CN207440104U - Detect the remaining immune chromatography test paper of glyphosate and kit - Google Patents
Detect the remaining immune chromatography test paper of glyphosate and kit Download PDFInfo
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- CN207440104U CN207440104U CN201721236734.5U CN201721236734U CN207440104U CN 207440104 U CN207440104 U CN 207440104U CN 201721236734 U CN201721236734 U CN 201721236734U CN 207440104 U CN207440104 U CN 207440104U
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Abstract
The utility model provides a kind of remaining immune chromatography test paper of detection glyphosate and kit, it is related to glyphosate detection technique field, the detection remaining immune chromatography test paper of glyphosate provided by the utility model, including being equipped with the bonding pad of glyphosate specific antibody and equipped with detection trace and the blotting membrane for compareing trace.The detection remaining immune chromatography test paper of glyphosate provided by the utility model is based on competitiveness enzyme-linked immunity principle, on the basis of glyphosate specific antibody is obtained, the highly sensitive detection of glyphosate is realized using the efficient amplification of enzyme linked immunoassay, can detect the glyphosate of the 1 following concentration of μ g/mL.The immune chromatography test paper of the utility model is easy to operate, it is sensitive it is special, result is accurate and visual, suitable for the remaining rapid screening of the several samples glyphosate such as water quality, food, application easy to spread.
Description
Technical field
The utility model is related to glyphosate detection technique field, more particularly, to a kind of detection remaining immune layer of glyphosate
Analyse test paper and kit.
Background technology
Glyphosate (Glyphosate) is Monsanto Chemicals to go out natural disposition water solubility in a kind of wide spectrum of exploitation in 1971
Herbicide, effectively inhibition that can be non-selective is a variety of annual or perennial weeds.The entitled N- phosphine carboxymerhyls of glyphosate chemistry
Glycine (N-Phosphonomethyl-glycine, PMG), trade name glyphosate, agriculture up to etc., chemical formula C3H8NO5P,
Molecular weight is 169.
With the rapid development of modern agriculture, the use of pesticide is more and more extensive, and pesticide residue is to environment, animals and plants and people
It threatens caused by body health and increasingly highlights.Glyphosate has become at present using one of most extensive, dosage maximum herbicide.Though
The toxicity of right glyphosate is relatively low, but long-term a large amount of uses can be constantly enriched in environmental and biological materials, and is entered by food chain
Human body causes potential threat to health.Existing research confirms that glyphosate has certain animal toxicity effect.Therefore, very
The residue limits of glyphosate are all included in relevant laws and regulations and standard by more countries and regions, the drinking water standard in China and the U.S.
In all provide the maximum residue limit of glyphosate as 0.7mg/L, the national food safety standard of China's revision in 2014《In food
Pesticide maximum residue limit》(GB2763-2014) maximum residue limit of the glyphosate in food has been made specifically in
Regulation.
In recent years, research hotspot is increasingly becoming for the remaining detection research of glyphosate, detection side more mature at present
Method includes gas-chromatography, liquid chromatogram and joint technology, but these conventional methods all exist in sample treatment, on analysis time
Some shortcomings are unsuitable for high-volume rapid screening and popularization and application.
Therefore, a kind of remaining rapid detection method of sensitive special, easy timesaving glyphosate of exploitation is particularly important.
In view of this, it is special to propose the utility model.
Utility model content
First of the utility model is designed to provide a kind of detection remaining immune chromatography test paper of glyphosate, this practicality
New second is designed to provide the kit for including above-mentioned immune chromatography test paper, in the prior art to alleviate
It all comes with some shortcomings in sample treatment, on analysis time, is unsuitable for high-volume rapid screening and popularization and application, and for
The detecting instrument of glyphosate is expensive, it is complicated for operation the technical issues of.
The utility model provides a kind of detection remaining immune chromatography test paper of glyphosate, and the immune chromatography test paper includes
Adsorption layer and protective film；
The adsorption layer includes the sample pad that connects successively, bonding pad, blotting membrane and water absorption pad, and the protective film includes the
One protective film and the second protective film；
The bonding pad includes glyphosate specific antibody, and the blotting membrane includes detection trace and control trace；
First protective film is arranged in the sample pad and bonding pad, and second protective film is arranged on the water absorption pad
On；
First protective film is different with the second protective film color.
Further, the immune chromatography test paper further includes support plate；
The support plate is arranged below the adsorption layer, for bonding the adsorption layer.
Further, it is additionally provided with mark line on the protective film.
Further, the mark line is arranged between the sample pad and the bonding pad.
Further, arrow and Max printed words are additionally provided on one side protective film of the mark line close to sample pad.
Further, the detection trace is glyphosate carrier protein couplet object, and the control trace resists for anti-mouse IgG
Body；
The detection trace compares the spaced and parallel setting of trace with described, and it is left that the detection trace is arranged on the control trace
Side.
Further, the support plate is waterproof material.
Further, the bonding pad is glass fibre cotton or nylon membrane.
Further, the blotting membrane is nitrocellulose filter, pure cellulose film or carboxylated cellulose film.
In addition, the utility model additionally provides a kind of detection remaining kit of glyphosate, including above-mentioned immunochromatography
Test paper.
The detection remaining immune chromatography test paper of glyphosate provided by the utility model, including being equipped with glyphosate specific antibody
Bonding pad and equipped with detection trace and compare trace blotting membrane.Detection glyphosate provided by the utility model is remaining immune
Chromatographic test paper has the advantages that：
(1) it is sensitive, special.Immune chromatography test paper provided by the utility model is based on competitive immunization reaction principle, passes through
The rabbit source polyclonal antibody of Au colloidal nanoparticles mark realizes the residual of detection glyphosate.Au colloidal nanoparticles and antibody point
Van der Waals force between son between charge is combined, and the affinity of antibody is influenced small.Therefore, which has stronger specificity
Higher sensitivity, range estimation detection limit can reach 1g/mL or so.
(2) it is easy, quick.Using immune chromatography test paper provided by the utility model, detection knot is judged without instrument
Test paper need to only be inserted into test sample liquid, testing result is can determine that in 5 minutes by fruit.
(3) it is directly perceived, accurate.Immune chromatography test paper provided by the utility model with show the trace of colloidal gold itself red make
Feminine gender and positive mark for detection, as a result can bore hole directly differentiate, be less prone to erroneous judgement.
(4) it is economical and practical.Immune chromatography test paper provided by the utility model to batch samples can detect simultaneously,
Testing cost and detection time can be effectively reduced, expensive instrument is not required to be detected, can effectively reduce installation cost.
(5) convenient for popularization.The utility model test paper disclosure satisfy that inspection and quarantine, environmental monitoring, defend since its is easy to operate
The detection demand of multiple units such as raw supervision, manufacturing enterprise, strong applicability enable in particular to promote the use of in grass-roots unit.This reality
New approach is provided with residue detection of the immune chromatography test paper of new offer for glyphosate in the substances such as water quality and food,
It can be that guarantee environment detection and food inspection play a significant role, good economic benefit can be generated.
Description of the drawings
It, below will be right in order to illustrate more clearly of specific embodiment of the present invention or technical solution of the prior art
Specific embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, it describes below
In attached drawing be the utility model some embodiments, for those of ordinary skill in the art, do not paying creativeness
On the premise of work, other attached drawings are can also be obtained according to these attached drawings.
Fig. 1 is that the side view structure for the remaining immune chromatography test paper of detection glyphosate that the utility model embodiment 1 provides is shown
It is intended to；
Fig. 2 is the top view for the detection remaining immune chromatography test paper of glyphosate that the utility model embodiment 1 provides.
Icon：1- support plates；2- sample pads；3- bonding pads；4- blotting membranes；5- water absorption pads；6- detects trace；7- control prints
Mark；The first protective films of 81-；The second protective films of 82-；9- mark lines.
Specific embodiment
The technical solution of the utility model is clearly and completely described below in conjunction with attached drawing, it is clear that described
Embodiment is the utility model part of the embodiment, instead of all the embodiments.Based on the embodiment in the utility model, sheet
Field those of ordinary skill all other embodiments obtained without making creative work, belong to this practicality
Novel protected scope.The person that is not specified actual conditions in embodiment, the condition suggested according to normal condition or manufacturer carry out.Institute
Production firm person is not specified with reagent or instrument, is the conventional products that can be obtained by commercially available purchase.
, it is necessary to explanation in the description of the utility model, such as occur term " " center ", " on ", " under ", " left side ",
" right side ", " vertical ", " level ", " interior ", " outer " etc., indicated by orientation or position relationship be based on orientation shown in the drawings or
Position relationship is for only for ease of description the utility model and simplifies description rather than instruction or imply signified device or member
Part must have specific orientation, with specific azimuth configuration and operation, therefore it is not intended that limitation to the utility model.
In addition, such as there is term " first ", " second ", " the 3rd " are only used for description purpose, and it is not intended that instruction or implying opposite
Importance.
The remaining detection method of glyphosate uses gas-chromatography, liquid chromatogram and joint technology, but these sides more at present
Method is relatively complicated in sample treatment, and analysis time is longer, and equipment cost and testing cost are higher, and it is fast to be unsuitable for high-volume
Fast examination and popularization and application.
To solve the above problems, the utility model provides a kind of detection remaining immune chromatography test paper of glyphosate, including
Adsorption layer and protective film；
Adsorption layer includes the sample pad 2 to connect successively, bonding pad 3, blotting membrane 4 and water absorption pad 5, and protective film, which includes first, to be protected
81 and second protective film 82 of cuticula；
Bonding pad 3 includes glyphosate specific antibody, and glyphosate specific antibody is used for special with the antigen in sample to be tested
The opposite sex combines；
Blotting membrane 4 includes detection trace 6 and control trace 7, for showing whether there is glyphosate residual in sample to be tested；
First protective film 81 is arranged in sample pad 2 and bonding pad 3, and the second protective film 82 is arranged on water absorption pad 5；
First protective film 81 is different with 82 color of the second protective film, and the first protective film 81 is white, is conducive to clearly differentiate
Mark line 9 on film, the second protective film 82 is the common colors such as blueness or red, for prompting handgrip part.
In one preferred embodiment, the detection trace 6 on blotting membrane 4 and trace 7 is compareed to be arranged in parallel hidden
The distance between shape straight line trace or for other analogous shape traces, two kinds of traces are 0.5cm.
Wherein, bonding pad 3 for example can be, but be not limited to glass fibre cotton or nylon membrane.Blotting membrane 4 for example can be,
But be not limited to cellulose membrane, it is preferable that cellulose membrane for example can be, but be not limited to nitrocellulose filter, pure cellulose film or
Carboxylated cellulose film.
In one preferred embodiment, adsorption layer further includes sample pad 2 and water absorption pad 5；
Sample pad 2 connects with the bonding pad 3, for being in contact with sample to be tested；
Water absorption pad 5 connects with the blotting membrane 4, for absorbing testing sample solution.
Wherein, sample pad 2 for example can be, but be not limited to glass fibre cotton or nylon membrane.Water absorption pad 5 for example can be,
But it is not limited to the pure white filter paper that absorbs water.
In one preferred embodiment, immune chromatography test paper further includes support plate 1；
Support plate 1 is arranged below the adsorption layer, for bonding the adsorption layer.
Wherein, support plate 1 does not absorb water and there is adhesive on surface.
There is sample mark line 9 in the position of the protective film corresponding with 3 intersection of bonding pad of sample pad 2.
In one preferred embodiment, glyphosate specific antibody is the resistance glyphosate of Au colloidal nanoparticles mark
Rabbit source polyclonal antibody.
In one preferred embodiment, it is glyphosate carrier protein couplet object to detect trace 6, and control trace 7 is anti-
Mouse IgG antibody.
The utility model additionally provides application of the above-mentioned immune chromatography test paper in detection glyphosate residual.
In addition, the utility model additionally provides the preparation method of above-mentioned immune chromatography test paper, including：
Glyphosate specific antibody is arranged on bonding pad 3, by detection trace 6 and control 7 specking of trace in blotting membrane 4
On；Fix sample pad 2, bonding pad 3, blotting membrane 4 and water absorption pad 5, and covering protection film successively in support plate 1；
Preferably, glyphosate specific antibody is the glyphosate rabbit source polyclonal antibody of Au colloidal nanoparticles mark, is examined
Survey trace 6 is glyphosate carrier protein couplet object, and control trace 7 is anti-mouse IgG antibody.
In one preferred embodiment, the carrier protein of glyphosate carrier protein couplet object for example can be, but not
It is limited to bovine serum albumin(BSA), chicken egg white or hemocyanin.
Glyphosate carrier protein couplet object preparation method includes：
Glyphosate solution, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides solution and carrier is respectively configured
Protein solution；Glyphosate solution and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution are mixed and reacted
Afterwards, add in carrier protein solution to mix and react, obtain reaction product；After reaction product is dialysed, centrifuging and taking supernatant obtains
Glyphosate carrier protein couplet object.
In a preferred embodiment, glyphosate carrier protein couplet object preparation method includes：
(a) 2mg glyphosates are weighed, are dissolved with 500 μ L phosphate buffers (PBS)；
(b) 2mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides are weighed and are dissolved in 500 μ L PBS, so
Afterwards under conditions of magnetic agitation, its liquid is slowly added to react 10min in the glyphosate solution that step (a) obtains dropwise left
It is right；
(c) 3mg carrier proteins are weighed and are dissolved in 1000 μ L PBS, are then slowly added to dropwise under conditions of magnetic agitation
In above-mentioned mixed liquor, 3h is stirred to react under the conditions of being protected from light at 4 DEG C, then reaction product is dialysed in PBS, last 5000r/
Min centrifugations 5min takes supernatant, obtains glyphosate carrier protein couplet object.
In one preferred embodiment, the preparation method of glyphosate rabbit source polyclonal antibody includes：
Conjugate is prepared, experimental rabbit is immunized as immunogene using conjugate, takes a blood sample and purifies after the completion of immune
Antibody obtains glyphosate rabbit source polyclonal antibody.
In a preferred embodiment, the preparation method of glyphosate rabbit source polyclonal antibody includes：
(a) conjugate is prepared and as immunogene according to above-mentioned conjugate preparation method；
(b) cleaning grade New Zealand White Rabbit is chosen, is immunized using the subcutaneous multi-point injection of neck, immunizing dose is 200 μ g/
ML/ only, is immunized 4 times altogether, every minor tick 2-4 weeks.First immunisation is helped completely after immunogene is diluted with PBS with isometric Freund
Agent mixes, and the fully emulsified 10min of 25000r/min are immunized New Zealand White Rabbit after solution becomes white emulsion, 2-4
Secondary to be immunized as booster immunization, booster immunization dosage is exempted from identical with head, and Freund's complete adjuvant only is changed to incomplete Freund's adjuvant.
(c) 10-30 days after the completion of immune programme, progress auricular vein or Culling heart blood, 2h under the conditions of being initially positioned at 37 DEG C,
6-8h under the conditions of being subsequently placed in 4 DEG C, 5000-10000r/min centrifuge 5-10min, take out supernatant.It is carried with saturated ammonium sulfate method
Pure antibody takes serum to add double PBS, then adds isometric saturated ammonium sulfate solution mixing, under the conditions of being placed in 4 DEG C overnight, then
5000-10000r/min centrifuges 5-10min, and a small amount of PBS of precipitation is dissolved, and then plus 1/2 volume saturated ammonium sulfate, mixing are put
Under the conditions of 4 DEG C overnight, then 5000-10000r/min centrifuges 5-10min, and a small amount of PBS of precipitation is dissolved, uses PBS dialysis
48~72h, changes liquid 5-10 times, and then 5000-10000r/min centrifuges 5-10min, and it is that glyphosate rabbit source is polyclonal to take supernatant
Antibody.
In one preferred embodiment, the preparation of the glyphosate rabbit source polyclonal antibody of Au colloidal nanoparticles mark
Method includes：
Colloidal gold solution is prepared, glyphosate rabbit source polyclonal antibody is added in colloidal gold solution and mixes and reacts, is centrifuged
After precipitated, precipitation is redissolved, centrifugation again obtains Au colloidal nanoparticles precipitation, by Au colloidal nanoparticles precipitation with four
Dobell's solution redissolves, and obtains the glyphosate rabbit source polyclonal antibody of Au colloidal nanoparticles mark.
In a preferred embodiment, the system of the glyphosate rabbit source polyclonal antibody of Au colloidal nanoparticles mark
Preparation Method includes：
The colloidal gold solution of 10~20nm of diameter is prepared first by sodium citrate, with the mark of the μ L/mL of 2 μ L/mL~3
Note amount adds in glyphosate rabbit source polyclonal antibody to be marked in the colloidal gold solution that pH is 8.5~9.5, after reacting 30min,
BSA the reactions 30min, 4 DEG C, 15000rpm centrifugation 20min of 10% (w/v) is added in, precipitation sodium tetraborate solution redissolves, again
Au colloidal nanoparticles precipitation sodium tetraborate solution is resuspended after centrifugation, obtains the glyphosate rabbit of Au colloidal nanoparticles mark
Source polyclonal antibody.
The detection remaining immune chromatography test paper of glyphosate provided by the utility model, the original that competitive immunization make use of to react
Reason, when the test lead of immune chromatography test paper, i.e., after sample pad 2 is inserted into testing sample solution, testing sample solution is made by siphon
It is spread together to blotting membrane 4 with the glyphosate in drive sample and the specific antibody in bonding pad 3, and eventually penetrates handle end
Water absorption pad 5.In diffusion process, the glyphosate in sample can be first combined with specific antibody, and then make specific antibody
It cannot be combined with the glyphosate carrier protein couplet analyte detection trace 6 of specking on blotting membrane 4, the glyphosate in sample is printed with detection
The glyphosate carrier protein couplet object competition of mark 6 is combined with glyphosate specific antibody, i.e. competitive immunoreaction pattern.If sample
Glyphosate in product is completely combined with specific antibody, does not show red detection 6 band of trace on cellulose membrane then；And blotting membrane 4
The anti-mouse IgG antibody control trace 7 of upper specking can still be combined with specific antibody, red control 7 band of trace of display, i.e., one
Red trace band, is expressed as the positive；Otherwise it when in sample solution without glyphosate, then cannot be combined with specific antibody, specificity
Antibody combines convenient for glyphosate carrier protein couplet object, and red trace band is shown at trace 6 detecting, and specking on blotting membrane 4
Anti-mouse IgG antibody control trace 7 can still be combined with specific antibody, show red control 7 band of trace, two red of formation
Band is expressed as feminine gender.
Immune chromatography test paper provided by the utility model based on competitive immunization reaction principle, passes through colloid gold nano
The rabbit source polyclonal antibody of grain mark realizes the residual of detection glyphosate, has stronger specific and higher sensitivity, mesh
It surveys detection limit and can reach 1g/mL or so.Using immune chromatography test paper provided by the utility model, judge to detect without instrument
As a result, only test paper need to be inserted into test sample liquid, testing result is can determine that in 5 minutes, it is easy, quick.Also, this practicality
The immune chromatography test paper of new offer is to show feminine gender and positive mark of the trace of colloidal gold itself red as detection, as a result
Can bore hole directly differentiate, be less prone to erroneous judgement, it is directly perceived, accurate.
On the basis of above-mentioned advantageous effect, immune chromatography test paper provided by the utility model can also be to batch samples
Progress detects simultaneously, can effectively reduce testing cost and detection time, and expensive instrument is not required to be detected, can effectively be subtracted
Few installation cost, it is economical and practical.Since its is easy to operate, inspection and quarantine, environmental monitoring, health supervision, manufacturing enterprise disclosure satisfy that
Etc. the detection demand of multiple units, strong applicability enables in particular to promote the use of in grass-roots unit.It is provided by the utility model immune
Residue detection of the chromatographic test paper for glyphosate in the substances such as water quality and food provides new approach, can be that guarantee environment is examined
It surveys and food inspection plays a significant role, good economic benefit can be generated.
With reference to specific embodiment and experimental example, the utility model is described in further detail.
Embodiment 1
A kind of detection remaining immune chromatography test paper of glyphosate is present embodiments provided, as depicted in figs. 1 and 2, including：
Support plate 1, adsorption layer and protective film.
Support plate 1 is plastic glue strip, and there is adhesive on surface, for bonding adsorption layer.
Adsorption layer includes the sample pad 2, bonding pad 3, blotting membrane 4 and the water absorption pad 5 that connect successively.
Wherein, sample pad 2 is glass fibre cotton, for being in contact with sample to be tested；Bonding pad 3 is glass fibre cotton, is inhaled
Glyphosate rabbit source with Au colloidal nanoparticles mark is mostly anti-；Blotting membrane 4 is cellulose membrane, is specially nitrocellulose filter；
Water absorption pad 5 is absorbent filter.
Specking has stealthy detection trace 6 and stealthy control trace 7 on blotting membrane 4.Stealth detection trace 6 is carried by glyphosate
Body protein conjugate solution is made；Stealth control trace 7 is made of goat anti-mouse igg antibody solution.Stealth detection trace 6 and hidden
Shape control trace 7 is arranged in parallel.
Protective film is divided into the first protective film 81 and the second protective film 82.
First protective film 81 is the sample end protective film being covered in above sample pad 2 and bonding pad 3；Second protective film 82 is
The protective film being covered in above water absorption pad 5；Set the first protective film 81 different with the color of the second protective film 82, to distinguish.
In the present embodiment, mark line 9 is additionally provided on the first protective film 81, for prompting test paper insertion sample solution most
Deep position.The mark line 9 is arranged on sample pad 2 close to the position of bonding pad 3, and specifically, which is arranged on sample pad 2 and knot
It closes and is inclined on corresponding first protective film 81 of 3 intersections of pad at 2 one side 0.5cm of sample pad.
In the present embodiment, arrow and Max printed words are printed on the protective film of 2 one side of sample pad in mark line 9.
When using the detection remaining immune chromatography test paper of glyphosate provided in this embodiment, comprise the following steps：
Detect water quality：It draws 1mL samples to be tested to add in 9mL PBS, abundant mixing 10min, 10000r/min centrifugation 5-
10min takes supernatant for monitoring.
Operating method：By the sample end of the detection remaining immune chromatography test paper of glyphosate provided in this embodiment, i.e. sample
Pad 2 is inserted into sample to be tested, and insertion depth is no more than mark line 9, takes out immune chromatography test paper within about 10~20 seconds, level is put
It puts, observation judgement testing result in 5min.
Result judgement：(a) it is negative, i.e., the red trace of display two (detection trace 6 and the control trace 7) on blotting membrane 4,
It represents in test sample without or with the glyphosate less than detection limit content；(b) it is positive, i.e., one is shown on cellulose membrane
Red trace (control trace 7), represents to contain the glyphosate for being more than detection limit in test sample；(c) fail, i.e., in cellulose membrane
Upper no red zone is shown, then shows that test paper has failed.
Embodiment 2
A kind of detection remaining immune chromatography test paper of glyphosate is present embodiments provided, with the utility model embodiment 1
The difference is that sample pad 2 is made of nylon membrane, blotting membrane 4 uses pure cellulose film.
Operating method and result judgement are the same as the utility model embodiment 1.
Embodiment 3
A kind of detection remaining immune chromatography test paper of glyphosate is present embodiments provided, with the utility model embodiment 1
The difference is that stealthy control trace 7 is made with rabbit anti-mouse IgG antibody solution on blotting membrane 4, blotting membrane 4 uses carboxylic
Cellulose film.
Operating method and result judgement are the same as the utility model embodiment 1.
Embodiment 4
A kind of detection remaining immune chromatography test paper of glyphosate is present embodiments provided, with the utility model embodiment 1
The difference is that sample pad 2 is made of nylon membrane, stealth control trace 7 is with rabbit anti-mouse IgG antibody solution in blotting membrane 4
On be made.
Operating method and result judgement are the same as the utility model embodiment 1.
Embodiment 5
The preparation of the detection remaining immune chromatography test paper of glyphosate of the offer of above-described embodiment 1 to 4 is be provided
Method, including：
A, the preparation of glyphosate carrier protein couplet object
The carrier protein of glyphosate carrier protein couplet object is bovine serum albumin(BSA), and conjugate is made by following methods：
(1) 2mg glyphosates are weighed, are dissolved with 500 μ L phosphate buffers (PBS)；
(2) 2mg 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides are weighed and are dissolved in 500 μ L PBS, so
Afterwards under conditions of magnetic agitation, its liquid is slowly added to dropwise to react 10min or so in first step solution；
(3) 3mg carrier proteins are weighed and are dissolved in 1000 μ L PBS, are then slowly added to dropwise under conditions of magnetic agitation
In above-mentioned mixed liquor, 3h is stirred to react under the conditions of being protected from light at 4 DEG C, then reaction product is dialysed in PBS, last 5000r/
Min centrifugations 5min takes supernatant, obtains glyphosate carrier protein couplet object.
B, the preparation of glyphosate rabbit source polyclonal antibody
(1) immunogene is prepared according to above-mentioned conjugate preparation method；
(2) cleaning grade New Zealand White Rabbit is chosen, is immunized using the subcutaneous multi-point injection of neck, immunizing dose is 200 μ g/
ML/ only, is immunized 4 times altogether, every minor tick 2-4 weeks.First immunisation is helped completely after immunogene is diluted with PBS with isometric Freund
Agent mixes, and the fully emulsified 10min of 25000r/min are immunized New Zealand White Rabbit after solution becomes white emulsion, 2-4
Secondary to be immunized as booster immunization, booster immunization dosage is exempted from identical with head, and Freund's complete adjuvant only is changed to incomplete Freund's adjuvant.
(3) 10-30 days after the completion of immune programme, progress auricular vein or Culling heart blood, 2h under the conditions of being initially positioned at 37 DEG C,
6-8h under the conditions of being subsequently placed in 4 DEG C, 5000-10000r/min centrifuge 5-10min, take out supernatant.It is carried with saturated ammonium sulfate method
Pure antibody takes serum to add double PBS, then adds isometric saturated ammonium sulfate solution mixing, under the conditions of being placed in 4 DEG C overnight, then
5000-10000r/min centrifuges 5-10min, and a small amount of PBS of precipitation is dissolved, and then plus 1/2 volume saturated ammonium sulfate, mixing are put
Under the conditions of 4 DEG C overnight, then 5000-10000r/min centrifuges 5-10min, and a small amount of PBS of precipitation is dissolved, uses PBS dialysis
48~72h, changes liquid 5-10 times, and then 5000-10000r/min centrifuges 5-10min, and it is that glyphosate rabbit source is polyclonal to take supernatant
Antibody.
C, the preparation of sample pad 2
The material preparations such as glass fibre cotton or nylon membrane of sample pad 2, are cut into the band of wide 1.5cm specifications, are put into
After impregnating 30min in confining liquid, it is dried for standby.
D, the preparation of bonding pad 3
The colloidal gold solution of 10~20nm of diameter is prepared first by sodium citrate, with the mark of the μ L/mL of 2 μ L/mL~3
Note amount adds in glyphosate rabbit source polyclonal antibody to be marked in the colloidal gold solution that pH is 8.5~9.5, after reacting 30min,
BSA the reactions 30min, 4 DEG C, 15000rpm centrifugation 20min of 10% (w/v) is added in, precipitation sodium tetraborate solution redissolves, again
Au colloidal nanoparticles precipitation sodium tetraborate solution is resuspended after centrifugation.By the diluted colloidal gold labeled monoclonal antibody spray of proper proportion
Point takes in the item of the materials such as glass fibre cotton, and 4 DEG C of low-temperature vacuum dryings are spare.
E, the preparation of blotting membrane 4
The nitrocellulose filter of blotting membrane 4, pure cellulose film or carboxylated cellulose film are cut into the band of wide 1.5cm specifications,
With spray film instrument, different position distinguishes specking glyphosate carrier protein couplet object and anti-mouse IgG antibody, two kinds of prints on blotting membrane 4
The distance between mark is 0.5cm, and stealthy detection trace 6 and control trace 7 are formed after 37 DEG C of drying.
F, the assembling of immune chromatography test paper
Sample pad 2, bonding pad 3, blotting membrane 4, water absorption pad 5 are pasted in support plate 1, each 2~3mm of overlapped thereto, so
By slitting to be cut into the test paper of 3~4mm wide.
The testing principle of immune chromatography test paper provided by the utility model is：
The principle reacted using competitive immunization, when the test lead of immune chromatography test paper, i.e. sample pad 2 is inserted into sample to be tested
After solution, testing sample solution drives the glyphosate in sample and the specific antibody in bonding pad 3 together by siphonage
It is spread to blotting membrane 4, and eventually penetrates the water absorption pad 5 of handle end.In diffusion process, glyphosate in sample can first with specifically
Property antibody be combined, and then prevent specific antibody from on blotting membrane 4 specking glyphosate carrier protein couplet analyte detection print
Mark 6 combines, the glyphosate in sample and the glyphosate carrier protein couplet object competition of detection trace 6 and glyphosate specific antibody
With reference to i.e. competitive immunoreaction pattern.If the glyphosate in sample is completely combined with specific antibody, if on cellulose membrane not
Red detection 6 band of trace of display；And the anti-mouse IgG antibody control trace 7 of specking still can be with specific antibody knot on blotting membrane 4
It closes, shows red control 7 band of trace, i.e., one red trace band is expressed as the positive；Otherwise when in sample solution without glyphosate,
It cannot then be combined with specific antibody, specific antibody combines convenient for glyphosate carrier protein couplet object, is shown at detection trace 6
Show red trace band, and the anti-mouse IgG antibody control trace 7 of specking can still be combined with specific antibody on blotting membrane 4, be shown
Red control 7 band of trace, forms two red zones and is expressed as feminine gender.
Experimental example 1
By glyphosate standard items be configured to 125ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL and
The solution of six concentration of 4000ng/mL, is then detected with immune chromatography test paper provided by the utility model, on blotting membrane 4
Detection trace 6 develop the color situation it is as shown in the table：
| Glyphosate concentration | Detect the colour developing situation of trace 6 |
| 125ng/mL | It is red |
| 250ng/mL | It is red |
| 500ng/mL | Faint red |
| 1000ng/mL | It is colourless |
| 2000ng/mL | It is colourless |
| 4000ng/mL | It is colourless |
As can be seen from the table, when glyphosate concentration is more than 1000ng/mL (1 μ g/mL), the detection print of immune chromatography test paper
Mark 6 does not develop the color；During less than 1000ng/mL (1 μ g/mL), the detection trace 6 of immune chromatography test paper starts aobvious red.This illustrates to try
Paper has preferable detection result, and range estimation detection limit can reach 1 μ g/mL.
Experimental example 2
Using other common pesticides imine phosphate, demeton, parathion, metrifonate, malathion and carrier proteins as
Detectable substance, each standard of physical product are each configured to 1 μ g/mL, 5 μ g/mL, 25 μ g/mL, the solution of 125 tetra- concentration of μ g/mL,
Then it is detected with immune chromatography test paper, the colour developing of detection trace 6 on blotting membrane 4 is red, the results showed that test paper has
Stronger specificity.
In conclusion the detection remaining immune chromatography test paper of glyphosate provided by the utility model, simplicity swift to operate, inspection
It is specific good to survey, and high sensitivity, testing result is intuitively accurate, and batch samples is suitble to detect simultaneously, effectively reduces detection
Expense and detection time, it is economical and practical, meet the detection demand of multiple detection fields, it should suitable for high-volume rapid screening and popularization
With.
Finally it should be noted that：Various embodiments above is only to illustrate the technical solution of the utility model rather than it is limited
System；Although the utility model is described in detail with reference to foregoing embodiments, those of ordinary skill in the art should
Understand：It can still modify to the technical solution recorded in foregoing embodiments either to which part or whole
Technical characteristic carries out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is not made to depart from this practicality newly
The scope of each embodiment technical solution of type.
Claims (10)
1. a kind of detection remaining immune chromatography test paper of glyphosate, which is characterized in that the immune chromatography test paper includes adsorption layer
And protective film；
The adsorption layer includes the sample pad to connect successively, bonding pad, blotting membrane and water absorption pad, and the protective film, which includes first, to be protected
Cuticula and the second protective film；
The bonding pad includes glyphosate specific antibody, and the blotting membrane includes detection trace and control trace；
First protective film is arranged in the sample pad and bonding pad, and second protective film is arranged on the water absorption pad；
First protective film is different with the second protective film color.
2. immune chromatography test paper according to claim 1, which is characterized in that the immune chromatography test paper further includes support
Plate；
The support plate is arranged below the adsorption layer, for bonding the adsorption layer.
3. immune chromatography test paper according to claim 2, which is characterized in that be additionally provided with mark line on the protective film.
4. immune chromatography test paper according to claim 3, which is characterized in that the mark line is arranged on the sample pad and institute
It states between bonding pad.
5. immune chromatography test paper according to claim 4, which is characterized in that in the mark line close to the one side of sample pad
Arrow and Max printed words are additionally provided on protective film.
6. immune chromatography test paper according to claim 5, which is characterized in that the detection trace is glyphosate carrier protein
Conjugate, the control trace is anti-mouse IgG antibody；
The detection trace compares the spaced and parallel setting of trace with described, and the detection trace is arranged on the left of the control trace.
7. immune chromatography test paper according to claim 6, which is characterized in that the support plate is waterproof material.
8. immune chromatography test paper according to claim 7, which is characterized in that the bonding pad is glass fibre cotton or nylon
Film.
9. immune chromatography test paper according to claim 8, which is characterized in that the blotting membrane is nitrocellulose filter, pure
Cellulose membrane or carboxylated cellulose film.
10. a kind of detection remaining kit of glyphosate, which is characterized in that be immunized including claim 1-9 any one of them
Chromatographic test paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721236734.5U CN207440104U (en) | 2017-09-25 | 2017-09-25 | Detect the remaining immune chromatography test paper of glyphosate and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201721236734.5U CN207440104U (en) | 2017-09-25 | 2017-09-25 | Detect the remaining immune chromatography test paper of glyphosate and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN207440104U true CN207440104U (en) | 2018-06-01 |
Family
ID=62294659
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201721236734.5U Active CN207440104U (en) | 2017-09-25 | 2017-09-25 | Detect the remaining immune chromatography test paper of glyphosate and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN207440104U (en) |
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2017
- 2017-09-25 CN CN201721236734.5U patent/CN207440104U/en active Active
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