CN109633182A - Colloidal-gold detecting-card and preparation method thereof and hormone residues detection method - Google Patents
Colloidal-gold detecting-card and preparation method thereof and hormone residues detection method Download PDFInfo
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- CN109633182A CN109633182A CN201811617293.2A CN201811617293A CN109633182A CN 109633182 A CN109633182 A CN 109633182A CN 201811617293 A CN201811617293 A CN 201811617293A CN 109633182 A CN109633182 A CN 109633182A
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- gold
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention discloses the hormone residues detection method of a kind of colloidal-gold detecting-card and preparation method thereof and application colloidal-gold detecting-card, wherein, the colloidal-gold detecting-card includes support plate, sample pad, gold conjugation pad, nitrocellulose filter and water absorption pad, the sample pad, the gold conjugation pad, the nitrocellulose filter and the water absorption pad are sequentially arranged in the plate face of the support plate along the direction of first end to the second end of the detection card, and the nitrocellulose filter is provided with detection line and nature controlling line；The gold conjugation pad is coated with the anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody of colloid gold label.Technical solution of the present invention can be realized while detect the residual of male sex hormone and female hormone in aquatic products, to promote its detection efficiency.
Description
Technical field
The present invention relates to technical field of food detection, in particular to a kind of colloidal-gold detecting-card and preparation method thereof and answer
With the hormone residues detection method of the colloidal-gold detecting-card.
Background technique
In culture fishery, male sex hormone (such as Methyltestosterone) and female hormone (such as diethylstilbestrol) are usually made
For the specific medicament of seed rearing and sexual control etc..But residual of these hormones in aquatic products seriously threatens human body
Health, and to the harm of human body be it is chronic, at a specified future date and cumulative, its harm is mainly shown as: natural hormone in interfering bodies
Balance, makes the physiological function of normal person get muddled, newborn teratogenesis is also resulted in when serious, influences the normal growth hair of children
It educates.
Currently, colloidal-gold detecting-card is widely used in aquatic products because it has many advantages, such as simple, quick, effective, high sensitivity
The detection of hormone residues in product.But most of existing colloidal-gold detecting-card can only detect unitem, that is, detection is male
Separate detection is wanted in sex hormone (Methyltestosterone) and detection female hormone (diethylstilbestrol), then detection efficiency is lower.
Summary of the invention
The main object of the present invention is to provide a kind of colloidal-gold detecting-card, it is intended to realize while detect male in aquatic products and swash
The residual of element and female hormone, to promote its detection efficiency.
To achieve the above object, colloidal-gold detecting-card proposed by the present invention, including support plate, sample pad, colloidal gold combine
Pad, nitrocellulose filter and water absorption pad, the sample pad, the gold conjugation pad, the nitrocellulose filter and the suction
Water cushion is sequentially arranged in the plate face of the support plate, the nitrocellulose along the direction of first end to the second end of the detection card
Film is provided with detection line and nature controlling line；The anti-Methyltestosterone monoclonal that the gold conjugation pad is coated with colloid gold label is anti-
Body and anti-diethylstilbestrol monoclonal antibody.
Optionally, the detection line includes the first detection line and the second detection line, first detection line, second inspection
Survey line and the nature controlling line are set in turn in the nitrocellulose filter backwards along the direction of the first end to the second end
The surface of the water absorption pad.
Optionally, first detection line is coated with Methyltestosterone-chicken ovalbumin conjugate, second detection
Line is coated with diethylstilbestrol-chicken ovalbumin conjugate, and the nature controlling line is coated with sheep anti-mouse igg antibody.
Optionally, the support plate is polyvinyl chloride panel；And/or the material of the sample pad is glass fibre；And/or
The material of the gold conjugation pad is polyester film；And/or the water absorption pad is filter paper.
The invention also provides a kind of preparation method of colloidal-gold detecting-card, the preparation method the following steps are included:
The mixture for fighting Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody carries out colloid gold label,
Obtain anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label:
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody of the colloid gold label is coated on gold conjugation pad；
In the same direction successively by sample pad, coated gold conjugation pad, be coated with the nitre of detection line and nature controlling line
Acid cellulose film and water absorption pad are attached at the plate face of support plate, after cutting and are fitted into template the colloidal-gold detecting-card is made.
Optionally, described " obtaining anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of the colloid gold label " step includes
Following steps:
Taking granular size is the colloidal gold solution of 20nm-40nm, and the pH value of colloidal gold solution is adjusted to 7-9；
The mixing of anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody is added into colloidal gold solution
Object is stirred, and adds polyethylene glycol, is stirred and is centrifuged, discard supernatant liquid, obtains sediment；
Phosphate buffered saline solution is added to clean sediment, and uses the phosphate-buffered salt containing chicken ovalbumin
Solution dissolves sediment, and anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of the colloid gold label is obtained after filtering.
Optionally, the detection line includes the first detection line and the second detection line, by Methyltestosterone-chicken ovalbumin
Conjugate is coated on first detection line, and diethylstilbestrol-chicken ovalbumin conjugate is coated on second detection line；
Sheep anti-mouse igg antibody is coated on the nature controlling line.
Optionally, the Methyltestosterone-chicken ovalbumin conjugate is prepared by the following steps to obtain:
By Methyltestosterone and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, Methyltestosterone-chicken ovalbumin conjugate is obtained；
And/or the diethylstilbestrol-chicken ovalbumin conjugate is prepared by the following steps to obtain:
By diethylstilbestrol and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, diethylstilbestrol-chicken ovalbumin conjugate is obtained.
The invention also provides a kind of hormone residues detection methods, are applied to colloidal-gold detecting-card, hormone residues detection
Method the following steps are included:
Sample to be measured is pre-processed, sample liquid to be checked is obtained；
Sample drop to be checked is added to the sample pad of colloidal-gold detecting-card, stands 10mim-20mim, observes nitrocellulose
Whether the detection line and nature controlling line of film develop the color；
If detection line and nature controlling line develop the color, testing result is determined as feminine gender, shows in sample without containing aquatic products hormone
Or aquatic products hormone-content is lower than detection limit；
If detection line does not develop the color, nature controlling line colour developing, testing result is determined as the positive, shows aquatic products hormone-content in sample
It is limited equal to or higher than detection；
If nature controlling line does not develop the color, testing result is invalid, shows that incorrect operation or detection block failure out of date.
Optionally, described " sample to be measured being pre-processed, the sample liquid to be checked is obtained " step the following steps are included:
Remove skin bone fat sample, with homogenizer homogeneous uniform sample；
This liquid is sampled in test tube, deionized water is added and mixes, and ethyl acetate extraction is added, concussion, centrifugation discard
Layer clear liquid, lower liquid nitrogen or air blow drying；
Phosphate buffered saline solution is added into the test tube of drying, rinses the residue on dissolution inboard wall of test tube, obtains sample
Liquid to be checked.
Technical solution of the present invention, colloidal-gold detecting-card include support plate, sample pad, gold conjugation pad, cellulose nitrate
Plain film and water absorption pad, sample pad, gold conjugation pad, nitrocellulose filter and water absorption pad along detection card first end to second end
Direction be successively attached at the plate face of support plate, nitrocellulose filter is provided with detection line and nature controlling line.When in test sample liquid
When hormone residues, sample liquid is added dropwise to the surface of sample pad, and then, sample liquid is successively permeated in gold conjugation pad and nitric acid
Cellulose membrane, finally by water absorption pad pipette samples liquid.When sample liquid infiltration is when nitrocellulose filter, sample liquid is successively by inspection
Survey line and nature controlling line judge hormone residues situation in test sample liquid by the way that whether detection line and nature controlling line develop the color, and judge
Whether the testing result for detecting card is effective.Also, gold conjugation pad is coated with the anti-Methyltestosterone Dan Ke of colloid gold label
Grand antibody and anti-diethylstilbestrol monoclonal antibody, then the colloidal-gold detecting-card can be realized while detect male sex hormone in aquatic products
With the residual of female hormone, compared to the prior art in can only carry out single hormone test, detection efficiency greatly improves.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
The structure shown according to these attached drawings obtains other attached drawings.
Fig. 1 is the schematic cross-sectional view of one embodiment of colloidal-gold detecting-card of the present invention；
Fig. 2 is the structural schematic diagram at another visual angle of nitrocellulose filter in Fig. 1；
Fig. 3 is the structural schematic diagram of the cartridge of colloidal-gold detecting-card of the present invention.
Drawing reference numeral explanation:
| Label | Title | Label | Title |
| 100 | Colloidal-gold detecting-card | 411 | First detection line |
| 100a | First end | 412 | Second detection line |
| 100b | Second end | 43 | Nature controlling line |
| 10 | Support plate | 50 | Water absorption pad |
| 20 | Sample pad | 60 | Cartridge |
| 30 | Gold conjugation pad | 61 | Well |
| 40 | Nitrocellulose filter | 62 | Observation window |
| 41 | Detection line |
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its
His embodiment, shall fall within the protection scope of the present invention.
It in addition, the technical solution between each embodiment of the present invention can be combined with each other, but must be general with this field
Based on logical technical staff can be realized, it will be understood that when the combination of technical solution appearance is conflicting or cannot achieve this
The combination of technical solution is not present, also not the present invention claims protection scope within.
The present invention proposes a kind of colloidal-gold detecting-card 100, for detecting the hormone residues in aquatic products.
Referring to Fig.1 and 2, in 100 1 embodiment of colloidal-gold detecting-card of the present invention, the colloidal-gold detecting-card 100 packet
Include support plate 10, sample pad 20, gold conjugation pad 30, nitrocellulose filter 40 and water absorption pad 50, sample pad 20, colloidal gold knot
Pad 30, nitrocellulose filter 40 and water absorption pad 50 is closed to be sequentially arranged in along the direction of first end 100a to the second end 100b of detection card
The plate face of support plate 10, nitrocellulose filter 40 are provided with detection line 41 and nature controlling line 43；Gold conjugation pad 30 is coated with glue
The anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody of body gold label.
Specifically, the cross-sectional shape for detecting card is generally rectangular, has opposite first end 100a and second end
100b.Along first end 100a to the direction of second end 100b, the plate face of support plate 10, which is successively sticked, sample pad 20, colloidal gold knot
Pad 30, nitrocellulose filter 40 and water absorption pad 50 are closed, nitrocellulose filter 40 is provided with detection line backwards to the surface of support plate 10
41 and nature controlling line 43.When specifically used, sample liquid is added dropwise to the surface of sample pad 20, and then, sample liquid is successively permeated in glue
Body gold bonding pad 30 and nitrocellulose filter 40, finally by 50 pipette samples liquid of water absorption pad.When sample liquid is permeated in cellulose nitrate
When plain film 40, sample liquid successively line 41 and nature controlling line 43 after testing are sentenced by the way that whether detection line 41 and nature controlling line 43 develop the color
Hormone residues situation in disconnected test sample liquid, and judge whether the testing result of detection card is effective.Due to gold conjugation pad 30
Be coated with the anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody of colloid gold label, when sample liquid infiltration in
When gold conjugation pad 30, aquatic products hormone (Methyltestosterone or diethylstilbestrol) therein energy and colloidal gold in flow process
The monoclonal antibody specific of label combines, it is suppressed that the combination of the antigen in the detection line 41 of antibody and nitrocellulose enzyme,
So as to cause the variation of 41 shade of detection line, and judged in sample liquid according to the variation of 41 shade of detection line
The residual condition of Methyltestosterone or diethylstilbestrol.In this way, the colloidal-gold detecting-card 100 can be realized while detect in aquatic products
The residual of male sex hormone and female hormone.
Accordingly, it is to be understood that, technical solution of the present invention, colloidal-gold detecting-card 100 includes support plate 10, sample pad
20, gold conjugation pad 30, nitrocellulose filter 40 and water absorption pad 50, sample pad 20, gold conjugation pad 30, nitrocellulose
Film 40 and water absorption pad 50 are successively attached to the direction of second end 100b the plate face of support plate 10 along the first end 100a of detection card,
Nitrocellulose filter 40 is provided with detection line 41 and nature controlling line 43.When hormone residues in test sample liquid, sample liquid is added dropwise
To the surface of sample pad 20, then, sample liquid is successively permeated in gold conjugation pad 30 and nitrocellulose filter 40, finally by inhaling
50 pipette samples liquid of water cushion.When sample liquid is permeated in nitrocellulose filter 40, sample liquid successively line 41 and Quality Control after testing
Line 43 judges hormone residues situation in test sample liquid by the way that whether detection line 41 and nature controlling line 43 develop the color, and judges to detect
Whether the testing result of card is effective.Also, anti-Methyltestosterone-hexene that gold conjugation pad 30 is coated with colloid gold label is female
Phenol monoclonal antibody, then the colloidal-gold detecting-card 100 can be realized while detect male sex hormone and female hormone in aquatic products
Residual, compared to the prior art in can only carry out single hormone test, detection efficiency greatly improves.
Optionally, the both ends of gold conjugation pad 30 are overlapped with sample pad 20 and nitrocellulose filter 40 respectively, and are absorbed water
One end of pad 50 is overlapped in nitrocellulose filter 40, in this way, the sample drop for being added dropwise to sample pad 20 can smoothly and directly
Successively permeable colloids gold bonding pad 30 and nitrocellulose filter 40 shorten drop infiltration journey, so as to shorten detection time,
Improve detection efficiency.Water absorption pad 50 can rapidly draw the drop after detection simultaneously.
Preferably, gold conjugation pad 30 and support plate 10 are arranged in angle, and gold conjugation pad 30 and sample pad 20
The height of bond end is higher than the height of gold conjugation pad 30 and 40 bond end of nitrocellulose filter.So setting, is being added dropwise
Sample drop and lay flat detection card after, due to the gravity of drop, further accelerate sample drop in colloidal gold knot
The process of osmosis in pad 30 is closed, the time of detection is significantly shortened, to improve detection efficiency.
Further, detection line 41 includes the first detection line 411 and the second detection line 412, the first detection line 411, second
Detection line 412 and nature controlling line 43 are set in turn in nitrocellulose filter 40 backwards along the direction of first end 100a to second end 100b
The surface of water absorption pad 50.First detection line 411, the second detection line 412 and nature controlling line 43 are arranged in parallel, and along detection card
Width direction extend.First detection line 411, the second detection line 412 and nature controlling line 43 are along first end 100a to second end 100b
Direction be sequentially distributed, and the spacing of nature controlling line 43 and the second detection line 412 be greater than the first detection line 411 and the second detection line
412 spacing.So setting, can be effectively prevented from interference of the detection line 41 to nature controlling line 43.
In one embodiment of the invention, the first detection line 411 is coated with Methyltestosterone-chicken ovalbumin conjugate,
Second detection line 412 is coated with diethylstilbestrol-chicken ovalbumin conjugate, and nature controlling line 43 is coated with sheep anti-mouse igg antibody.The
For one detection line 411 for detecting Methyltestosterone residual therein, the second detection line 412 is residual for detecting diethylstilbestrol therein
It stays, nature controlling line 43 is used to judge whether the testing result of detection card to be effective.In this way, the colloidal-gold detecting-card 100 can be realized inspection
Survey the residual of Methyltestosterone and diethylstilbestrol in aquatic products.
Optionally, the material of support plate 10 is polyvinyl chloride.Support plate 10 is the polyvinyl chloride for being coated with adhesive sticker on one side
(PVC) plate, and adhesive sticker is to paste sample pad 20, gold conjugation pad 30, nitrocellulose filter 40 and water absorption pad 50.
Optionally, the material of sample pad 20 is glass fibre, and sample pad 20 is made of glass fibre.
Optionally, the material of gold conjugation pad 30 is polyester film.Polyester film is colorless and transparent film, chemical-resistant resistance
Property it is preferable and more environmentally friendly.
Optionally, water absorption pad 50 is filter paper.Filter paper water absorbing properties are preferable, and cost is relatively low.Of course, water absorption pad 50 is also
Other objects with water absorbing properties can be used.
In addition, colloidal-gold detecting-card 100 further includes cartridge 60, accommodating chamber, and the one of cartridge 60 are formed with inside cartridge 60
Surface is also provided with observation window 62 and well 61, and well 61 is connected with accommodating chamber.Sample pad 20, gold conjugation pad
30, nitrocellulose filter 40 and water absorption pad 50 are mounted in the accommodating chamber, and sample pad 20 is at least partly shown by well 61
Expose, nitrocellulose filter 40 is manifested by observation window 62.When needing test sample, sample drop is passed through into well 61 simultaneously
It is added dropwise to sample pad 20, the colour developing feelings of the detection line 41 and nature controlling line 43 on nitrocellulose filter 40 are then observed by observation window 62
Condition.
The invention also provides a kind of preparation method of colloidal-gold detecting-card 100, which is such as preceding institute
The colloid stated, specific structure are shown in above-described embodiment, and this is no longer going to repeat them.
The preparation method of colloidal-gold detecting-card 100 the following steps are included:
The mixture for fighting Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody carries out colloid gold label,
Obtain anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label；
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label is coated on gold conjugation pad 30；
In the same direction successively by sample pad 20, coated gold conjugation pad 30, be coated with detection line 41 and Quality Control
The nitrocellulose filter 40 and water absorption pad 50 of line 43 are attached at the plate face of support plate 10, after cutting and are fitted into template colloid is made
Gold detection card 100.
Specifically, anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody are sufficiently mixed, and
Mixture is subjected to colloid gold label, obtains anti-Methyltestosterone-diethylstilbestrol monoclonal antibody containing colloid gold label.It will
Anti- Methyltestosterone-diethylstilbestrol the monoclonal antibody for containing colloid gold label is coated on gold conjugation pad 30 backwards to support
The surface of plate 10.In the same direction successively by sample pad 20, be coated with anti-Methyltestosterone-diethylstilbestrol list of colloid gold label
The gold conjugation pad 30 of clonal antibody, the nitrocellulose filter 40 and water absorption pad 50 for being coated with detection line 41 and nature controlling line 43
It is attached at the one side that support plate 10 is coated with adhesive sticker.It should be understood that sample pad 20, gold conjugation pad 30, nitrocellulose
Film 40 and water absorption pad 50 are glued in support plate 10, of course, can also be by double-sided adhesive or glue come their splicing.Then,
The plate posted is cut into the item of 3mm-5mm wide with cutting machine, for example is cut into the item of 4mm wide, is fitted into template and colloid is made
Gold detection card 100, places into sealed storage in the aluminium foil bag with desiccant, spare to make.
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label is coated on gold conjugation pad by the present invention
30, when sample liquid is permeated in gold conjugation pad 30, aquatic products hormone (Methyltestosterone or diethylstilbestrol) therein is being flowed
It can be in conjunction with the monoclonal antibody specific of colloid gold label during dynamic, it is suppressed that the detection line of antibody and nitrocellulose enzyme
The combination of antigen on 41, so as to cause the variation of 41 shade of detection line, and according to the change of 41 shade of detection line
Change the residual condition to judge Methyltestosterone or diethylstilbestrol in sample liquid.In this way, the colloidal-gold detecting-card being prepared
100 can be realized while detecting the residual of male sex hormone and female hormone in aquatic products.
Further, the step that " obtains anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label " include with
Lower step:
Taking granular size is the colloidal gold solution of 20nm-40nm, and the pH value of colloidal gold solution is adjusted to 7-9；
The mixing of anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody is added into colloidal gold solution
Object is stirred, and adds PEG 20000, is stirred and is centrifuged, discard supernatant liquid, obtains sediment；
Phosphate buffered saline solution is added to clean sediment, and uses the phosphate-buffered salt containing chicken ovalbumin
Solution dissolves sediment, and anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label is obtained after filtering.
Specifically, colloidal gold preparation process are as follows: take 0.01% aqueous solution of chloraurate, heating is boiled, and is added as needed
1% trisodium citrate continues to boil 10min, and colloidal gold can be made after cooling, and the grain diameter of colloidal gold is 20nm-40nm.
And 0.1mol/L solution of potassium carbonate is added, its pH value is adjusted to 7-9, for example, pH value is adjusted to 8.Anti- methyl is added while stirring
The mixture of stosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody stirs 15min-30min, then is added dropwise
25mol/L PEG 20000 stirs 10min-20min.Then it is centrifuged 15min under the revolving speed of 2000r/min, discarded
Clear liquid, and the PBS buffer solution that pH7.4 is added generally, contains 0.4mg/ in the buffer solution for sediment undergoes washing 2-3 times
ML polyethanol.It is eventually adding phosphate buffered saline solution (pH7.4) the dissolution sediment for having chicken ovalbumin containing 2%, and is used
After 0.22 μm of sterilizing filter filtering, anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label can be obtained, in
4 DEG C save backup.
In an embodiment of invention, detection line 41 includes the first detection line 411 and the second detection line 412, by methyl testis
Ball ketone-chicken ovalbumin conjugate is coated on the first detection line 411, and diethylstilbestrol-chicken ovalbumin conjugate is coated with
In the second detection line 412；Sheep anti-mouse igg antibody is coated on nature controlling line 43.First detection line 411 is for detecting methyl therein
Stosterone residual, the second detection line 412 are used to judge the inspection of detection card for detecting diethylstilbestrol residual therein, nature controlling line 43
Whether effective survey result.In this way, the colloidal-gold detecting-card 100 being prepared can be realized while detect methyl testis in aquatic products
The residual of ketone and diethylstilbestrol.
In an embodiment of invention, Methyltestosterone-chicken ovalbumin conjugate is prepared by the following steps to obtain:
By Methyltestosterone and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, Methyltestosterone-chicken ovalbumin conjugate is obtained.
Specifically, by Methyltestosterone (MT) and hydrochloric acid carboxymethyl azanol hybrid reaction, haptens MT-3- carboxymethyl is obtained
Oxime, then reacted MT-3- carboxymethyl oxime with the primary amino group of chicken ovalbumin by mixed anhydride method, amide cross-bond is formed,
Methyltestosterone-chicken ovalbumin conjugate antigen is made.Methyltestosterone obtained-chicken ovalbumin the conjugate can
UV scanning is carried out at 200nm-300nm wavelength, its coupling ratio, i.e. mole of MT and carrier protein are calculated by calculation method
Than.
In an embodiment of invention, diethylstilbestrol-chicken ovalbumin conjugate is prepared by the following steps to obtain:
By diethylstilbestrol and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, diethylstilbestrol-chicken ovalbumin conjugate is obtained.
Specifically, by diethylstilbestrol and hydrochloric acid carboxymethyl azanol hybrid reaction, haptens diethylstilbestrol-carboxymethyl is obtained
Oxime, then reacted diethylstilbestrol-carboxymethyl oxime with the primary amino group of chicken ovalbumin by mixed anhydride method, form amide crosslinking
Diethylstilbestrol-chicken ovalbumin conjugate antigen is made in key.Diethylstilbestrol obtained-chicken ovalbumin the conjugate can
UV scanning is carried out at 200nm-300nm wavelength, its coupling ratio, i.e. mole of MT and carrier protein are calculated by calculation method
Than.
Present invention improves over antigen coupling proteins to be changed existing small using chicken ovalbumin as carrier protein
The interference of bovine serum albumin bring non-specific binding to improve the sensitivity of its detection, while also reducing reality
The false positive of sample detection.
The invention also provides a kind of hormone residues detection methods, are applied to foregoing colloidal-gold detecting-card 100, should
Detection method includes the following steps for hormone residues:
Sample to be measured is pre-processed, sample liquid to be checked is obtained；
Sample drop to be checked is added to the sample pad 20 of colloidal-gold detecting-card 100, stands a period of time, observes cellulose nitrate
Whether the detection line 41 and nature controlling line 43 of plain film 40 develop the color；
If detection line 41 and nature controlling line 43 develop the color, testing result is determined as feminine gender, shows in sample without containing aquatic products
Hormone or aquatic products hormone-content are lower than detection limit；
If detection line 41 does not develop the color, nature controlling line 43 develops the color, and testing result is determined as the positive, shows aquatic products hormone in sample
Content is equal to or higher than detection limit；
If nature controlling line 43 does not develop the color, testing result is invalid, shows that incorrect operation or detection block failure out of date.
Specifically, sample liquids to be measured are pre-processed first, obtains sample liquid to be checked.Then using pipettor or
Sample prepare liquid is passed through well 61 and is added dropwise in sample pad 20 by dropper, after standing 10min-20min, can observe nitric acid
Whether the detection line 41 and nature controlling line 43 of cellulose membrane 40 develop the color situation.If detection line 41 colour developing be deeper than nature controlling line 43 colour developing or
Close with the colour developing of nature controlling line 43, testing result is determined as feminine gender, shows in sample without containing aquatic products hormone or aquatic products hormone
(Methyltestosterone or diethylstilbestrol) content is lower than detection limit；If detection line 41 does not develop the color, no matter whether nature controlling line 43 develops the color, inspection
Surveying result judgement is the positive, shows that (the Methyltestosterone or diethylstilbestrol) content of aquatic products hormone in sample is equal to or higher than detection
Limit；If nature controlling line 43 does not develop the color, testing result is invalid, shows that incorrect operation or detection block failure out of date.It then needs to adopt
It is detected again with new detection card.
Hormone residues detection method of the present invention is simple, quick, effective and sensitivity is higher, and can be realized while examining
Survey the residual of Methyltestosterone and diethylstilbestrol.
In one embodiment of this invention, step " is pre-processed, obtain sample liquid to be checked " to sample to be measured includes
Following steps:
Remove skin bone fat sample, with homogenizer homogeneous uniform sample；
This liquid is sampled in test tube, deionized water is added and mixes, and ethyl acetate extraction is added, concussion, centrifugation discard
Layer clear liquid, lower liquid nitrogen or air blow drying；
Phosphate buffered saline solution is added into the test tube of drying, rinses the residue on dissolution inboard wall of test tube, obtains sample
Liquid to be checked.
Specifically, firstly, removing the sample for fat of boning, and uniform sample liquid is obtained with homogenizer homogeneous.
Then a certain amount of sample liquid is weighed in centrifuge tube, and deionized water is added or distilled water mixes well, adds ethyl acetate
It extracts, vibrates 5min, carry out centrifugation 10min more than the revolving speed of 4000r/min at room temperature, it is invisible spectro after discarding supernatant liquid
Lower liquid under 65 DEG C of nitrogen or air draught using drying up.Phosphate buffered saline solution finally is added into the test tube of drying,
It rinses and dissolves the residue on inboard wall of test tube, sample liquid to be checked can be obtained.The method of the pretreatment sample to be tested operates
It is relatively simple, and a sample can detect the residual of Methyltestosterone and diethylstilbestrol simultaneously after pretreatment, detection
Efficiency greatly improves.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all at this
Under the inventive concept of invention, using equivalent structure transformation made by description of the invention and accompanying drawing content, or directly/use indirectly
It is included in other related technical areas in scope of patent protection of the invention.
Claims (10)
1. a kind of colloidal-gold detecting-card, for detecting the hormone residues in aquatic products, which is characterized in that the colloidal-gold detecting-card
Including support plate, sample pad, gold conjugation pad, nitrocellulose filter and water absorption pad, the sample pad, the colloidal gold are combined
Pad, the nitrocellulose filter and the water absorption pad are sequentially arranged in described along the direction of first end to the second end of the detection card
The plate face of support plate, the nitrocellulose filter are provided with detection line and nature controlling line；The gold conjugation pad is coated with colloid
The anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody of gold label.
2. colloidal-gold detecting-card as described in claim 1, which is characterized in that the detection line includes the first detection line and second
Detection line, first detection line, second detection line and the nature controlling line are along the first end to the side of the second end
To being set in turn in the nitrocellulose filter backwards to the surface of the water absorption pad.
3. colloidal-gold detecting-card as claimed in claim 2, which is characterized in that first detection line is coated with methyl testis
Ketone-chicken ovalbumin conjugate, second detection line are coated with diethylstilbestrol-chicken ovalbumin conjugate, the matter
Control line is coated with sheep anti-mouse igg antibody.
4. colloidal-gold detecting-card as claimed any one in claims 1 to 3, which is characterized in that the support plate is polychlorostyrene second
Alkene plate；
And/or the material of the sample pad is glass fibre；
And/or the material of the gold conjugation pad is polyester film；
And/or the water absorption pad is filter paper.
5. a kind of preparation method of colloidal-gold detecting-card, which is characterized in that the preparation method the following steps are included:
The mixture for fighting Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody carries out colloid gold label, obtains
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody of colloid gold label:
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody of the colloid gold label is coated on gold conjugation pad；
In the same direction successively by sample pad, coated gold conjugation pad, the nitric acid for being coated with detection line and nature controlling line are fine
It ties up plain film and water absorption pad is attached at the plate face of support plate, after cutting and be fitted into template the colloidal-gold detecting-card is made.
6. the preparation method of colloidal-gold detecting-card as claimed in claim 5, which is characterized in that described " to obtain colloid gold label
Anti- Methyltestosterone-diethylstilbestrol monoclonal antibody " step the following steps are included:
Taking granular size is the colloidal gold solution of 20nm-40nm, and the pH value of colloidal gold solution is adjusted to 7-9；
The mixture of anti-Methyltestosterone monoclonal antibody and anti-diethylstilbestrol monoclonal antibody is added into colloidal gold solution, stirs
Mixing is mixed, polyethylene glycol is added, stir and is centrifuged, liquid is discarded supernatant, obtains sediment；
Phosphate buffered saline solution is added to clean sediment, and uses the phosphate buffered saline solution containing chicken ovalbumin
Sediment is dissolved, anti-Methyltestosterone-diethylstilbestrol monoclonal antibody of the colloid gold label is obtained after filtering.
7. the preparation method of colloidal-gold detecting-card as claimed in claim 5, which is characterized in that the detection line includes the first inspection
Methyltestosterone-chicken ovalbumin conjugate is coated on first detection line by survey line and the second detection line, and hexene is female
Phenol-chicken ovalbumin conjugate is coated on second detection line；Sheep anti-mouse igg antibody is coated on the nature controlling line.
8. the preparation method of colloidal-gold detecting-card as claimed in claim 7, which is characterized in that the Methyltestosterone-ovum gallinaceum
Pure protein conjugate is prepared by the following steps to obtain:
By Methyltestosterone and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, Methyltestosterone-chicken ovalbumin conjugate is obtained；
And/or the diethylstilbestrol-chicken ovalbumin conjugate is prepared by the following steps to obtain:
By diethylstilbestrol and hydrochloric acid carboxymethyl azanol hybrid reaction, reactant is obtained；
By reactant and chicken ovalbumin hybrid reaction, diethylstilbestrol-chicken ovalbumin conjugate is obtained.
9. a kind of hormone residues detection method is applied to colloidal-gold detecting-card, which is characterized in that the hormone residues detection method packet
Include following steps:
Sample to be measured is pre-processed, sample liquid to be checked is obtained；
Sample drop to be checked is added to the sample pad of colloidal-gold detecting-card, 10mim-20mim is stood, observes nitrocellulose filter
Whether detection line and nature controlling line develop the color；
If detection line and nature controlling line develop the color, testing result is determined as feminine gender, shows in sample without containing aquatic products hormone or water
Product hormone-content is lower than detection limit；
If detection line does not develop the color, nature controlling line colour developing, testing result is determined as the positive, shows that aquatic products hormone-content is equal in sample
Or it is higher than detection limit；
If nature controlling line does not develop the color, testing result is invalid, shows that incorrect operation or detection block failure out of date.
10. hormone residues detection method as claimed in claim 9, which is characterized in that described " to be located in advance to sample to be measured
Reason, obtain sample liquid to be checked " step the following steps are included:
Remove skin bone fat sample, with homogenizer homogeneous uniform sample；
This liquid is sampled in test tube, deionized water is added and mixes, and ethyl acetate extraction is added, it is clear to discard upper layer for concussion, centrifugation
Liquid, lower liquid nitrogen or air blow drying；
Phosphate buffered saline solution is added into the test tube of drying, rinses the residue on dissolution inboard wall of test tube, it is to be checked to obtain sample
Liquid.
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