.
Preferably, the reagent strip 1 is molded to be formed using high molecular material.
Preferably, the high molecular material includes acrylonitrile-butadiene-styrene copolymer, polymethyl methacrylate
In polyethylene any one or at least two combination, preferably polymethyl methacrylate.
Preferably, detection reagent is housed, the detection reagent is loaded in sealing liquid storing bag in the liquid storage chamber 4.
Preferably, the sealing liquid storing bag is using any one in plastic film, aluminium foil or aluminium plastic composite material or extremely
Few two kinds of combination is prepared, and is preferably prepared using aluminium plastic composite material.
Sealing liquid storing bag of the invention is using the sealing means sealing being easy to tear, under the pressure effect of sealing plug, storage
The rupture of liquid bag, realizes the release of detection reagent.
Preferably, the detection reagent include A β -40 capture antibody coupling magnetic particle, A β -42 capture antibody coupling magnetic it is micro-
Grain, cleaning solution, A β -40 detect the combination of any one or at least two in antibody, A β -42 detection antibody or reaction solution, excellent
It is selected as A β -40 capture antibody coupling magnetic particle, A β -42 capture antibody coupling magnetic particle, cleaning solution, A β -40 detection antibody, A β -
The combination of 42 detection antibody and reaction solution.
Preferably, the partial size of the magnetic particle is 0.1-10 μm, such as can be 0.1 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4
μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm.
Preferably, the detecting instrument includes power pressurizing unit, magnetic particle mobile controller or signal supervisory instrument
In any one or at least two combination, preferably power pressurizing unit, magnetic particle mobile controller and signal detection
The combination of device.
In the present invention, by taking the detection process of A β -40 as an example, the course of work of the reagent strip is as follows:
(1) A β -40 antibody coupling magnetic particle solution, cleaning solution, A β -40 detection antibody-solutions and reaction solution is captured to distinguish
It is packed into liquid storing bag sealing, is respectively placed in corresponding liquid storage chamber 4；
(2) when reagent strip 1 stores and transports, sealing plug 3 is inserted into liquid storage chamber 4, makes buckle 6 and storage on sealing plug 3
The first card slot connection in sap cavity 4, sealing plug 3 are in liquid storage position；
(3) in an experiment, reagent strip 1 is put into first and is tested and analyzed in instrument, the power pressurizing unit of instrument squeezes envelope
Mouth plug 3, connect the buckle 6 on sealing plug 3 with the second card slot in liquid storage chamber, and liquid storing bag rupture, detection reagent passes through miniflow
Channel 8 flows into reaction chamber 2；
(4) then to sample to be tested is added in the first reaction chamber of reagent strip 1, antibody coupling magnetic particle is captured with A β -40
It is incubated for；
(5) the magnetic particle mobile controller of instrument by the first reaction chamber magnetic particle enrichment after, by reaction chamber it
Between 9 top of partition space, magnetic particle is transferred to the second reaction chamber, carries out magnetic particle cleaning；
(6) same steps are used, magnetic particle is transferred to adjacent reaction chamber, the signal detection dress of ultimate analysis instrument respectively
Acquisition signal is set, output analysis result after software processing.
Second aspect, the present invention provides a kind of reagent strips as described in relation to the first aspect in detection A β -40, by 1-20
Replacing, missing or adding for amino acid residue and obtain there is the active amino acid sequence of A β -40, A β -42 or by 1-20
A replacing, missing or adding for amino acid residue and obtain with the application in the active amino acid sequence of A β -42.
The third aspect, the present invention provides the methods of a kind of joint-detection A β -40 and A β -42, and the method includes following
Step:
(1) using the values of chemiluminescence of reagent strip detection A β -40 and A β -42 as described in relation to the first aspect；
(2) values of chemiluminescence of A β -40 is subjected to the joint inspection of A β -40 and A β -42 divided by the values of chemiluminescence of A β -42
It surveys.
Compared with prior art, the invention has the following beneficial effects:
(1) reagent strip of the invention utilizes microflow control technique, is connected to liquid storage chamber and reaction chamber using microchannel, not only mentions
High detection efficiency, and reduce the consumption of sample and reagent, be conducive to be loaded into realization A β-on automatic detection device
The automation of 40 and/or A β -42 controllably detects；
(2) detection reagent in reagent strip of the invention is integrated in the reagent strip, using sealing plug to liquid storing bag
The detection reagent being sealed in liquid storing bag is discharged into reaction chamber by squeezing action, avoids reagent contamination, reduce transport at
This；
(3) reagent strip of the invention is loaded on detecting instrument, other than sample to be tested is added, all experimental implementations,
The transfer of release, magnetic particle including detection reagent and the detection of signal, are automatically performed by analysis instrument, easy to operate easy
Row eliminates experimental error caused by open system pollution that may be present and manual operation, in A β -40 and/or A β -42 early stage
There is important application in detection.
Detailed description of the invention
Fig. 1 is the southeast isometric view of reagent strip of the present invention, wherein 1- reagent strip, 2- reaction chamber, 3- sealing plug, 4-
Liquid storage chamber, 5- locating slot；
Fig. 2 is the right side view of reagent strip of the present invention, wherein 3- sealing plug, 5- locating slot；
Fig. 3 is the isometric schematic cross-sectional view in the local southeast of reagent strip of the present invention, wherein 6- buckle, 7- card slot, 8- miniflow are logical
Road, 9- partition；
Fig. 4 is the isometric schematic cross-sectional view in the whole southeast of reagent strip of the present invention, wherein 10- magnetic particle liquid storage chamber, 11- the
One cleaning solution liquid storage chamber, 12- the second cleaning solution liquid storage chamber, 13- detect antibody stock chamber, 14- third cleaning solution liquid storage chamber, 15-
4th cleaning solution liquid storage chamber, 16- chemiluminescence reaction liquid liquid storage chamber, 17- sample incubation chamber, 18- first clean chamber, 19- second
Chamber is cleaned, 20- detects antibody incubation chamber, and 21- third cleans chamber, and 22- the 4th cleans chamber, 23- chemiluminescence detection chamber；
Fig. 5 is the standard curve using reagent strip of the present invention detection A β -40 concentration；
Fig. 6 is the standard curve using reagent strip of the present invention detection A β -42 concentration；
Fig. 7 is the chemiluminescence result using the A β -40/A β -42 of reagent strip of the present invention detection sample.
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The assembling of 1 reagent strip of embodiment
The southeast isometric view of the reagent strip of the present embodiment as shown in Figure 1, right side view as shown in Fig. 2, part east
The isometric schematic cross-sectional view in south is as shown in figure 3, the isometric schematic cross-sectional view in the whole southeast is as shown in Figure 4.
The reagent strip 1 includes the liquid storage cavity layer set gradually from top to bottom, flow channel layer and reaction cavity layer；
The liquid storage cavity layer is provided with seven liquid storage chambers 4, respectively magnetic particle liquid storage chamber 10, the first cleaning solution liquid storage chamber
11, the second cleaning solution liquid storage chamber 12, detection antibody stock chamber 13, third cleaning solution liquid storage chamber 14, the 4th cleaning solution liquid storage chamber 15
With chemiluminescence reaction liquid liquid storage chamber 16；
The reaction cavity layer is provided with seven reaction chambers 2, and it is clear to clean chamber 18, second for respectively sample incubation chamber 17, first
It washes chamber 19, detection antibody incubation chamber 20, third cleaning chamber the 21, the 4th and cleans chamber 22 and chemiluminescence detection chamber 23；
The flow channel layer is provided with seven microchannels 8, for being connected to liquid storage chamber and corresponding reaction chamber；
The length of the reaction cavity layer is greater than the length of the liquid storage cavity layer；
The reagent strip 1 further includes seven sealing plugs, is loaded into corresponding liquid storage chamber；
A buckle 6 is provided on the outer surface of the sealing plug 3, on the inner surface of the liquid storage chamber 4 there are two settings
Highly different card slots 7 limits the position of sealing plug 3 by connecting with buckle 6；
The reaction cavity layer is provided with partition 9, and reaction chamber interlayer is divided into reaction chamber 17,18,19,20,21,22 and 23,
The height of the partition 9 is less than the height of the reaction cavity layer；
The upper surface of the partition 9 is inclined-plane；
The bottom surface of the reagent strip 1 is provided with locating slot 5, for the reagent strip 1 to be fixed on detecting instrument；
The volume of the liquid storage chamber 4 is 10-500 μ L；
Preferably, the volume of the reaction chamber 2 is 10-500 μ L.
By taking the detection process of A β -40 as an example, the course of work of reagent strip of the invention is as follows:
(1) A β -40 is captured into antibody coupling magnetic particle solution, the first cleaning solution, the second cleaning solution, A β -40 and detects antibody
Solution, third cleaning solution, the 4th cleaning solution and chemiluminescence reaction liquid are respectively charged into liquid storing bag sealing, are respectively placed in magnetic particle
Liquid storage chamber 10, the first cleaning solution liquid storage chamber 11, the second cleaning solution liquid storage chamber 12, detection antibody stock chamber 13, the storage of third cleaning solution
In sap cavity 14, the 4th cleaning solution liquid storage chamber 15 and chemiluminescence reaction liquid liquid storage chamber 16；
(2) when reagent strip 1 stores and transports, sealing plug is inserted into liquid storage chamber, makes buckle 6 and liquid storage chamber on sealing plug
Interior the first card slot connection, sealing plug are in liquid storage position；
(3) in an experiment, reagent strip 1 is put into first and is tested and analyzed in instrument, the power pressurizing unit of instrument squeezes envelope
Mouth plug, ruptures liquid storing bag, detection reagent flows into reaction chamber by microchannel；
(4) it then to sample to be tested is added in the sample incubation chamber 17 of reagent strip, is incubated for magnetic particle；
(5) the magnetic particle mobile controller of instrument is incubated after the magnetic particle enrichment in sample incubation chamber 17 by sample
A β -40 capture antibody coupling magnetic particle is transferred to first by the space for educating the partition top between chamber 17 and the first cleaning chamber 18
Chamber 18 is cleaned, magnetic particle cleaning is carried out；
(6) same steps are used, A β -40 capture antibody coupling magnetic particle is transferred to the second cleaning chamber 19, detection respectively
Antibody incubation chamber 20, third cleaning chamber the 21, the 4th clean chamber 22 and chemiluminescence detection chamber 23, the signal inspection of ultimate analysis instrument
The chemiluminescence signal of device acquisition chemiluminescence detection chamber 23 is surveyed, output analysis result after software processing.
The detection of embodiment 2A β -40 and A β -42 standard items
The reagent strip of the present embodiment is formed using polymethyl methacrylate (PMMA) injection molding, and liquid storing bag is using plastic-aluminum combined
Material is prepared.
Respectively by the anti-A β -40 magnetic particle solution of 50 μ L, 100 the first cleaning solutions of μ L, 100 the second cleaning solutions of μ L, 100 μ L alkalinity
Anti- A β -40 antibody of phosphatase enzyme mark, 100 μ L third cleaning solutions, the 4th cleaning solution of 100 μ L and 100 μ L alkaline phosphatase substrates
After AMPPD reaction solution injects liquid storing bag, is sealed using welding technique, be respectively placed in magnetic particle liquid storage chamber 10, the first cleaning solution
Liquid storage chamber 11, the second cleaning solution liquid storage chamber 12, detection antibody stock chamber 13, third cleaning solution liquid storage chamber 14, the storage of the 4th cleaning solution
In sap cavity 15 and chemiluminescence reaction liquid liquid storage chamber 16, the reagent strip is obtained.
The preparation of A β -40 standard items includes that the artificial synthesized gene constructed recombinant vector of A β -40 is imported host strain, screening
It carries out Protein expression and purification afterwards to obtain, A β -40 standard items are obtained using TBS or PBS buffer solution dilution mother liquor, and concentration is respectively
400pg/mL, 200pg/mL, 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL, 6.25pg/mL and 0pg/mL.
In the present embodiment, the sealed membrane of 8 reagent strips is torn off for the detection of primary achievable 8 reagent strips of detecting instrument,
The 100 μ L of A β -40 standard items of various concentration is added into magnetic particle liquid storage chamber 10, is put into detecting instrument starting detection, power squeezes
The reagent of liquid storage chamber is added in corresponding reaction chamber by pressure device；
It is 37 DEG C that the temperature control console of instrument, which keeps temperature locating for reagent strip, A β -40 standard items and is modified with capture at this time
0.1 μm of magnetic particle of antibody reacts 10-15min at a suitable temperature；
Then, start magnetic particle transfer device, the magnetic particle absorption of instrument is with transfer device by sample by sample incubation chamber
Respectively by the first cleaning chamber and the second cleaning chamber, magnetic particle cleaning is carried out；
The magnetic particle cleaned is transferred to detection antibody incubation chamber, capture has the magnetic particle and alkaline phosphatase of A β -40
Anti- A β -40 antibody is marked to mix, 37 DEG C of reaction 10-15min；
Start magnetic particle transfer device, sample is incubated for chamber by detection and led to respectively by the magnetic particle absorption of instrument and transfer device
Third cleaning chamber and the 4th cleaning chamber are crossed, magnetic particle cleaning is carried out；
The magnetic particle cleaned is transferred to chemiluminescence detection chamber, carries out chemiluminescence detection.
Detection chemiluminescence detection is carried out to A β -42 standard items using same reagent strip and method.
The values of chemiluminescence of A β -40 standard items is as shown in table 1 and Fig. 5, equation of linear regression y=1.6882x+
240.73 relative coefficient R2=0.991；The values of chemiluminescence of A β -42 standard items is as shown in table 1 and Fig. 6, linear regression side
Journey is y=1.2913x+265.39, relative coefficient R2=0.9919；A β -40 concentration and A β -42 concentration and values of chemiluminescence
Between have good linear relationship, illustrate the reliability and accuracy of reagent strip and instrument.
Table 1
| A β -40 concentration (pg/mL) | Luminous value | A β -42 concentration (pg/mL) | Luminous value |
| 400 | 931.42 | 400 | 799.31 |
| 200 | 539.86 | 200 | 499.86 |
| 100 | 419.13 | 100 | 371.52 |
| 50 | 319.81 | 50 | 323.74 |
| 25 | 307.49 | 25 | 304.12 |
| 12.5 | 277.31 | 12.5 | 293.9 |
| 6.25 | 257.72 | 6.25 | 287.64 |
| 0 | 213.09 | 0 | 268 |
The detection of embodiment 3A β -40/A β -42 clinical sample
The reagent strip of the present embodiment is formed using acrylonitrile-butadiene-styrene copolymer (ABS) injection molding, and liquid storing bag is adopted
It is prepared with aluminium plastic composite material.
Respectively by the anti-A β -40 magnetic particle solution of 50 μ L, 100 the first cleaning solutions of μ L, 100 the second cleaning solutions of μ L, 100 μ L alkalinity
Anti- A β -40 antibody of phosphatase enzyme mark, 100 μ L third cleaning solutions, the 4th cleaning solution of 100 μ L and 100 μ L alkaline phosphatase substrates
After AMPPD reaction solution injects liquid storing bag, is sealed using welding technique, be respectively placed in magnetic particle liquid storage chamber 10, the first cleaning solution
Liquid storage chamber 11, the second cleaning solution liquid storage chamber 12, detection antibody stock chamber 13, third cleaning solution liquid storage chamber 14, the storage of the 4th cleaning solution
In sap cavity 15 and chemiluminescence reaction liquid liquid storage chamber 16, A β -40 detection reagent item is obtained；
Respectively by the anti-A β -42 magnetic particle solution of 50 μ L, 100 the first cleaning solutions of μ L, 100 the second cleaning solutions of μ L, 100 μ L alkalinity
Anti- A β -42 antibody of phosphatase enzyme mark, 100 μ L third cleaning solutions, the 4th cleaning solution of 100 μ L and 100 μ L alkaline phosphatase substrates
After AMPPD reaction solution injects liquid storing bag, is sealed using welding technique, be respectively placed in magnetic particle liquid storage chamber 10, the first cleaning solution
Liquid storage chamber 11, the second cleaning solution liquid storage chamber 12, detection antibody stock chamber 13, third cleaning solution liquid storage chamber 14, the storage of the 4th cleaning solution
In sap cavity 15 and chemiluminescence reaction liquid liquid storage chamber 16, A β -42 detection reagent item is obtained.
3000rpm/min at whole blood sample each 16,4 DEG C of Alzheimer's and normal person is chosen to be centrifuged
10min collects blood plasma or serum, -80 DEG C of preservations；
Sample is diluted 5 times using PBS or TBS buffer, 50 μ L is drawn and reagent strip is added, be put into instrument and examined
It surveys, as a result as shown in table 2 (A), table 2 (B), table 2 (C) and Fig. 7, it can be seen that compared to human normal plasma A β -40/A β's -42
The ratio of ratio, Alzheimer's plasma A β -40/A β -42 significantly improves, this and alzheimer's disease reported in the literature
Patient's body A β -42 aggregation, deposition, A β -42 content reduce, and the raised result of concentration proportion of A β -40/A β -42 is identical, explanation
The testing result of the present embodiment reagent strip has referential.
Table 2 (A)
Table 2 (B)
Table 2 (C)
In conclusion reagent strip of the invention utilizes microflow control technique, liquid storage chamber and reaction chamber are connected to using microchannel,
Detection efficiency is not only increased, and reduces the consumption of sample and reagent, is conducive to be loaded on automatic detection device
Realize that the automation of molecule controllably detects；Detection reagent is integrated in reagent strip, using sealing plug to the squeezing action of liquid storing bag
The detection reagent being sealed in liquid storing bag is discharged into reaction chamber, reagent contamination is avoided, reduces transportation cost；Reagent strip
It is loaded on detecting instrument, other than sample to be tested is added, all experimental implementations, release, magnetic particle including detection reagent
Transfer and signal detection, be automatically performed by analysis instrument, it is easy to operation, it is that may be present to eliminate open system
Experimental error caused by pollution and manual operation；The detection of A β -40/A β -42, the A of patient are carried out using reagent strip of the invention
β -40/A β -42 ratio is significantly higher than normal person, reduces with A β -42 reported in the literature aggregation, deposition, A β -42 content, A β -40/A
The raised result of the concentration proportion of β -42 is identical, illustrates that the testing result of the present embodiment reagent strip has referential.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of reagent strip, which is characterized in that the reagent strip (1) includes the liquid storage cavity layer set gradually from top to bottom, runner
Layer and reaction cavity layer；
The liquid storage cavity layer is provided at least one liquid storage chamber (4)；
The reaction cavity layer is provided at least one reaction chamber (2)；
The flow channel layer is provided with microchannel (8), for being connected to liquid storage chamber (4) and reaction chamber (2)；
Detection reagent is housed in the liquid storage chamber (4)；
The detection reagent includes A β -40 capture antibody coupling magnetic particle, A β -42 capture antibody coupling magnetic particle, cleaning solution, A
β -40 detects the combination of any one or at least two in antibody, A β -42 detection antibody or reaction solution.
2. reagent strip according to claim 1, which is characterized in that the reagent strip (1) further includes at least one sealing plug
(3), it is loaded into liquid storage chamber (4)；
Preferably, at least one buckle (6), the inner surface of the liquid storage chamber (4) are provided on the outer surface of the sealing plug (3)
On be provided at least two card slots (7), by connect with buckle (6) limit sealing plug (3) position；
Preferably, the length of the reaction cavity layer is not less than the length of the liquid storage cavity layer.
3. reagent strip according to claim 1 or 2, which is characterized in that the reaction cavity layer is provided with partition (9), described
The height of partition (9) is less than the height of the reaction cavity layer；
Preferably, the upper surface of the partition (9) includes any one in plane, inclined-plane or curved surface, preferably inclined-plane.
4. reagent strip according to claim 1-3, which is characterized in that the bottom surface of the reagent strip (1) is provided with
Locating slot (5), for the reagent strip (1) to be fixed on detecting instrument.
5. reagent strip according to claim 1-4, which is characterized in that the volume of the liquid storage chamber (4) is 10-
500μL；
Preferably, the volume of the reaction chamber (2) is 10-500 μ L.
6. reagent strip according to claim 1-5, which is characterized in that the reagent strip (1) uses macromolecule material
Material injection molding is formed；
Preferably, the high molecular material includes acrylonitrile-butadiene-styrene copolymer, polymethyl methacrylate or poly-
In ethylene any one or at least two combination, preferably polymethyl methacrylate.
7. reagent strip according to claim 1-6, which is characterized in that the detection reagent is loaded on sealing liquid storing bag
In；
Preferably, the sealing liquid storing bag uses any one in plastic film, aluminium foil or aluminium plastic composite material or at least two
The combination of kind is prepared, and is preferably prepared using aluminium plastic composite material；
Preferably, the detection reagent includes that A β -40 captures antibody coupling magnetic particle, A β -42 captures antibody coupling magnetic particle, clear
The combination of washing lotion, A β -40 detection antibody, A β -42 detection antibody and reaction solution；
Preferably, the partial size of the magnetic particle is 0.1-10 μm.
8. reagent strip according to claim 1-7, which is characterized in that the detecting instrument includes that power squeezes dress
Set, in magnetic particle mobile controller or signal supervisory instrument any one or at least two combination, preferably power squeeze
The combination of pressure device, magnetic particle mobile controller and signal supervisory instrument.
9. a kind of if the described in any item reagent strips of claim 1-8 are in detection A β -40, taking by 1-20 amino acid residue
Generation, missing or addition and obtain with the active amino acid sequence of A β -40, A β -42 or by 1-20 amino acid residue
The application having in the active amino acid sequence of A β -42 for replacing, missing or adding and obtaining.
10. a kind of method of joint-detection A β -40 and A β -42, which is characterized in that the described method comprises the following steps:
(1) using the values of chemiluminescence such as the described in any item reagent strip detection A β -40 and A β -42 of claim 1-8；
(2) values of chemiluminescence of A β -40 is subjected to the joint-detection of A β -40 and A β -42 divided by the values of chemiluminescence of A β -42.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811000652.XA CN109142716A (en) | 2018-08-30 | 2018-08-30 | The detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811000652.XA CN109142716A (en) | 2018-08-30 | 2018-08-30 | The detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109142716A true CN109142716A (en) | 2019-01-04 |
Family
ID=64829182
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811000652.XA Pending CN109142716A (en) | 2018-08-30 | 2018-08-30 | The detection reagent item of joint-detection A β -40 and A β -42 a kind of and its application |
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| Country | Link |
|---|---|
| CN (1) | CN109142716A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334480A (en) * | 2020-05-18 | 2020-06-26 | 苏州仁端生物医药科技有限公司 | Hybridoma cell strain secreting anti-A β 1-42 monoclonal antibody and application thereof |
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