CN102539732B - Lateral flow device - Google Patents
Lateral flow device Download PDFInfo
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- CN102539732B CN102539732B CN201010609718.2A CN201010609718A CN102539732B CN 102539732 B CN102539732 B CN 102539732B CN 201010609718 A CN201010609718 A CN 201010609718A CN 102539732 B CN102539732 B CN 102539732B
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- diuron
- antibody
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- hapten
- conversion zone
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Abstract
The present invention relates to lateral flow device, comprising: (a) substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample and are partially moved to described conversion zone from described sample reception；B () is positioned at the diuron hapten of primary importance；And (c) is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance, described hapten and described anti-diuron antibody are arranged such that, when described fluid sample is applied in described sample reception part, to make described hapten, described antibody and described fluid sample at described conversion zone to converge from described sample reception part to described moving of conversion zone by this fluid sample.The invention still further relates to use this device to detect the method that whether there is diuron in fluid sample.Apparatus and method of the present invention allows to obtain scene at sample and detects sample rapidly.
Description
Technical field
The present invention relates to a kind of lateral flow device for detecting diuron, use this lateral flow device detection enemy's grass
Grand method and comprise described for detecting the test kit of the lateral flow device of diuron.
Background technology
Diuron (3-(3,4-Dichlorobenzene base)-1,1-dimethyl urea) is a member in aryl urea herbicide families, its
It is developed in early 1950s, and has been widely used agricultural since the sixties in 20th century.
Diuron is widely used in agricultural in many countries, these countries include the U.S., Australia, Europe and in
State.Herbicide uses as soil apholate (soil sterilent) on non-crops soil, industrial land, and on a small quantity
For the straw or like vegetable in vegetable, Rhizoma Solani tuber osi, Cotton Gossypii, Semen Maydis, beans, frumentum and orchard, vineyard and broad leaved weed are entered
The selective forward and backward emergent control of row.
About diuron, the toxicity people of the mankind are still known little about it.But, the diuron of Environmental Protection Agency suggestion
Health level in water is set to 10/1000000000ths parts (10 μ g/L).
Therefore, it is necessary to the residue monitored in commodity and Environment space.Additionally, in the U.S., meat and most of commodity
In the legal tolerance limit of diuron residue be 2/1000000ths to million/1000000th parts (mg/kg), and at such as Fructus Musae, Fructus Persicae and
In the commodity such as nut the most as little as 100 parts.
The standard method of analysis of detection diuron includes: spectrophotography, gas chromatography/mass spectrometry method (GC/MS) and efficient
Liquid chromatography (HPLC).But, methods of these detection diuron need expensive instrument, and need to have passed through instrument and make
Detect with the those of skill in the art of training.Additionally, the place of many diuron to be detected be typically Environment space or
It it is the place that can not have easy access to of detecting instrument.As a result, sample must be transported to the detection sampled field residing for detecting instrument
Institute, the most just can be measured.Accordingly, it would be desirable to the method for cheap, portable and effective detection diuron, the method allows at sample
Product obtain scene and detect sample rapidly.
Summary of the invention
A first aspect of the present invention provides the lateral flow device for detecting the diuron in sample, including:
A () substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample
It is partially moved to described conversion zone from described sample reception；
B () is positioned at the diuron hapten of primary importance；And
C () is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance,
Described hapten and described anti-diuron antibody are arranged such that when described fluid sample is applied in described sample
During product receiving portion, described half is made to resist from described sample reception part to described moving of conversion zone by this fluid sample
Antibody former, described and described fluid sample converge (combine) at described conversion zone.
A second aspect of the present invention provides the method that whether there is diuron in detection fluid sample, including:
-described fluid sample is put on lateral flow device, this device includes:
A () substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample
It is partially moved to described conversion zone from described sample reception；
B () is positioned at the diuron hapten of primary importance；And
C () is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance,
Described hapten and described antibody are arranged such that when described fluid sample is applied in described sample reception portion
Timesharing, makes described hapten, described by this fluid sample from described sample reception part to described moving of conversion zone
Antibody and described fluid sample converge at described conversion zone；
-provide time enough so that described hapten, described anti-diuron antibody and described fluid sample are described
Conversion zone converges；And
-observe described conversion zone to determine whether described anti-diuron antibody is combined with described diuron hapten.
When in fluid sample without diuron, described anti-diuron antibody is tied with described hapten at described conversion zone
Close, and when in test sample containing diuron, described anti-diuron antibody is at described conversion zone and described haptenic knot
Close and reduce.
A third aspect of the present invention provides the test kit including the lateral flow device described in first aspect.
Accompanying drawing explanation
Fig. 1 be each distribution mode for components of an embodiment of lateral flow device top view (on) and side view
Under ().
Fig. 2 is the perspective view of each distribution mode for components of an embodiment of lateral flow device.
Fig. 3 is obtained by using lateral flow device analysis diuron positive (left) and diuron negative sample (right)
The schematic diagram of result.
Fig. 4 A is for being shown with envelope antigen (hapten 4C-OVA or hapten 6C-OVA) being at war with property indirect ELISA
The figure of result.
Fig. 4 B is the figure of the result illustrating competitive Salmonella, and this figure demonstrates along with the concentration of coated antibody increases,
The combination of antibody is suppressed.
Fig. 4 C is the figure of the result being shown in the competitive ELISA under different pH.
Fig. 4 D is the result of antibody being at war with property Salmonella being shown with producing for hapten 4C-KLH
Figure.
Detailed description of the invention
In an aspect, the present invention relates to the lateral flow device for detecting the diuron in sample, including:
A () substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample
It is partially moved to described conversion zone from described sample reception；
B () is positioned at the diuron hapten of primary importance；And
C () is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance,
Described hapten and described anti-diuron antibody are arranged such that when described fluid sample is applied in described sample
During product receiving portion, described half is made to resist from described sample reception part to described moving of conversion zone by this fluid sample
Antibody former, described and described fluid sample converge at described conversion zone.
Primary importance is usually in substrate or intrabasement position.The second position is usually in substrate or intrabasement position
Put.Generally, primary importance is in substrate or intrabasement position, and the second position is in substrate or intrabasement position.The
Two positions and primary importance are separate.
When in fluid sample without diuron, anti-diuron antibody is combined with hapten at conversion zone, and when tested
Time in sample containing diuron, anti-diuron antibody reduces with haptenic combination at conversion zone.
As used herein, lateral flow device is a kind of device for detecting analyte, and wherein reagent is by mobile
Converge through substrate (generally by means of capillarity).
This lateral flow device uses competitive immunometric assay method detection diuron.In this respect, this device is in use
Make use of the ability that the diuron in sample can suppress this antibody to be combined with anti-diuron antibodies with hapten, from
And provide the assay method for detecting the diuron in sample.
Hapten and anti-diuron antibody are positioned in this device the position being separated from each other, and when being applied to sample reception
The fluid sample of part is when this sample reception is partially moved to conversion zone, and this hapten and anti-diuron antibody converge.?
This respect, fluid sample makes to be positioned at those reagent of conversion zone upstream from sample reception part to the movement of conversion zone
(such as, anti-diuron antibody) is transported to conversion zone.Conversion zone includes being positioned in substrate or intrabasement region, if
Without diuron in sample, the most anti-diuron antibody converges with hapten in this region, so that antibody can be with hapten
In conjunction with.In one embodiment, diuron hapten is positioned at conversion zone, and by anti-diuron antibody and fluid sample
Hapten, anti-diuron antibody and fluid sample is made to converge through substrate to moving of conversion zone.In this embodiment
In, sample makes the diuron (if present) in anti-diuron antibody and sample be transported to instead through the movement of substrate
Answer region.
Described device can include can be with the control antibodies of anti-diuron antibodies.Generally, conversion zone includes comparison
Antibody.Therefore, in one embodiment, conversion zone includes the enemy's grass being positioned at primary importance (herein also referred to as test zone)
Grand hapten and be positioned at the control antibodies of control site separate with this primary importance (herein also referred to as control zone), its
Middle fluid sample makes anti-diuron antibody at primary importance and diuron half from sample reception part to the movement of conversion zone
Antigen converges, and anti-diuron antibody is converged with control antibodies in control site.In this embodiment, fluid sample
Movement through substrate makes anti-diuron antibody and diuron (if present) be transported to conversion zone, in this reaction
Region, if anti-diuron antibody is not combined with diuron, then this anti-diuron antibody can be with the hapten phase of test zone
In conjunction with, and control antibodies is in control zone and anti-diuron antibodies.Whether diuron is there is regardless of in sample, anti-by comparison
The anti-diuron antibody that body combines all can be detected in control zone.
Generally, fluid sample moves through substrate by means of capillarity.
In one embodiment, diuron hapten is represented by Formulas I:
Wherein:
Protein in the group that X is formed by selecting free bovine serum albumin (BSA) and ovalbumin (OVA)；And
N is 3 to 5.
This diuron hapten has various form, including: X is OVA and n is 5；X is OVA and n is
3；X is BSA and n is 3；And X is BSA and n is 5.Generally, X be BSA and n value be 5.
So that the detection sensitivity of diuron reaches to be capable of detecting when the degree of as little as part per billion part of diuron,
X is usually BSA, and n is usually 5.Inventor it was unexpectedly found that, compared with when using other diuron hapten,
By the wherein X that uses shown in formula I in lateral flow device be BSA and n is the hapten of 5, lateral flow device
Sensitivity is greatly improved.In this respect, it was found by the inventors that by use wherein X shown in formula I be BSA also
And n is the hapten of 5, the amount of the diuron of in sample as little as part per billion part can be detected.
Inventor is also contemplated by, for sensitivity decrease up to when 10/1000000000ths parts, it is possible to use such as Formulas I institute
The following any hapten shown: X is BSA and n is 3；X is OVA and n is 3；X is OVA and n is 5.Therefore, invention Crinis Carbonisatus
Existing, by changing the haptenic chemical constitution of diuron used in this device, the dress with different sensitivity can be prepared
Put.The haptenic amount of diuron of primary importance it is positioned at by increase, it is possible to so that the sensitivity decrease of this device.
Anti-diuron antibodies diuron and diuron hapten.When diuron and anti-diuron antibodies,
This anti-diuron antibody cannot in conjunction with diuron hapten, or, the anti-diuron haptenic ability of antibodies diuron
Reduce.Generally, anti-diuron antibody produces for diuron immunogen.Diuron immunogen can be for shown in Formula II
Compound:
Wherein:
X1By the protein in the group that choosing free BSA, OVA and keyhole limpet hemocyanin (KLH) are formed；And
N is 3 to 5.
These diuron original various forms of immunity, including: X is BSA and n is 3；X is BSA and n is
5；X is KLH and n is 3；And X is KLH and n is 5.
Generally, X is KLH and n is 3.
Described substrate can be any material allowing liquid to move to conversion zone from sample reception part.In a kind of shape
In formula, substrate is multi-disc film, and these films contact with each other, thus allow liquid to move between film.Such as, sample reception part can
Thinking sample reception pad, it contacts with antibody reagent pad, and antibody reagent pad and then (conversion zone is positioned at this can with permeable membrane
On permeable membrane) contact.Generally, sample reception pad, antibody reagent pad and permeable membrane are arranged to allow liquid from sample reception
Pad and flow to permeable membrane via antibody reagent pad.Described substrate may also include generally and permeable membrane be positioned at antibody reagent pad
The liquid storage pad that contacts of the region of far-end.Described liquid storage pad is arranged to receive hapten, antibody and fluid sample in reaction
Region converge after fluid sample.Permeable membrane can be to allow liquid to move to another position from a position (generally
Move by means of capillarity) any film.Sample reception pad, antibody reagent pad and permeable membrane can be all by following
The fibrous membrane that material is made, described material for example, glass fibre, cellulose, polyester, Cotton Gossypii, spun polyethylene and nitrocellulose
Element.Described sample reception pad is usually glass fibre.Described antibody pad is usually glass fibre.Glass fibre can be from (such as) position
Gold-Bio company limited in Shanghai City, China obtains.Described permeable membrane is usually nitrocellulose filter.
Any label allowing detection antibody can be used to carry out antibody described in labelling.It is, for example possible to use Radix Cochleariae officinalis peroxide
Compound enzyme, phosphatase, fluorescence labels (fluorescent tag), colloid gold label thing are marked.Generally, gold colloidal is used
Label carrys out antibody described in labelling.Colloid gold label thing can directly be observed, without adding other reagent, therefore keeping away
Exempt to need to use extra developing agent.Thus, use colloid gold label thing can allow to enter quickly and easily in this device
Row detection.
In order to contribute to the arrangement of the embodiment understanding that the present invention, Fig. 1 illustrate lateral flow device (11)
Side view.In this embodiment, substrate (12) includes sample reception pad (13), antibody reagent pad (14), permeable membrane
And liquid storage pad (17) (15).Sample reception pad (13) is generally by such as Cotton Gossypii, glass fibre, cellulose, polyester, nitrocellulose
The fibrous materials such as element or nylon are constituted.Sample reception pad (13) contacts with antibody reagent pad (14).Antibody reagent pad (14) is permissible
Formed by arbitrarily pervious material such as such as Cotton Gossypii, glass fibre, cellulose, polyester, nitrocellulose or nylon etc..Antibody reagent
Pad (14) comprises labeled anti-diuron antibody.Anti-diuron antibody for for diuron immunogen (as shown in above-mentioned Formula II
Compound) and the antibody that produces.Generally, anti-diuron antibody is for the wherein X shown in Formula II1It is 3 for KLH and n
Immunogen and produce.Any label allowing to detect anti-diuron antibody in this device can be used to carry out this antibody of labelling.
Described label can be metal particle, enzyme, chromogenic substrate, chromophore, fluorescence or chemiluminescent molecule.For example, it is possible to peppery
Antibody described in root peroxidase (HRP), phosphatase or colloid gold label.Generally, with antibody described in colloid gold label.By labelling
Thing is well known in the art with the method for antibody coupling.By the method for colloidal gold labeled monoclonal antibody at (such as) document Peng etc.
(2007) Intern.J.Environ.Anal.Chem.87:275-283 is described.Antibody reagent pad (14) and Permeability
The end thereof contacts of film (15).Permeable membrane has the conversion zone (16) being positioned at antibody reagent pad downstream.Permeable membrane can be by
Arbitrarily pervious material such as such as Cotton Gossypii, glass fibre, polyester, cellulose, nitrocellulose or nylon etc. is formed.Permeable membrane
(15) generally formed by nitrocellulose.Permeable membrane is generally elongated, for example, the form of band.Permeability liquid storage pad
(17) generally contact with the other end of permeable membrane.Liquid storage pad is generally by such as Cotton Gossypii, glass fibre, polyester, cellulose, nitrification
The sorptive material such as cellulose or nylon is formed.
Conversion zone (16) is positioned between antibody reagent pad and liquid storage pad (17), and it includes test zone (18), logical
Often also include control zone (19).Test zone generally comprises the diuron hapten being arranged on this film or in this film.Enemy's grass
Grand hapten is generally arranged to pattern or the shape that can be easily detected by the user of this device.Such as, enemy's grass
Grand hapten can be arranged across the form of the line or belt (18) of permeable membrane.Diuron hapten is normally held in this
On film.The method being fixed on permeable membrane by antigen is well known in the art.Such as, can be by such as document Peng etc.
(2007), as described in Intern.J.Environ.Anal.Chem.87:275-283, the hapten on permeable membrane is done
Dry, thus this hapten is fixed on permeable membrane.The method being fixed on permeable membrane by antigen is special in (such as) U.S.
Profit No.7,736,890 is also described.
Control zone (19) is usually located at the downstream (closer to liquid storage pad) of test zone, and comprises for combining against the enemy
The control antibodies of the grand antibody of grass.Such as, if anti-diuron antibody is rabbit antibody, control antibodies is then that anti-rabbit antibody is (such as goat-anti
Rabbit antibody).Control antibodies is generally arranged to pattern or the shape that can be easily detected by the user of this device.Example
As, control antibodies can be arranged across the form of the line or belt (19) of permeable membrane.Control antibodies is normally held in can
On permeable membrane.The method being fixed on permeable membrane by antibody is well known in the art.Such as, can be by such as document Peng
As described in (2007) Intern.J.Environ.Anal.Chem.87:275-283, the comparison on permeable membrane is resisted
Soma is dry, thus control antibodies is fixed on permeable membrane.
Fig. 2 is to illustrate the sample reception pad (13) of lateral flow device, antibody reagent pad (14), permeable membrane (15) and storage
Fluid cushion (17) is relative to the figure of an embodiment of the arrangement of shell.Permeable membrane (15) is disposed in generally by plastics
On the solid support parts (30) formed.Permeable membrane is fixed in this support member by logical typical binders.Sample reception pad
(13) and the end thereof contacts of antibody reagent pad (14) and permeable membrane (15), another of liquid storage pad (17) and permeable membrane (15)
End in contact.Support member (30) has the projection (32) extended on surface from it, and this projection limits the transverse shifting of substrate.Institute
Stating the case member (33) that device includes generally being formed by plastics, it is positioned at the top of this film, and matches with support member (30)
Close, thus case member is locked on substrate and the correct position of surrounding.Case member includes sample window (34) and observes
Window (35).Sample window (34) is aligned and arranged on the top of sample reception pad (13), thus allows to be applied by sample by sample window
In sample pad.Observation window (35) is arranged on the top of conversion zone (16), thus is allowed in applying observed result after sample.
In use, by sample (such as food, beverage or environmental sample) to be tested is mixed in a liquid, thus make
Standby fluid sample, described liquid is usually water or is buffer, such as phosphate buffer (PBS, pH 7.5).By fluid sample
It is applied in sample reception pad (13) by sample window (34).Fluid sample by sample reception pad (13) be flowed into comprise against the enemy
On the antibody reagent pad (14) of the grand antibody of grass.In antibody reagent pad, fluid sample mixes with anti-diuron antibody, and liquid
The mixture of sample and anti-diuron antibody flows into permeable membrane by means of capillarity towards the direction of conversion zone (16)
(15) in.In conversion zone (16), sample contacts with test zone (18) with anti-diuron antibody.In this region, if against the enemy
The grand antibody of grass not yet combines diuron, then this antibody can be combined with the diuron hapten being fixed on test zone.This side
Face, if containing diuron in sample, the most anti-diuron antibody can be in conjunction with the diuron in sample, thus itself and test zone
The haptenic combination of diuron can hindered or emulative suppression.In this case, can't detect at test zone
The combination of antibody, or the combination of at least antibody can weaken.If without diuron in sample, the most anti-diuron antibody can be freely
Ground is combined with the diuron hapten of test zone.Therefore, in this case, anti-diuron can be detected at test zone
Antibody and the haptenic combination of diuron.Then, the mixture of sample and antibody flows to control zone by means of capillarity,
At this region, anti-diuron antibody is combined by control antibodies.As a result, it is possible to detect in control zone and be combined with control antibodies
Anti-diuron antibody.Anti-diuron antibody is combined this by control antibodies and shows, the anti-diuron antibody of detectable amount by means of
Capillarity has moved through permeable membrane from antibody reagent pad.
The anti-diuron antibody being combined with diuron hapten can be detected by detection antibody labeling thing.Therefore, make
With in the embodiment of the anti-diuron antibody of colloid gold label, can the gold of visual detection and anti-diuron antibodies, and
Without adding developing agents.Alternatively, if anti-diuron antibody has such as horseradish peroxidase or fluorescent labeling
The label of thing etc, then can use suitable developing agents or observation condition to detect the antibody being attached on hapten.No
The developing agents of isolabeling thing and observation condition are to it known in the art, and can be easily commercially available from (such as) Millipore
Company's (being positioned at Massachusetts, United States), Promega company or BD Biosciences company.
Fig. 3 illustrates and applies a sample to the example of the rear result obtained on this device.Observation window (35) can be passed through observe
Antibody and haptenic combination.Such as, diuron hapten and control antibodies are being applied for crossing the form of the line of calibration tape
Time, if without diuron in sample, then antibody had not only been combined with test zone but also and the result of control zone combination can be revealed as
Cross two visual lines (18) and (19) of calibration tape.If containing diuron in sample, then can only see control line
(19), or p-wire not as control line the most obvious.
Only referring to following not limiting example, present invention is described below.
Example
A. the haptenic synthesis of diuron
In order to greatest extent to target molecule Selective recognition in addition, will act as immunogenic diuron hapten design is
At these aspects of structure, electrical properties and hydrophobic property and diuron the most completely the same (Harrison etc., 1991).Use Asia
Methyl carbochain at the end and middle nitrogen-atoms of urea part by diuron molecule derivatization so that it is abundant with carrier protein
Separately (Karu etc. (1994) J.Agric.Food Chem.42:301-309；Goodrow and Hammock (1998) Analytica
Chimica Acta, 376:83-91).
According to the method described in (1994) the J.Agric.Food Chem.42:301-309 such as document Karu, use N-
Described hapten is connected on protein by N-Hydroxysuccinimide reaction.
Prepare haptenic reactions steps as follows:
Step one: the preparation of spacerarm
Step 2: haptenic preparation
Step 3: the preparation of Acibenzolar
(0.17mmol hapten 4C/6C and 0.22mmol NHS is added into 2.2mL and does the DCC of THF and 0.22mmol
In)
Step 4: the preparation of haptenic protein conjugate
Hapten 4C-BSA:n=3, protein=BSA
Hapten 4C-KLH:n=3, protein=KLH
Hapten 6C-BSA:n=5, protein=BSA
Hapten 6C-KLH:n=5, protein=KLH
Hapten 4C-OVA:n=3, protein=OVA
Hapten 6C-OVA:n=5, protein=OVA
B. the preparation of polyclonal antibody
By 8 buck (1.5kg) for immunity.2 rabbit hapten 4C-KLH conjugate immunity, use hapten for 2
4C-BSA immunity, 2 immune with hapten 6C-KLH, and 2 immune with hapten 6C-BSA.
Conjugate (300 μ L) and physiological saline solution (1700 μ L) are mixed with 2mL complete Freund's adjuvant and (exempts from for the first time
Epidemic disease), thus inject with the dosage of every rabbit 1mg.A half-value dose was used to carry out booster injection every two weeks.
In using diuron and haptenic suppression test, the antibody produced for hapten immunogen is surveyed
Examination.The hapten used is hapten 6C-OVA.The result of titre and suppression test is as shown in table 1.
Table 1
ND: the undetermined because rabbit is dead
: suppression ratio is less than 20%.
Owing to using the antibody produced for hapten 4C-KLH to obtain the maximal percentage inhibition to diuron, therefore select
Select this antibody for lateral flow device.
C. antibody sensitivity evaluation
In order to determine the detection limit that diuron measures, use the antibody produced for hapten 4C-KLH, by competition
Property indirect ELISA obtain standard curve.Then use the higher competitive Salmonella of sensitivity obtain accurate detection limit and
Sensitivity.
Competitive indirect ELISA: by diuron 1mg/mL solution (at being stored in-20 DEG C) in methanol is carried out dilute
Release and prepare standard substance, and dilute in PBS.Use buffering at PBS of 6 kinds of concentration in the range of 0.04-10 μ g/L
Standard curve prepared by diuron in liquid.Fig. 4 A illustrates that the antibody of described antihapten 4C-KLH is coated by diuron from different
The standard curve of the inhibitory action of the combination of antigen (hapten 4C-OVA and hapten 6C-OVA).Being coated of both types is anti-
Former show equal excellent effect (suppression ratio under 0.48 μ g/L is 50%, and under 0.04 μ g/L and 0.05 μ g/L
Suppression ratio be respectively 15% (Fig. 4 A)).
Competitive Salmonella: use diuron 4C-horseradish peroxidase oxidase (HRP) conjugate in this test
As enzyme tracer.Use coated antibody (2 μ g/ holes, 1 μ g/ hole, 0.5 μ g/ hole, 0.25 μ g/ hole, the 0.1 μ g/ of 5 kinds of concentration
Hole).Result is as shown in Figure 4 B.Along with the change of the concentration of coated antibody, IC50The change that value is the biggest.Select 0.25 μ g/
The concentration in hole is as the concentration (Fig. 4 B) of coated antibody in final analysis.
About the pH value of PBS, in the competitive test using the antibody produced for hapten 4C-KLH
Test under the conditions of the pH of 4.5,5.5,6.5,7.5,8.5,9.5.Result is as shown in Figure 4 C.It has been found that acidity is the strongest
Condition is more conducive to competitive reaction.But, owing to sour environment may adversely affect by antagonist, thus select pH7.5
(Fig. 4 C).
Prepared by the diuron in PBS using six kinds of concentration in the range of 0.001ppb to 10ppb subsequently
Standard curve.Fig. 4 D illustrates that the intermediate point of the standard curve of diuron corresponds to 0.18ppb, and the suppression ratio of 15% corresponds to
0.03ppb。
Finding after optimizing further, Salmonella is the sensitiveest, and it produces the suppression of 50% under conditions of 0.18ppb
Rate, compared with 0.48 μ g/L in indirect ELISA test, the former is sensitiveer.
D. the optimization of belt one-step method test (One-Step Strip Assay)
The antibody produced for hapten 4C-KLH is used to carry out horizontal mobility test on nitrocellulose strips.Use
The sample of the diuron containing known quantity is fine to using different envelope antigen, colloid gold particle size, sample solvent and nitrifications
The sensitivity of the test that dimension element film is done detects.By calibration tape as depicted in figs. 1 and 2 and as already mentioned above cloth
Put.Goat anti-rabbit antibody is used as control antibodies.According to the method described in (2007) such as document Peng by diuron hapten, partly resist
Antibody and the control antibodies of former 4C-KLH put on permeable membrane.The preparation of test component and the result general introduction obtained
As follows.
I. gold colloidal
The selection of gold grain size: according to 1982 (5nm) institutes such as document Frens 1973 (＞ 10nm) and Tschopp
Stating, preparation has the gold colloidal of different-grain diameter.According to (2007) such as document Peng Suo Shu, will produce for hapten 4C-KLH
Antibody colloid gold label.Various sizes of gold colloidal is tested.Result shows the glue that mean diameter is 17nm (G17)
Body gold best results.
Ii. envelope antigen
Haptenic selection: according to documentDeng (2004), M.H.Goodrow, B.D.Hammock (1998) institute
The method stated, uses NHS or EDC reaction to be combined with OVA and BSA respectively by hapten 4C and hapten 6C, is coated anti-with preparation
Former.In the case of using the hapten 6C being combined by NHS reaction with BSA, can detect that the diuron of concentration as little as 1ppb.
Iii. film
Obtain following nitrocellulose (NC) film, and the situation that they are used as film in testing detect:
● HF090,6cm x 30cm (8 μm) and HFB18002 2.5cm x 100 meters (5 μm), it derives from and is positioned at the U.S.
The Millipore company in Bedford city；
● YN1801F, it derives from this locality；
● CNPF-SN12,25mm (8 μm) and CNPF-SN12,25mm (5 μm), it derives from MDI company and (is positioned at India
Ambala Cantt city).
● to deriving from Millipore 135s (8 μm) and 180s (5 μm), MDI (8 μm and 5 μm) and YN 180s (China
Manufacture) NC film detect.
Select Millipore 180s, because while the speed of its release mark antibody is slightly slower than 135s, but but have
Higher sensitivity.
Iv. sample solvent
Following sample solvent is detected:
■ deionized water, PBS, PBST
0.1% (v/v) methanol
■ PBS/PBST+1% (v/v) methanol
10% (v/v) methanol
0.1% (v/v) ethanol
■ PBS/PBST+1% (v/v) ethanol
10% (v/v) ethanol
0.1% (v/v) isopropanol
■ PBS/PBST+1% (v/v) isopropanol
10% (v/v) isopropanol
The sample being dissolved in PBS produces more preferable color intensity gradient.Therefore select PBS as sample solvent.
It has been found that be required for casein, skimmed milk or other sealers to close nitrocellulose filter.
V. brief summary
Anti-diuron antibody: antihapten 4C-KLH
Gold grain size: G17
Stabilizer: BSA and PEG
Envelope antigen: BSA-hapten 6C
NC film: untight HFB 18002 (Millipore company)；
Sample solvent: PBS
Control antibodies: goat anti-rabbit igg
Use said components, in the sample containing as little as part per billion part (ppb) diuron of concentration, detected enemy
Grass is grand.
In the above description of described claims and the present invention, unless due to clear and definite language or necessity
Implying and context is required otherwise, otherwise word " comprises " or its modification (such as " including " or " having ") is all with containing of including
Justice uses, i.e. in the different embodiment of the present invention, which defines and should there is described feature, but it is not excluded that also
There is or be added with other feature.
Claims (20)
1. for detecting a lateral flow device for the diuron in sample, including:
A () substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample from institute
State sample reception and be partially moved to described conversion zone；
B () is positioned at the diuron hapten of primary importance；And
C () is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance,
Wherein said primary importance is in substrate or intrabasement position, and the wherein said second position is in substrate or intrabasement
Position,
Wherein said conversion zone includes described primary importance, is wherein applied in described sample reception portion when described fluid sample
Timesharing, makes described hapten, described against the enemy by this fluid sample and anti-diuron antibody to described moving of conversion zone
The grand antibody of grass and described fluid sample converge at described conversion zone,
Wherein said anti-diuron antibody is labeled, thus allows to detect described antibody with described haptenic combination,
Wherein said diuron hapten is the compound shown in Formulas I:
Wherein:
X is bovine serum albumin (BSA) and n is 3 or 5；Or X is ovalbumin (OVA) and n is 3 or 5,
And wherein said device is 10/1000000000ths parts for the detection sensitivity of the described diuron in described sample.
2. the device described in claim 1, wherein when in described fluid sample without diuron, described anti-diuron antibody exists
Described conversion zone is combined with described hapten, and when in described fluid sample containing diuron, described anti-diuron antibody
Reduce with described haptenic combination at described conversion zone.
3. the device described in claim 1 or 2, wherein said fluid sample moves through described base by means of capillarity
The end.
4. the device described in claim 1, wherein said conversion zone includes the diuron hapten being positioned at described primary importance
And it being positioned at the control antibodies of control site, described control antibodies can be with described anti-diuron antibodies, wherein said liquid
Body sample makes described anti-diuron antibody in described primary importance from described sample reception part to the movement of described conversion zone
Converge with described diuron hapten, and make described anti-diuron antibody converge with described control antibodies in described control site
Close.
5. the device described in claim 1, wherein X is BSA, and n is 5.
6. the device described in claim 1, wherein said anti-diuron antibody is to produce for the compound shown in Formula II:
Wherein:
X1By the protein in the group that choosing free BSA, OVA and keyhole limpet hemocyanin (KLH) are formed；And
N is 3 to 5.
7. the device described in claim 6, wherein X1For KLH, and n is 3.
8. the device described in claim 1, wherein said substrate is permeable membrane.
9. the device described in claim 8, wherein said permeable membrane is nitrocellulose.
10. the device described in claim 1, wherein colloid gold particle is used as label.
11. 1 kinds are detected the method that whether there is diuron in fluid sample, including:
-described fluid sample is put on lateral flow device, this device includes:
A () substrate, this substrate includes that sample reception part and conversion zone, described substrate are configured to allow for fluid sample from institute
State sample reception and be partially moved to described conversion zone；
B () is positioned at the diuron hapten of primary importance；And
C () is positioned at the anti-diuron antibody of the second position, the described second position is separate with described primary importance,
Wherein said primary importance is in substrate or intrabasement position, and the wherein said second position is in substrate or intrabasement
Position,
Wherein said conversion zone includes described primary importance,
Wherein when described fluid sample is applied in described sample reception part, by this fluid sample and anti-diuron antibody
Make described hapten, described anti-diuron antibody and described fluid sample in described reaction to described moving of conversion zone
Region is converged；
Wherein said anti-diuron antibody is labeled, thus allows to detect described antibody with described haptenic combination,
Wherein said diuron hapten is the compound shown in Formulas I:
Wherein: X is bovine serum albumin (BSA) and n is 3 or 5；Or X is ovalbumin (OVA) and n is 3 or 5,
And wherein said device is 10/1000000000ths parts for the detection sensitivity of the described diuron in described sample；
-provide time enough so that described hapten, described anti-diuron antibody and described fluid sample are in described reaction
Region is converged；And
-observe described conversion zone to determine whether described anti-diuron antibody is combined with described diuron hapten.
Method described in 12. claim 11, wherein when in described fluid sample without diuron, described anti-diuron antibody
Be combined with described hapten at described conversion zone, and when in described fluid sample containing diuron, described anti-diuron resists
Body reduces with described haptenic combination at described conversion zone.
Method described in 13. claim 12, wherein said fluid sample moves through described base by means of capillarity
The end.
Method described in 14. claim 11, wherein said conversion zone includes that the diuron half being positioned at described primary importance is anti-
Former and be positioned at the control antibodies of control site, described control antibodies can be wherein said with described anti-diuron antibodies
Fluid sample makes described anti-diuron antibody at described first from described sample reception part to the movement of described conversion zone
Put and converge with described diuron hapten, and make described anti-diuron antibody converge with described control antibodies in described control site
Close.
Method described in 15. claim 11, wherein X is BSA, and n is 5.
Method described in 16. claim 11, wherein said anti-diuron antibody is to produce for the compound shown in Formula II
:
Wherein:
X1By the protein in the group that choosing free BSA, OVA and keyhole limpet hemocyanin (KLH) are formed；And
N is 3 to 5.
Method described in 17. claim 16, wherein X1For KLH, and n is 3.
Method described in 18. claim 11, wherein said substrate is permeable membrane.
Method described in 19. claim 18, wherein said permeable membrane is nitrocellulose.
Method described in 20. claim 11, wherein colloid gold particle is used as label.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010609718.2A CN102539732B (en) | 2010-12-20 | 2010-12-20 | Lateral flow device |
| AU2011349119A AU2011349119B2 (en) | 2010-12-20 | 2011-12-20 | Lateral flow device for the detection of diuron |
| PCT/AU2011/001645 WO2012083356A1 (en) | 2010-12-20 | 2011-12-20 | Lateral flow device for the detection of diuron |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201010609718.2A CN102539732B (en) | 2010-12-20 | 2010-12-20 | Lateral flow device |
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| CN102539732B true CN102539732B (en) | 2016-08-17 |
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| CN114317451B (en) * | 2022-01-18 | 2023-08-22 | 江南大学 | Hybridoma cell strain secreting diuron monoclonal antibody, and preparation method and application thereof |
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