CN106645683A - Orange II colloidal gold testing card and preparation method thereof - Google Patents
Orange II colloidal gold testing card and preparation method thereof Download PDFInfo
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- CN106645683A CN106645683A CN201610898665.8A CN201610898665A CN106645683A CN 106645683 A CN106645683 A CN 106645683A CN 201610898665 A CN201610898665 A CN 201610898665A CN 106645683 A CN106645683 A CN 106645683A
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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
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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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Abstract
The invention provides an orange II colloidal gold testing card. The testing card is mainly used for quickly detecting whether the orange II ingredient is illegally added into a food or not, and comprises a substrate, filter testing paper, immune colloidal gold paper, an immune cellulose nitrate film and absorbing paper, wherein the filter testing paper, the immune colloidal gold paper, the immune cellulose nitrate film and the absorbing paper are connected sequentially end to end and are fixed on the substrate; the immune colloidal gold paper is coated with a colloidal gold labeled second species animal protein and anti-orange II monoclonal antibody; and the immune cellulose nitrate film is provided with a detection line coated with orange II-bovine serum albumin composition and an IgG quality control line coated with anti-second species animal protein. The orange II colloidal gold testing card has the beneficial effects of strong specificity, high sensitivity and the like, is simple and convenient to operate, requires an application environment temperature of 4-35 DEG C, and is applicable to quick detection on orange II ingredient in foods for individuals, factories, food hygiene quality detection departments, customs and the like.
Description
Technical field
Belong to field of detection of food safety, and in particular to the detection method of illegal additive, particularly gold orange II in food
Test paper detecting method.
Background technology
Gold orange II, also known as Acid Orange II, No. two oranges, orange orange or Acidity golds, popular name Acid orange Ⅱ, chemical entitled 2- naphthalenes
Phenol azo P-TOLUENE SULFO ACID 99's sodium, is a kind of abiotic synthetic coloring matter, is soluble in ethanol, acetonitrile etc..It is a kind of chemical dyestuff, mainly
For leather, textile dyeing, forbid being used as food additives.But it has the spy such as bright in colour, coloring stabilized, inexpensive
Point, some illegal retailers use it for production of crude drugs and processing, serious harm consumer health.
Acid orange Ⅱ has stronger toxicity and carcinogenicity, and domestic and international pertinent regulations are forbidden for food color.However, near
The ground such as Nian Lai, Guangdong, Sichuan have been reported that this kind of dyestuff is abused in the food such as cured is burnt by some producers, there is serious food
Potential safety hazard.At present, the method for inspection of gold orange II mostly is high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, RP-HPLC
Chromatography etc..The method such as high performance liquid chromatography and LC-MS chromatogram is state-of-the-art modern analysis means, be can be good at point
From detection gold orange II, and a very low test limit is reached, sensitivity is very high.Using standard control method, can be very
Accurately detect the content of gold orange II in sample.
In addition, also finding no the fast inspection detection card of gold orange II on the market, according to above-mentioned experimental technique, operating process is all
More complicated, experimental period length, experimental cost are high, and are not particularly suited for Site Detection.
The content of the invention
It is an object of the invention to provide whether a kind of colloidal-gold detecting-card of gold orange II, be mainly used in non-in quick detection food
Method adds the composition of gold orange II.
The technical solution used in the present invention is as follows：A kind of colloidal-gold detecting-card of gold orange II, including reagent strip, the reagent strip
Including substrate, filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter and blotting paper；The filter sample paper, immune colloid gold
The scraps of paper, immune nitrocellulose filter and blotting paper tandem array and are fixed on the substrate successively；The immune colloid gold paper
The second kind animal protein and the monoclonal antibody of anti-gold orange II of colloid gold label, the immune celluloid are coated with piece
Film is provided with and is coated with the detection line of gold orange II-bovine serum albumin(BSA) compound and is coated with anti-second kind animal protein
The nature controlling line of IgG.
Preferably, the immune colloid gold scraps of paper have colloid gold particle, and colloid gold particle size is in 30nm or so.
Preferably, the collaurum is prepared by trisodium citrate reduction method, and trisodium citrate reduction method is：Prepare
Concentration is 0.01% HAuCl4Aqueous solution 100ml, water-bath or oil bath to 95-100 DEG C, control temperature constant, back stirring
Side adds 1% trisodium citrate (Na3C6H5O7·2H2O) aqueous solution 1.80ml, continues agitating heating 20 minutes or so, and ultrasound is shaken
It is cooled to that keeping temperature in 4.0 DEG C of water-baths is constant after swinging (frequency is in 20-30kHz) 2-3 minutes, that is, 30nm or so particle diameter is obtained
Colloidal gold solution；
Wherein, the various aqueous solution are configured and boils off ionized water using double steamings or three.
Limpid transparent using collaurum obtained in said method, grain size is homogeneous, without agglutinating particle, does not find substantially non-
Spheroidal particle.
Preferably, the immune colloid gold scraps of paper be through pretreatment all-glass paper, the pretreatment of the pretreatment
Liquid includes 1～1.5%Tween-80,6～8% disaccharide or polysaccharide, and solvent is water.
It is a further object of the present invention to provide a kind of preparation method of the colloidal-gold detecting-card of gold orange II, comprises the following steps：
1) trisodium citrate reduction method prepares collaurum；
2) with step 1) collaurum for preparing, the anti-monoclonal antibody of gold orange II is marked, obtain second of colloid gold label
Category animal protein and the monoclonal antibody of anti-gold orange II, i.e., required immune colloid gold；
3) preparation of the immune colloid gold scraps of paper：Pretreated glass fibrous paper；Dilution step 2) colloid gold label that obtains
Second kind animal protein and the monoclonal antibody of anti-gold orange II, obtain immune colloid gold solution；With the immune colloid gold solution
The pretreated all-glass paper of coating, the adaptive immune collaurum scraps of paper；
4) IgG of II-bovine serum albumin(BSA) of gold orange compound and anti-second kind animal protein is sprayed on into respectively nitric acid fine
On the position of plain film detection line (T lines) of dimension and nature controlling line (C lines), dry for standby is obtained immunity nitrocellulose filter；
5) filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter, blotting paper are pasted onto successively on offset plate, are cut
Tailor to obtain reagent strip；
6) finally reagent strip loaded into plastic casing, that is, obtains the colloidal-gold detecting-card of gold orange II.
Wherein, preferably, step 2) in, the ratio of the monoclonal antibody of anti-gold orange II of colloid gold label is：To collaurum
In add the anti-monoclonal antibody of gold orange II according to the ratio of 18-20 μ g antibody/(ml collaurums), prepare immune colloid gold
(anti-II monoclonal antibody of gold orange-collaurum).
Preferably, step 3) in, per 30-35ml pretreatment fluids immersion all-glass paper 261mm × 220mm × 0.5mm, do
After dry, then with immune colloid gold solution spraying all-glass paper, spray on every 261mm × 220mm × 0.5mm all-glass papers
20-25ml immune colloid gold solution, is dried, and the immune colloid gold scraps of paper are obtained.
Preferably, step 4) in, the anti-second kind animal protein being sprayed on nitrocellulose filter nature controlling line (C lines)
The concentration of IgG is 0.3～0.5mg/ml, and the II-bovine serum albumin(BSA) of gold orange being sprayed in nitrocellulose filter detection line (T lines) is multiple
Compound concentration is 0.4～0.6mg/ml.
Preferably, it is coated with II-the bovine serum albumin of goat anti-rabbit igg and gold orange of 1ml respectively per the long nitrocellulose filters of 1m
The spacing of white complex solution, detection line and nature controlling line is 5.0mm.
The present invention's employs trisodium citrate reduction method, obtains collaurum molecule in spherical of uniform 30nm
Grain, is of moderate size due to colloid gold particle, overall homogeneous so as to which, effect higher with the joint efficiency of monoclonal antibody is more stable.
The operation principle of the colloidal-gold detecting-card of gold orange of the present invention II is：Using the antibody-antigene specificity knot of high special
Close reaction and immune film chromatographic technique, the composition of gold orange II for suppressing method to occur in qualitative detection food by Immune competition.
Detection reagent bar in detection card is to realize the key that gold orange II detects, in reagent strip detection line (T lines) coating
Gold orange II-bovine serum albumin(BSA) compound；The anti-gold orange of colloid gold label is fixed with the immune colloid gold scraps of paper of reagent strip
II monoclonal antibody.When sample extracting solution from well add, penetrate on the sample-adding pad of reagent strip, tested sample first with glass
The monoclonal antibody of gold orange II of the colloid gold label on glass tunica fibrosa is combined, and continues to chromatograph to immune nitric acid by capillarity
Cellulose membrane, sequentially by specking on immune nitrocellulose filter the detection line of gold orange II-bovine serum albumin(BSA) compound and
The nature controlling line of the IgG of the anti-second kind animal protein of specking.If containing gold orange II in sample extracting solution, they will be with gold orange
Limited antigen binding site on II-bovine serum albumin(BSA) compound competition collaurum-antibody of anti-gold orange II, when the concentration of gold orange II
When reaching more than the threshold concentration of product design, they will occupy all or part of antigen in the monoclonal antibody of anti-gold orange II
Binding site, so it is prevented that II-the ox blood of gold orange of the monoclonal antibody of anti-gold orange II of colloid gold label and detection line is pure
Albumen composition is combined, and detection line can not capture enough colloid gold particles and the red ribbon shallow compared with nature controlling line or nothing occurs
Red ribbon occurs, and testing result is positive.If the concentration without gold orange II or gold orange II in sample extracting solution is less than threshold value
Concentration, then anti-II monoclonal antibody of gold orange-collaurum run to detection line in company with sample extracting solution, detection line captures collaurum
Particle and deep compared with nature controlling line or equally deep red ribbon is presented, testing result is negative.
Nature controlling line (C lines) on reagent strip is coated with the IgG of anti-second kind animal protein, to indicate detection card reaction
Whether system work is normal, and (the second kind animal protein on the immune colloid gold scraps of paper, the operation with sample on paper slip is
Nature controlling line position is reached, the second kind animal protein is combined with the IgG of anti-second kind animal protein, you can occurred red
Colour band).The appearance of nature controlling line is unrelated with the presence of gold orange II.The appearance of nature controlling line colour band shows：1. sample introduction is sufficient 2.
Sample normal operation on paper slip.
The present invention detects that card sample treatment is：Testing sample epidermis or sample liquid are weighed in reaction vessel, plus
Enter appropriate solvent, fully shaking is mixed, and makes gold orange II as much as possible molten in solvent.The solvent can be water, ethanol, methyl alcohol
Or one or more in other solvents.Pure water is wherein preferably, because water the safest (protection operator), extraction effect
Ideal, so can fully extract the gold orange II in food by 3～5 times of preferably the taken amount of food of volume of the solvent,
Again without the content dilution by gold orange II too much with reduction detection effect.
The using method of colloidal gold strip：
1. detection card is placed on level table.
2., with sample-adding suction pipe pipette samples extract, then drop 5 drips (about 120ul) sample extracting solution to the sample-adding of detection card
Kong Zhong.Often detect that a different sample notes using different suction pipes.
3. result is observed：The 5-8 minute sentence read results after sample is added dropwise.
The determination methods of testing result：
It is negative：Occur two colour bands in result watch window, T lines (detection line, near well one end) colour developing is than C line
It is deep or equally deep, represent and exist without gold orange II in sample.
It is positive：In result watch window, T lines are substantially shallow than C line or T lines are without colour developing, represent in sample there is the presence of gold orange II.
It is invalid：Nature controlling line (C lines) is occurred without.In any case, C lines all should be formed, and represented sample-adding and operated correct.C lines
Do not occur showing that test result is uncertain, it may be possible to which misoperation or agent plate fail, should reform.
The present invention beneficial effect such as have high specificity, sensitivity high, simple to operation, be using required environment temperature
4-35 DEG C, being suitable for individual and factory, food hygiene quality testing department, customs etc. carries out the quick inspection of the composition of gold orange II to food
Survey.
The present invention beneficial effect such as have high specificity, sensitivity high, simple to operation, be using required environment temperature
4-35 DEG C, being suitable for individual and factory, food hygiene quality testing department, customs etc. carries out the quick inspection of the composition of gold orange II to food
Survey, and the inventive method suffers from obvious advantage on detecting step and testing cost.
Description of the drawings
Fig. 1 is the reagent strip schematic diagram of the colloidal-gold detecting-card of gold orange II, in figure：1. sample paper is filtered；2. the immune colloid gold scraps of paper；
3. detection line；4. nature controlling line；5. immune nitrocellulose filter；6. blotting paper；7. substrate.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this
It is bright, and unrestricted the scope of the present invention.
As shown in figure 1, a kind of colloidal-gold detecting-card of gold orange II, including reagent strip, the reagent strip includes substrate 7, filter sample
Paper 1, the immune colloid gold scraps of paper 2, immune nitrocellulose filter 5 and blotting paper 6；It is described to filter sample paper 1, the immune colloid gold scraps of paper 2, exempt from
Epidemic disease nitrocellulose filter 5 and blotting paper 6 tandem array and are fixed on the substrate 7 successively；On the immune colloid gold scraps of paper 2
The second kind animal protein and the monoclonal antibody of anti-gold orange II of colloid gold label are coated with, on the immune nitrocellulose filter
The detection line 3 for being coated with gold orange II-bovine serum albumin(BSA) compound is provided with the IgG's for being coated with anti-second kind animal protein
Nature controlling line 4.
It is below embodiments of the invention：
Embodiment 1：The preparation of the colloidal-gold detecting-card of gold orange II；
1. the preparation of collaurum：Trisodium citrate reduction method：
Prepare the HAuCl that concentration is 0.01%4Aqueous solution 100ml, water-bath or oil bath control temperature constant to 99 DEG C, it
Add 1% trisodium citrate (Na while stirring afterwards3C6H5O7·2H2O) aqueous solution 1.80ml, continues 20 minutes left sides of agitating heating
The right side, sonic oscillation, frequency is 25kHz, and it is constant to be cooled to keeping temperature in 4.0 DEG C of water-baths after 3 minutes, that is, 30nm or so grain is obtained
The colloidal gold solution in footpath；
Wherein, the various aqueous solution are configured and boils off ionized water using double steamings or three.
2. the preparation of immune colloid gold；
2.1 the monoclonal antibody of anti-gold orange II is diluted to into concentration with the phosphate buffer of 0.1M pH8.0 is 1.0mg/ml
Antibody-solutions.
2.2 colloidal gold solutions for taking 1000ml, the phosphate buffers of 0.1M pH 8.0 that 100ml is added inward mix 3 points
Clock.Under fast stirring, then slowly the monoclonal antibody solution 20ml of anti-gold orange II of dilution is added thereto.5 points of room temperature reaction
Clock is simultaneously slowly stirred frequently.
2.3 reaction terminate after, rapidly join in above-mentioned reactant liquor the 10% of 20ml bovine serum albumin(BSA) (BSA) it is molten
Liquid, room temperature reaction 5 minutes is simultaneously slowly stirred frequently.
2.4 are centrifuged the 8000 turns/min of immune colloid gold for preparing 20 minutes, retain precipitation, and collect supernatant 10000
Turn/min be centrifuged 30 minutes, abandon supernatant.Collect borate buffer of the centrifugation twice containing 0.1%BSA to redissolve to OD540
10.
3. immune colloid gold is made of paper standby；
The preparation of 3.1 pretreatment fluids：Accurately weigh 10ml Tween-80,70g soluble starches, plus purified water to be settled to
1000ml。
The preparation of 3.2 metal spraying buffer solutions：800ml purified waters are taken, 150ml 1.0M Tris liquid is added inward, adjusted with hydrochloric acid
Section pH to 8.0.Accurately weigh 3.0g PEG 20000s, 2.0g bovine serum albumin(BSA)s, 2.0g skim milks, 5.0g caseins
Solution and 0.6g sodium azide are added in solution, fully dissolving, are well mixed, plus purified water is to cumulative volume 1000ml.
3.3 monoclonal antibodies of anti-gold orange II that colloid gold label is diluted with metal spraying buffer solution, to solution O D540It is worth for 1.5,
Adaptive immune colloidal gold solution.
3.4 soak all-glass paper with pretreatment fluid, per 30ml pretreatment fluids immersion all-glass paper 261mm × 220mm
× 0.5mm, after immersion 30min, is dried at 37 DEG C；Immune colloid gold solution spraying all-glass paper is used again, per 261mm ×
25ml immune colloid gold solution is sprayed on 220mm × 0.5mm all-glass papers, is dried, the immune colloid gold scraps of paper are obtained.
4. the preparation of immune nitrocellulose filter；
Goat anti-rabbit igg phosphate buffer is diluted to 0.4mg/ml by 4.1, and nature controlling line (C lines) solution is obtained.
4.2 gold orange II-bovine serum albumin(BSA) compound phosphate buffer is diluted to into protein concentration is 0.5mg/ml,
Prepared detection line (T lines) solution.
4.3, with point film machine specking C lines, T line solution, are obtained immunity nitrocellulose filter.The long nitrocellulose filter point per 1m
The spacing of the C lines and T line solution, detection line and nature controlling line that are not coated with 1ml is 5.0mm.
5. as shown in figure 1, filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter, blotting paper are pasted onto successively
On offset plate, cutting is into the reagent strip that width is 4mm.
6. detection reagent bar is loaded and detection detection card is obtained final product in plastic casing.
The threshold value of the colloidal-gold detecting-card of gold orange II of present invention design is 1 μ g/ml.
Embodiment 2：The sensitivity experiment of the colloidal-gold detecting-card of gold orange II；
1. detection method：
(1) colloidal-gold detecting-card of gold orange II prepared by Example 1, detection card is placed on level table.
(2) with sample-adding suction pipe pipette samples, then drop 5 drips (about 120ul) sample in the well of detection card.Often detect
A different sample uses different suction pipes.
(3) result is observed：The 5-10 minute sentence read results after sample is added dropwise.
2. detection sample：
Configuration gold orange II concentration is 0 μ g/ml, 0.1 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, Quality Control reference material make
For sample, each concentration is repeated 3 times.
3. testing result：
Concentration is 0 μ g/ml, and the sample standard deviation of 0.1 μ g/ml, 0.5 μ g/ml shows negative, 1 μ g/ml, the sample standard deviation of 2 μ g/ml
Show positive.The detection sensitivity of present invention detection card is 1 μ g/ml, and the colloidal-gold detecting-card sensitivity of gold orange II meets detection
Require.
Embodiment 3：The specificity experiments of the colloidal-gold detecting-card of gold orange II.
1. detection method：
(1) colloidal-gold detecting-card of gold orange II obtained in Example 1, detection card is placed on level table.
(2) drawn for detecting the detection sample that cross reaction is tested with sample-adding suction pipe, then drop 5 drips (about 120ul) sample
Product are in the well of detection card.Often detect that a different sample uses different suction pipes.
(3) result is observed：The 5-10 minute sentence read results after sample is added dropwise.
2. detection sample：
The detection card of the present invention is detected with the Basic Orange of variable concentrations, Basic Orange II, lemon yellow, sunset yellow etc.,
See whether cross reaction.
3. testing result：
As a result, it was confirmed that the detection card of the present invention detection of gold orange II will not be subject to Basic Orange, Basic Orange II, lemon yellow,
The interference of sunset yellow etc., i.e., the colloidal-gold detecting-card of gold orange II of the invention, will not be with the dyestuff of similar effect under finite concentration
Cross reaction is produced, specificity is good.
Embodiment 4：The repeatability of the colloidal-gold detecting-card of gold orange II and stability experiment；
First, detection card batch in and batch between repeated experiment；
1. experimental technique：
The colloidal-gold detecting-card of gold orange II of same batch and different batches is detected into respectively 0.1,0.5,1,2 μ g/ml standard items, often
Individual concentration is repeated 3 times, the repeatability of observation detection card.
2. experimental result：Empirical tests, the colloidal-gold detecting-card of gold orange II batch in and batch between repeatability be 100%, false positive
Rate and false negative rate are 0.
2nd, the stability experiment of detection card：
1. experiment purpose：
By the colloidal-gold detecting-card sealing preserve of gold orange II, and deposit under 4 DEG C and room temperature (25 DEG C or so), observation difference is deposited
Temperature is put to detecting the impact of card stability.
2. experimental technique：
The detection card for being stored in 4 DEG C took out 4 parts times per 20 days, and the standard of 0.1,0.5,1,2 μ g/ml concentration is detected respectively
Product；The detection card for being stored in room temperature (25 DEG C) took out 4 parts times per 10 days, and the standard of 0.1,0.5,1,2 μ g/ml concentration is detected respectively
Product.
3. experimental result：
Empirical tests, reagent strip can be preserved 24 months at 4 DEG C, can be preserved 18 months at room temperature, in the preservable time limit
Interior, detection card can reach the detection sensitivity of 1 μ g/ml.
Claims (10)
1. the colloidal-gold detecting-card of gold orange II, it is characterised in that：It is described detection card include substrate, filter sample paper, the immune colloid gold scraps of paper,
Immune nitrocellulose filter and blotting paper, wherein, the filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter and suction
Water paper tandem array and is fixed on substrate successively；The second kind of colloid gold label is coated with the immune colloid gold scraps of paper
Animal protein and the monoclonal antibody of anti-gold orange II, the immune nitrocellulose filter is provided with that to be coated with II-ox blood of gold orange pure
The detection line of albumen composition and be coated with anti-second kind animal protein IgG nature controlling line.
2. the colloidal-gold detecting-card of gold orange according to claim 1 II, it is characterised in that：Described anti-second kind animal egg
White IgG is goat anti-rabbit igg.
3. the colloidal-gold detecting-card of gold orange according to claim 1 II, it is characterised in that：The second described kind animal protein
For the protein of non-antibody source animal, the such as one kind in bovine serum albumin(BSA), chicken ovalbumin or human serum albumins, this
It is bright to be specially bovine serum albumin(BSA).
4. the colloidal-gold detecting-card of gold orange according to claim 1 II, it is characterised in that：The immune colloid gold scraps of paper have
Colloid gold particle, colloid gold particle size is generally circular in shape in 30nm or so, and size is of substantially equal, uniformity, without aggegation,
The collaurum is prepared by trisodium citrate reduction method.
5. the colloidal-gold detecting-card of gold orange according to claim 4 II, it is characterised in that：The colloidal-gold detecting-card of the gold orange II
Including the all-glass paper for carrying out pre-processing through pretreatment fluid, the pretreatment fluid of the pretreatment includes 1～1.5%
Tween-80,6～8% disaccharide, polysaccharide, solvent is water.
6. the preparation method of the colloidal-gold detecting-card of gold orange II as described in claim 1-5 any one, it is characterised in that：Including
Following steps：
1) trisodium citrate reduction method prepares collaurum；
2) with step 1) collaurum for preparing, mark the second kind animal protein and the monoclonal antibody of anti-gold orange II obtain colloid
The second kind animal protein and the monoclonal antibody of anti-gold orange II of gold mark；
3) preparation of the immune colloid gold scraps of paper：Pretreated glass fibrous paper；Dilution step 2) obtain colloid gold label second
Kind animal protein and the monoclonal antibody of anti-gold orange II, obtain immune colloid gold solution；It is coated with the immune colloid gold solution
Pretreated all-glass paper, the adaptive immune collaurum scraps of paper；
4) IgG of II-bovine serum albumin(BSA) of gold orange compound and anti-second kind animal protein is sprayed on into respectively celluloid
On the position of film detection line and nature controlling line, dry for standby is obtained immunity nitrocellulose filter；
5) filter sample paper, the immune colloid gold scraps of paper, immune nitrocellulose filter, blotting paper are pasted onto successively on offset plate, cutting system
Obtain reagent strip；
6) reagent strip is loaded into plastic casing, that is, obtains the colloidal-gold detecting-card of gold orange II.
7. the preparation method of the colloidal-gold detecting-card of gold orange as claimed in claim 6 II, it is characterised in that：The trisodium citrate
Reducing process is：
Prepare the HAuCl that concentration is 0.01%4Aqueous solution 100ml, water-bath or oil bath control temperature constant, afterwards to 95-100 DEG C
1% trisodium citrate (Na is added while stirring3C6H5O7·2H2O) aqueous solution 1.80ml, continues agitating heating 20 minutes or so,
Keeping temperature in 4.0 DEG C of water-baths is cooled to after sonic oscillation (frequency is in 20-30kHz) 2-3 minutes constant, that is, 30nm or so is obtained
The colloidal gold solution of particle diameter；
Wherein, the various aqueous solution are configured and boils off ionized water using double steamings or three.
8. the preparation method of the colloidal-gold detecting-card of gold orange as claimed in claim 6 II, it is characterised in that：The step 3) immunity
The collaurum scraps of paper are prepared as：
A. the monoclonal antibody of anti-gold orange II of colloid gold label is diluted with metal spraying buffer solution, OD is obtained540It is worth the immune glue for 1.5
Body gold solution；
B. all-glass paper is soaked with pretreatment fluid, after being dried, then with the pretreated glass fibers of immune colloid gold solution spraying
Dimension paper, is dried, and the immune colloid gold scraps of paper are obtained.
9. the preparation method of the colloidal-gold detecting-card of gold orange as claimed in claim 8 II, it is characterised in that：In step A
Metal spraying buffer formulation is as follows：10～15%1.0M Tris liquid, 0.2～0.5% PEG 20000,0.1～0.2% ox blood
Pure albumen, 0.1～0.3% skim milk, 0.4～0.6% casein, and 0.02～0.08% sodium azide, are adjusted with hydrochloric acid
PH is saved to 8.0 ± 0.05, balance of water.
10. the preparation method of the colloidal-gold detecting-card of gold orange as claimed in claim 6 II, it is characterised in that：The step 4) in,
The concentration of the IgG of the anti-second kind animal protein being sprayed on nitrocellulose filter nature controlling line (C lines) is 0.3～0.5mg/ml,
II-bovine serum albumin(BSA) of the gold orange complex concentration being sprayed in nitrocellulose filter detection line (T lines) is 0.4～0.6mg/ml.
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Address after: 511000 Guangzhou High-tech Industrial Development Zone, Guangzhou, Guangdong Province, No. 3 Lanyue Road, Science City, No. G204, G District, Guangzhou International Business Incubator Applicant after: Guangzhou Annuo Polytron Technologies Inc Address before: 511000 Guangzhou High-tech Industrial Development Zone, Guangzhou, Guangdong Province, No. 3 Lanyue Road, Science City, No. G204, G District, Guangzhou International Business Incubator Applicant before: GUANGZHOU ANNUO FOOD SCIENCE AND TECHNOLOGY CO., LTD. |
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Application publication date: 20170510 |